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Genomic Analysis of Vulcanisaeta thermophila Type Strain CBA1501T Isolated from Solfataric Soil
Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment
Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea. Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea.
The complete salmonid IGF IR gene repertoire and its transcriptional response to disease
The insulin-like growth factor (IGF) receptor (IGF-IR) is necessary for IGF signalling and has essential roles in cellular growth. In teleost fish, two distinct IGF-IR duplicates are conserved called IGF-IRa and IGF-IRb. However, while a salmonid-specific whole genome duplication (ssWGD) is known to have expanded several key genes within the IGF axis, its impact on the IGF-IR repertoire remains unresolved. Using bioinformatic and phylogenetic approaches, we establish that salmonids retain two IGF-IRa paralogues from ssWGD and a single IGF-IRb copy. We measured the tissue-specific and developmental transcriptional regulation of each IGF-IR gene, revealing tight co-expression between the IGF-IRa paralogues, but expression divergence comparing IGF-IRa and IGF-IRb genes. We also examined the regulation of each IGF-IR gene in fish challenged by bacterial and viral infections, adding to recent reports that the IGF axis has roles linking growth and immunity. While whole salmonid fry showed a small upregulation of IGF-IR expression during both types of infection, bacterial challenge caused striking downregulation of IGF-IRa1 and IGF-IRa2 in head kidney and spleen of adult fish, alongside genes coding IGF hormones, highlighting a strong repression of IGF-signalling in primary immune tissues. The reported immune-responsive regulation of IGF-IR genes adds to an emerging body of evidence that supports important cross-talk between master growth and immune pathways in vertebrates. The insulin-like growth factor (IGF) receptor (IGF-IR) is necessary for IGF signalling and has essential roles in cellular growth. In teleost fish, two distinct IGF-IR duplicates are conserved called IGF-IRa and IGF-IRb. However, while a salmonid-specific whole genome duplication (ssWGD) is known to have expanded several key genes within the IGF axis, its impact on the IGF-IR repertoire remains unresolved. Using bioinformatic and phylogenetic approaches, we establish that salmonids retain two IGF-IRa paralogues from ssWGD and a single IGF-IRb copy. We measured the tissue-specific and developmental transcriptional regulation of each IGF-IR gene, revealing tight co-expression between the IGF-IRa paralogues, but expression divergence comparing IGF-IRa and IGF-IRb genes. We also examined the regulation of each IGF-IR gene in fish challenged by bacterial and viral infections, adding to recent reports that the IGF axis has roles linking growth and immunity. While whole salmonid fry showed a small upregulation of IGF-IR expression during both types of infection, bacterial challenge caused striking downregulation of IGF-IRa1 and IGF-IRa2 in head kidney and spleen of adult fish, alongside genes coding IGF hormones, highlighting a strong repression of IGF-signalling in primary immune tissues. The reported immune-responsive regulation of IGF-IR genes adds to an emerging body of evidence that supports important cross-talk between master growth and immune pathways in vertebrates.
Differential Functional Constraints Cause Strain Level Endemism in Polynucleobacter Populations
Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients. Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients.ABSTRACT The adaptation of bacterial lineages to local environmental conditions creates the potential for broader genotypic diversity within a species, which can enable a species to dominate across ecological gradients because of niche flexibility. The genus Polynucleobacter maintains both free-living and symbiotic ecotypes and maintains an apparently ubiquitous distribution in freshwater ecosystems. Subspecies-level resolution supplemented with metagenome-derived genotype analysis revealed that differential functional constraints, not geographic distance, produce and maintain strain-level genetic conservation in Polynucleobacter populations across three geographically proximal riverine environments. Genes associated with cofactor biosynthesis and one-carbon metabolism showed habitat specificity, and protein-coding genes of unknown function and membrane transport proteins were under positive selection across each habitat. Characterized by different median ratios of nonsynonymous to synonymous evolutionary changes (dN/dS ratios) and a limited but statistically significant negative correlation between the dN/dS ratio and codon usage bias between habitats, the free-living and core genotypes were observed to be evolving under strong purifying selection pressure. Highlighting the potential role of genetic adaptation to the local environment, the two-component system protein-coding genes were highly stable (dN/dS ratio, < 0.03). These results suggest that despite the impact of the habitat on genetic diversity, and hence niche partition, strong environmental selection pressure maintains a conserved core genome for Polynucleobacter populations. IMPORTANCE Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients. ABSTRACT The adaptation of bacterial lineages to local environmental conditions creates the potential for broader genotypic diversity within a species, which can enable a species to dominate across ecological gradients because of niche flexibility. The genus Polynucleobacter maintains both free-living and symbiotic ecotypes and maintains an apparently ubiquitous distribution in freshwater ecosystems. Subspecies-level resolution supplemented with metagenome-derived genotype analysis revealed that differential functional constraints, not geographic distance, produce and maintain strain-level genetic conservation in Polynucleobacter populations across three geographically proximal riverine environments. Genes associated with cofactor biosynthesis and one-carbon metabolism showed habitat specificity, and protein-coding genes of unknown function and membrane transport proteins were under positive selection across each habitat. Characterized by different median ratios of nonsynonymous to synonymous evolutionary changes (dN/dS ratios) and a limited but statistically significant negative correlation between the dN/dS ratio and codon usage bias between habitats, the free-living and core genotypes were observed to be evolving under strong purifying selection pressure. Highlighting the potential role of genetic adaptation to the local environment, the two-component system protein-coding genes were highly stable (dN/dS ratio, < 0.03). These results suggest that despite the impact of the habitat on genetic diversity, and hence niche partition, strong environmental selection pressure maintains a conserved core genome for Polynucleobacter populations. IMPORTANCE Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients.
Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea enriched microbiome characterised by lactic acid formation and utilisation
Background Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep. Results Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings. Conclusions Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0201-2) contains supplementary material, which is available to authorized users. Background Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep. Results Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings. Conclusions Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0201-2) contains supplementary material, which is available to authorized users.
Tandem Duplication Events in the Expansion of the Small Heat Shock Protein Gene Family in Solanum lycopersicum (cv. Heinz 1706)
In plants, fruit maturation and oxidative stress can induce small heat shock protein (sHSP) synthesis to maintain cellular homeostasis. Although the tomato reference genome was published in 2012, the actual number and functionality of sHSP genes remain unknown. Using a transcriptomic (RNA-seq) and evolutionary genomic approach, putative sHSP genes in the Solanum lycopersicum (cv. Heinz 1706) genome were investigated. A sHSP gene family of 33 members was established. Remarkably, roughly half of the members of this family can be explained by nine independent tandem duplication events that determined, evolutionarily, their functional fates. Within a mitochondrial class subfamily, only one duplicated member, Solyc08g078700, retained its ancestral chaperone function, while the others, Solyc08g078710 and Solyc08g078720, likely degenerated under neutrality and lack ancestral chaperone function. Functional conservation occurred within a cytosolic class I subfamily, whose four members, Solyc06g076570, Solyc06g076560, Solyc06g076540, and Solyc06g076520, support ∼57% of the total sHSP RNAm in the red ripe fruit. Subfunctionalization occurred within a new subfamily, whose two members, Solyc04g082720 and Solyc04g082740, show heterogeneous differential expression profiles during fruit ripening. These findings, involving the birth/death of some genes or the preferential/plastic expression of some others during fruit ripening, highlight the importance of tandem duplication events in the expansion of the sHSP gene family in the tomato genome. Despite its evolutionary diversity, the sHSP gene family in the tomato genome seems to be endowed with a core set of four homeostasis genes: Solyc05g014280, Solyc03g082420, Solyc11g020330, and Solyc06g076560, which appear to provide a baseline protection during both fruit ripening and heat shock stress in different tomato tissues. In plants, fruit maturation and oxidative stress can induce small heat shock protein (sHSP) synthesis to maintain cellular homeostasis. Although the tomato reference genome was published in 2012, the actual number and functionality of sHSP genes remain unknown. Using a transcriptomic (RNA-seq) and evolutionary genomic approach, putative sHSP genes in the Solanum lycopersicum (cv. Heinz 1706) genome were investigated. A sHSP gene family of 33 members was established. Remarkably, roughly half of the members of this family can be explained by nine independent tandem duplication events that determined, evolutionarily, their functional fates. Within a mitochondrial class subfamily, only one duplicated member, Solyc08g078700, retained its ancestral chaperone function, while the others, Solyc08g078710 and Solyc08g078720, likely degenerated under neutrality and lack ancestral chaperone function. Functional conservation occurred within a cytosolic class I subfamily, whose four members, Solyc06g076570, Solyc06g076560, Solyc06g076540, and Solyc06g076520, support ∼57% of the total sHSP RNAm in the red ripe fruit. Subfunctionalization occurred within a new subfamily, whose two members, Solyc04g082720 and Solyc04g082740, show heterogeneous differential expression profiles during fruit ripening. These findings, involving the birth/death of some genes or the preferential/plastic expression of some others during fruit ripening, highlight the importance of tandem duplication events in the expansion of the sHSP gene family in the tomato genome. Despite its evolutionary diversity, the sHSP gene family in the tomato genome seems to be endowed with a core set of four homeostasis genes: Solyc05g014280, Solyc03g082420, Solyc11g020330, and Solyc06g076560, which appear to provide a baseline protection during both fruit ripening and heat shock stress in different tomato tissues.
The Evolution of the FT/TFL1 Genes in Amaranthaceae and Their Expression Patterns in the Course of Vegetative Growth and Flowering in Chenopodium rubrum
The FT/TFL1 gene family controls important aspects of plant development: MFT-like genes affect germination, TFL1-like genes act as floral inhibitors, and FT-like genes are floral activators. Gene duplications produced paralogs with modified functions required by the specific lifestyles of various angiosperm species. We constructed the transcriptome of the weedy annual plant Chenopodium rubrum and used it for the comprehensive search for the FT/TFL1 genes. We analyzed their phylogenetic relationships across Amaranthaceae and all angiosperms. We discovered a very ancient phylogenetic clade of FT genes represented by the CrFTL3 gene of C. rubrum. Another paralog CrFTL2 showed an unusual structural rearrangement which might have contributed to the functional shift. We examined the transcription patterns of the FT/TFL1 genes during the vegetative growth and floral transition in C. rubrum to get clues about their possible functions. All the genes except for the constitutively expressed CrFTL2 gene, and the CrFTL3 gene, which was transcribed only in seeds, exhibited organ-specific expression influenced by the specific light regime. The CrFTL1 gene was confirmed as a single floral activator from the FT/TFL1 family in C. rubrum. Its floral promoting activity may be counteracted by CrTFL1. C. rubrum emerges as an easily manipulated model for the study of floral induction in weedy fast-cycling plants lacking a juvenile phase. The FT/TFL1 gene family controls important aspects of plant development: MFT-like genes affect germination, TFL1-like genes act as floral inhibitors, and FT-like genes are floral activators. Gene duplications produced paralogs with modified functions required by the specific lifestyles of various angiosperm species. We constructed the transcriptome of the weedy annual plant Chenopodium rubrum and used it for the comprehensive search for the FT/TFL1 genes. We analyzed their phylogenetic relationships across Amaranthaceae and all angiosperms. We discovered a very ancient phylogenetic clade of FT genes represented by the CrFTL3 gene of C. rubrum. Another paralog CrFTL2 showed an unusual structural rearrangement which might have contributed to the functional shift. We examined the transcription patterns of the FT/TFL1 genes during the vegetative growth and floral transition in C. rubrum to get clues about their possible functions. All the genes except for the constitutively expressed CrFTL2 gene, and the CrFTL3 gene, which was transcribed only in seeds, exhibited organ-specific expression influenced by the specific light regime. The CrFTL1 gene was confirmed as a single floral activator from the FT/TFL1 family in C. rubrum. Its floral promoting activity may be counteracted by CrTFL1. C. rubrum emerges as an easily manipulated model for the study of floral induction in weedy fast-cycling plants lacking a juvenile phase.
16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife
Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses. Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs. IMPORTANCE Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses. ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs. IMPORTANCE Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.
Thermus and the Pink Discoloration Defect in Cheese
Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin. Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.ABSTRACT A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin. ABSTRACT A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.
Small RNA and degradome deep sequencing reveals drought‐and tissue‐specific micrornas and their important roles in drought‐sensitive and drought‐tolerant tomato genotypes
Summary Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post‐transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue‐specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought‐responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought‐related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress‐ and tissue development‐related genes, including DRP (dehydration‐responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD‐ZIP (homeodomain‐leucine zipper), MYB and NAC‐domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought‐tolerant tomato breeding. Summary Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post‐transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue‐specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought‐responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought‐related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress‐ and tissue development‐related genes, including DRP (dehydration‐responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD‐ZIP (homeodomain‐leucine zipper), MYB and NAC‐domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought‐tolerant tomato breeding.
Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator
Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean. Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean.
Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota
Seaweed is receiving an increasing amount of attention as a “sea vegetable”. The microbiota of coastal populations may acquire seaweed associated enzymes through marine food. Several agarases have been found in non-marine environments; however, their origin is unknown. In this study, a hypothetical protein, Aga1, was identified as an agarase from an inland soil agar-degrading bacterium, Paenibacillus sp. SSG-1.Having low similarity to known glycoside hydrolases, Aga1 may be a distant member of the glycoside hydrolase family 86. Aga1 has good pH stability (pH 3–11) and is stable in the presence of various metal ions. Aga1 is an exo-type β-agarase that produces NA 4 (neoagarotetraose) and NA 6 (neoagarohexaose) as its main products. In addition, Aga1 may be a cell-surface-binding protein. The bioinformatic analysis showed aga1 may have been transfered together with its surrounding genes, from marine bacteria to soil bacteria via human microbiota. The use of seaweed as food and the disposal of human faeces or saliva were the most likely reasons for this gene transfer pathway. Notably, the results also indicated that microbes from inland humans may degrade agar and that these microbes may have acquired seaweed associated genes because of increased seaweed in diets. Seaweed is receiving an increasing amount of attention as a “sea vegetable”. The microbiota of coastal populations may acquire seaweed associated enzymes through marine food. Several agarases have been found in non-marine environments; however, their origin is unknown. In this study, a hypothetical protein, Aga1, was identified as an agarase from an inland soil agar-degrading bacterium, Paenibacillus sp. SSG-1.Having low similarity to known glycoside hydrolases, Aga1 may be a distant member of the glycoside hydrolase family 86. Aga1 has good pH stability (pH 3–11) and is stable in the presence of various metal ions. Aga1 is an exo-type β-agarase that produces NA 4 (neoagarotetraose) and NA 6 (neoagarohexaose) as its main products. In addition, Aga1 may be a cell-surface-binding protein. The bioinformatic analysis showed aga1 may have been transfered together with its surrounding genes, from marine bacteria to soil bacteria via human microbiota. The use of seaweed as food and the disposal of human faeces or saliva were the most likely reasons for this gene transfer pathway. Notably, the results also indicated that microbes from inland humans may degrade agar and that these microbes may have acquired seaweed associated genes because of increased seaweed in diets.
Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis
Author Summary Ectodermal dysplasias refer to a large group of inherited disorders characterized by developmental defects in tissues of ectodermal origin. The study of these conditions has been instrumental in the discovery of biological pathways involved in the regulation of epithelial tissue morphogenesis. In this report, through the delineation of the molecular basis of a novel form of autosomal recessive ectodermal dysplasia, we identified a new key player in ectodermal development. We detected a number of mutations in TSPEAR co-segregating with abnormal hair and tooth development in three families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR was found to be strongly expressed in murine hair and tooth. Using a reporter assay, we showed that it regulates Notch activity. Accordingly, NOTCH1 expression was altered in patient skin, and NOTCH1, as well as many of its known targets, was down-regulated in TSPEAR deficient keratinocytes. Moreover, Tspear silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch pathway. As such, these new data are likely to lead to further investigations aimed at characterizing the role of Notch signaling pathway in other forms of ectodermal dysplasias as well as acquired hair and tooth pathologies. Author Summary Ectodermal dysplasias refer to a large group of inherited disorders characterized by developmental defects in tissues of ectodermal origin. The study of these conditions has been instrumental in the discovery of biological pathways involved in the regulation of epithelial tissue morphogenesis. In this report, through the delineation of the molecular basis of a novel form of autosomal recessive ectodermal dysplasia, we identified a new key player in ectodermal development. We detected a number of mutations in TSPEAR co-segregating with abnormal hair and tooth development in three families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR was found to be strongly expressed in murine hair and tooth. Using a reporter assay, we showed that it regulates Notch activity. Accordingly, NOTCH1 expression was altered in patient skin, and NOTCH1, as well as many of its known targets, was down-regulated in TSPEAR deficient keratinocytes. Moreover, Tspear silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch pathway. As such, these new data are likely to lead to further investigations aimed at characterizing the role of Notch signaling pathway in other forms of ectodermal dysplasias as well as acquired hair and tooth pathologies.Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway. Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway.
Whole Genome Sequence of a Beak and Feather Disease Virus Isolate from a Fledgling Red Capped Parrot (Purpureicephalus spurius)
The complete genome sequence of beak and feather disease virus (BFDV) from a fledgling red-capped parrot (Purpureicephalus spurius) was assembled and characterized. The genome consists of 1,995 nucleotides and encodes two major proteins in opposing directions. This is the first evidence of BFDV infectivity and a complete genome sequence for this novel host. The complete genome sequence of beak and feather disease virus (BFDV) from a fledgling red-capped parrot (Purpureicephalus spurius) was assembled and characterized. The genome consists of 1,995 nucleotides and encodes two major proteins in opposing directions. This is the first evidence of BFDV infectivity and a complete genome sequence for this novel host.
Geography and Location Are the Primary Drivers of Office Microbiome Composition
Our study highlights several points that should impact the design of future studies of the microbiology of BEs. First, projects tracking changes in BE bacterial communities should focus sampling efforts on surveying different locations in offices and in different cities but not necessarily different materials or different offices in the same city. Next, disturbance due to repeated sampling, though detectable, is small compared to that due to other variables, opening up a range of longitudinal study designs in the BE. Next, studies requiring more samples than can be sequenced on a single sequencing run (which is increasingly common) must control for run effects by including some of the same samples in all of the sequencing runs as technical replicates. Finally, detailed tracking of indoor and material environment covariates is likely not essential for BE microbiome studies, as the normal range of indoor environmental conditions is likely not large enough to impact bacterial communities. Our study highlights several points that should impact the design of future studies of the microbiology of BEs. First, projects tracking changes in BE bacterial communities should focus sampling efforts on surveying different locations in offices and in different cities but not necessarily different materials or different offices in the same city. Next, disturbance due to repeated sampling, though detectable, is small compared to that due to other variables, opening up a range of longitudinal study designs in the BE. Next, studies requiring more samples than can be sequenced on a single sequencing run (which is increasingly common) must control for run effects by including some of the same samples in all of the sequencing runs as technical replicates. Finally, detailed tracking of indoor and material environment covariates is likely not essential for BE microbiome studies, as the normal range of indoor environmental conditions is likely not large enough to impact bacterial communities.ABSTRACT In the United States, humans spend the majority of their time indoors, where they are exposed to the microbiome of the built environment (BE) they inhabit. Despite the ubiquity of microbes in BEs and their potential impacts on health and building materials, basic questions about the microbiology of these environments remain unanswered. We present a study on the impacts of geography, material type, human interaction, location in a room, seasonal variation, and indoor and microenvironmental parameters on bacterial communities in offices. Our data elucidate several important features of microbial communities in BEs. First, under normal office environmental conditions, bacterial communities do not differ on the basis of surface material (e.g., ceiling tile or carpet) but do differ on the basis of the location in a room (e.g., ceiling or floor), two features that are often conflated but that we are able to separate here. We suspect that previous work showing differences in bacterial composition with surface material was likely detecting differences based on different usage patterns. Next, we find that offices have city-specific bacterial communities, such that we can accurately predict which city an office microbiome sample is derived from, but office-specific bacterial communities are less apparent. This differs from previous work, which has suggested office-specific compositions of bacterial communities. We again suspect that the difference from prior work arises from different usage patterns. As has been previously shown, we observe that human skin contributes heavily to the composition of BE surfaces. IMPORTANCE Our study highlights several points that should impact the design of future studies of the microbiology of BEs. First, projects tracking changes in BE bacterial communities should focus sampling efforts on surveying different locations in offices and in different cities but not necessarily different materials or different offices in the same city. Next, disturbance due to repeated sampling, though detectable, is small compared to that due to other variables, opening up a range of longitudinal study designs in the BE. Next, studies requiring more samples than can be sequenced on a single sequencing run (which is increasingly common) must control for run effects by including some of the same samples in all of the sequencing runs as technical replicates. Finally, detailed tracking of indoor and material environment covariates is likely not essential for BE microbiome studies, as the normal range of indoor environmental conditions is likely not large enough to impact bacterial communities. ABSTRACT In the United States, humans spend the majority of their time indoors, where they are exposed to the microbiome of the built environment (BE) they inhabit. Despite the ubiquity of microbes in BEs and their potential impacts on health and building materials, basic questions about the microbiology of these environments remain unanswered. We present a study on the impacts of geography, material type, human interaction, location in a room, seasonal variation, and indoor and microenvironmental parameters on bacterial communities in offices. Our data elucidate several important features of microbial communities in BEs. First, under normal office environmental conditions, bacterial communities do not differ on the basis of surface material (e.g., ceiling tile or carpet) but do differ on the basis of the location in a room (e.g., ceiling or floor), two features that are often conflated but that we are able to separate here. We suspect that previous work showing differences in bacterial composition with surface material was likely detecting differences based on different usage patterns. Next, we find that offices have city-specific bacterial communities, such that we can accurately predict which city an office microbiome sample is derived from, but office-specific bacterial communities are less apparent. This differs from previous work, which has suggested office-specific compositions of bacterial communities. We again suspect that the difference from prior work arises from different usage patterns. As has been previously shown, we observe that human skin contributes heavily to the composition of BE surfaces. IMPORTANCE Our study highlights several points that should impact the design of future studies of the microbiology of BEs. First, projects tracking changes in BE bacterial communities should focus sampling efforts on surveying different locations in offices and in different cities but not necessarily different materials or different offices in the same city. Next, disturbance due to repeated sampling, though detectable, is small compared to that due to other variables, opening up a range of longitudinal study designs in the BE. Next, studies requiring more samples than can be sequenced on a single sequencing run (which is increasingly common) must control for run effects by including some of the same samples in all of the sequencing runs as technical replicates. Finally, detailed tracking of indoor and material environment covariates is likely not essential for BE microbiome studies, as the normal range of indoor environmental conditions is likely not large enough to impact bacterial communities.
Draft Genome Sequences of Four Salmonella enterica Strains Isolated from Turkey Associated Sources
We report the draft genomes of four Salmonella enterica isolates evaluated for the contribution of plasmids to virulence. Strains SE163A, SE696A, and SE710A carry plasmids demonstrated to facilitate plasmid-associated virulence, while SE819 is less virulent and has been used as a recipient for conjugation experiments to assess plasmid-encoded virulence mechanisms. We report the draft genomes of four Salmonella enterica isolates evaluated for the contribution of plasmids to virulence. Strains SE163A, SE696A, and SE710A carry plasmids demonstrated to facilitate plasmid-associated virulence, while SE819 is less virulent and has been used as a recipient for conjugation experiments to assess plasmid-encoded virulence mechanisms.
Draft Genome Sequence of Streptomyces sp. SPMA113, a Prajinamide Producer
We report here the draft genome sequence of Streptomyces sp. SPMA113 isolated from soil in Thailand. This strain produces a new modified peptide, prajinamide, which has adipocyte differentiation activity. The genome harbors at least 30 gene clusters for synthases of polyketide and nonribosomal peptide, suggesting its potential to produce diverse secondary metabolites. We report here the draft genome sequence of Streptomyces sp. SPMA113 isolated from soil in Thailand. This strain produces a new modified peptide, prajinamide, which has adipocyte differentiation activity. The genome harbors at least 30 gene clusters for synthases of polyketide and nonribosomal peptide, suggesting its potential to produce diverse secondary metabolites.
Draft Genome Sequence of Marine Derived Bacillus subtilis TP B0611, a Producer of Bacilosarcins and Amicoumacins
Here, we report the draft genome sequence of Bacillus subtilis TP-B0611, which produces the isocoumarin-type compounds bacilosarcin and amicoumacin. The genome encodes three nonribosomal peptide synthetase (NRPS) gene clusters and one hybrid polyketide synthase (PKS)/NRPS gene cluster. The hybrid PKS/NRPS gene cluster was identified to be responsible for the biosynthesis of bacilosarcins and amicoumacins. Here, we report the draft genome sequence of Bacillus subtilis TP-B0611, which produces the isocoumarin-type compounds bacilosarcin and amicoumacin. The genome encodes three nonribosomal peptide synthetase (NRPS) gene clusters and one hybrid polyketide synthase (PKS)/NRPS gene cluster. The hybrid PKS/NRPS gene cluster was identified to be responsible for the biosynthesis of bacilosarcins and amicoumacins.
Whole Genome Sequence of Pseudomonas xanthomarina Strain UASWS0955, a Potential Biological Agent for Agricultural and Environmental Uses
We report here the whole-genome shotgun sequence of the strain UASWS0955 of the species Pseudomonas xanthomarina, isolated from sewage sludge. This genome was obtained with an Illumina MiniSeq and is the second genome registered for this species, which is considered as a promising resource for agriculture and bioremediation of contaminated soils. We report here the whole-genome shotgun sequence of the strain UASWS0955 of the species Pseudomonas xanthomarina, isolated from sewage sludge. This genome was obtained with an Illumina MiniSeq and is the second genome registered for this species, which is considered as a promising resource for agriculture and bioremediation of contaminated soils.
Draft Genome Sequence of Arenibacter sp. Strain C 21, an Iodine Accumulating Bacterium Isolated from Surface Marine Sediment
Arenibacter sp. strain C-21, isolated from surface marine sediment of Japan, accumulates iodine in the presence of glucose and iodide (I-). We report here the draft genome sequence of this strain to provide insight into the molecular mechanism underlying its iodine-accumulating ability. Arenibacter sp. strain C-21, isolated from surface marine sediment of Japan, accumulates iodine in the presence of glucose and iodide (I-). We report here the draft genome sequence of this strain to provide insight into the molecular mechanism underlying its iodine-accumulating ability.
Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells
Spatial and temporal control of MT dynamics is important for proper spindle assembly and chromosome segregation. The kinesin-8 Kif18B spatially regulates astral MT dynamics. Not all members of a single kinesin superfamily control MT dynamics in a similar fashion. Spatial and temporal control of MT dynamics is important for proper spindle assembly and chromosome segregation. The kinesin-8 Kif18B spatially regulates astral MT dynamics. Not all members of a single kinesin superfamily control MT dynamics in a similar fashion.The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle. The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.
Influence of Gene Expression on Hardness in Wheat
Puroindoline (Pina and Pinb) genes control grain texture or hardness in wheat. Wild-type/soft alleles lead to softer grain while a mutation in one or both of these genes results in a hard grain. Variation in hardness in genotypes with identical Pin alleles (wild-type or mutant) is known but the molecular basis of this is not known. We now report the identification of wheat genotypes with hard grain texture and wild-type/soft Pin alleles indicating that hardness in wheat may be controlled by factors other than mutations in the coding region of the Pin genes. RNA-Seq analysis was used to determine the variation in the transcriptome of developing grains of thirty three diverse wheat genotypes including hard (mutant Pin) and soft (wild type) and those that were hard without having Pin mutations. This defined the role of pin gene expression and identified other candidate genes associated with hardness. Pina was not expressed in hard wheat with a mutation in the Pina gene. The ratio of Pina to Pinb expression was generally lower in the hard non mutant genotypes. Hardness may be associated with differences in Pin expression and other factors and is not simply associated with mutations in the PIN protein coding sequences. Puroindoline (Pina and Pinb) genes control grain texture or hardness in wheat. Wild-type/soft alleles lead to softer grain while a mutation in one or both of these genes results in a hard grain. Variation in hardness in genotypes with identical Pin alleles (wild-type or mutant) is known but the molecular basis of this is not known. We now report the identification of wheat genotypes with hard grain texture and wild-type/soft Pin alleles indicating that hardness in wheat may be controlled by factors other than mutations in the coding region of the Pin genes. RNA-Seq analysis was used to determine the variation in the transcriptome of developing grains of thirty three diverse wheat genotypes including hard (mutant Pin) and soft (wild type) and those that were hard without having Pin mutations. This defined the role of pin gene expression and identified other candidate genes associated with hardness. Pina was not expressed in hard wheat with a mutation in the Pina gene. The ratio of Pina to Pinb expression was generally lower in the hard non mutant genotypes. Hardness may be associated with differences in Pin expression and other factors and is not simply associated with mutations in the PIN protein coding sequences.
Draft genome sequence of the marine Rhodobacteraceae strain O3.65, cultivated from oil polluted seawater of the Deepwater Horizon oil spill
The marine alphaproteobacterium strain O3.65 was isolated from an enrichment culture of surface seawater contaminated with weathered oil (slicks) from the Deepwater Horizon (DWH) oil spill and belongs to the ubiquitous, diverse and ecological relevant Roseobacter group within the Rhodobacteraceae. Here, we present a preliminary set of physiological features of strain O3.65 and a description and annotation of its draft genome sequence. Based on our data we suggest potential ecological roles of the isolate in the degradation of crude oil within the network of the oil-enriched microbial community. The draft genome comprises 4,852,484 bp with 4,591 protein-coding genes and 63 RNA genes. Strain O3.65 utilizes pentoses, hexoses, disaccharides and amino acids as carbon and energy source and is able to grow on several hydroxylated and substituted aromatic compounds. Based on 16S rRNA gene comparison the closest described and validated strain is Phaeobacter inhibens DSM 17395, however, strain O3.65 is lacking several phenotypic and genomic characteristics specific for the genus Phaeobacter. Phylogenomic analyses based on the whole genome support extensive genetic exchange of strain O3.65 with members of the genus Ruegeria, potentially by using the secretion system type IV. Our physiological observations are consistent with the genomic and phylogenomic analyses and support that strain O3.65 is a novel species of a new genus within the Rhodobacteraceae. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0201-7) contains supplementary material, which is available to authorized users. The marine alphaproteobacterium strain O3.65 was isolated from an enrichment culture of surface seawater contaminated with weathered oil (slicks) from the Deepwater Horizon (DWH) oil spill and belongs to the ubiquitous, diverse and ecological relevant Roseobacter group within the Rhodobacteraceae. Here, we present a preliminary set of physiological features of strain O3.65 and a description and annotation of its draft genome sequence. Based on our data we suggest potential ecological roles of the isolate in the degradation of crude oil within the network of the oil-enriched microbial community. The draft genome comprises 4,852,484 bp with 4,591 protein-coding genes and 63 RNA genes. Strain O3.65 utilizes pentoses, hexoses, disaccharides and amino acids as carbon and energy source and is able to grow on several hydroxylated and substituted aromatic compounds. Based on 16S rRNA gene comparison the closest described and validated strain is Phaeobacter inhibens DSM 17395, however, strain O3.65 is lacking several phenotypic and genomic characteristics specific for the genus Phaeobacter. Phylogenomic analyses based on the whole genome support extensive genetic exchange of strain O3.65 with members of the genus Ruegeria, potentially by using the secretion system type IV. Our physiological observations are consistent with the genomic and phylogenomic analyses and support that strain O3.65 is a novel species of a new genus within the Rhodobacteraceae. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0201-7) contains supplementary material, which is available to authorized users.
Moleculo Long Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes
Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes.ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available. ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available.
Is the Evolution of Salmonella enterica subsp. enterica Linked to Restriction Modification Systems?
The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica. The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica.ABSTRACT Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica. ABSTRACT Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica.
Long Term Evolution of Burkholderia multivorans during a Chronic Cystic Fibrosis Infection Reveals Shifting Forces of Selection
Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression. Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.ABSTRACT Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o− epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient’s lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle. IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression. ABSTRACT Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o− epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient’s lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle. IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.
Ancient recombination events and the origins of hepatitis E virus
Background Hepatitis E virus (HEV) is an enteric, single-stranded, positive sense RNA virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. Tracing the evolutionary ancestry of HEV has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry. Results To reveal close protein relatives of the Hepeviridae family, similarity searching of the GenBank database was carried out using a complete Orthohepevirus A, HEV genotype I (GI) ORF1 protein sequence and individual proteins. The closest non-Hepeviridae homologues to the HEV ORF1 encoded polyprotein were found to be those from the lepidopteran-infecting Alphatetraviridae family members. A consistent relationship to this was found using a phylogenetic approach; the Hepeviridae RdRp clustered with those of the Alphatetraviridae and Benyviridae families. This puts the Hepeviridae ORF1 region within the “Alpha-like” super-group of viruses. In marked contrast, the HEV GI capsid was found to be most closely related to the chicken astrovirus capsid, with phylogenetic trees clustering the Hepeviridae capsid together with those from the Astroviridae family, and surprisingly within the “Picorna-like” supergroup. These results indicate an ancient recombination event has occurred at the junction of the non-structural and structure encoding regions, which led to the emergence of the entire Hepeviridae family. The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses, whilst its non-structural region is related to viruses of the Potyviridae; a large family of plant-infecting viruses with a flexible filamentous rod-shaped virion. Thus, we identified a separate inter-viral family recombination event, again at the non-structural/structural junction, which likely led to the creation of the Astroviridae. Conclusions In summary, we have shown that new viral families have been created though recombination at the junction of the genome that encodes non-structural and structural proteins, and such recombination events are implicated in the genesis of important human pathogens; HEV, astrovirus and rubella virus. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0785-y) contains supplementary material, which is available to authorized users. Background Hepatitis E virus (HEV) is an enteric, single-stranded, positive sense RNA virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. Tracing the evolutionary ancestry of HEV has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry. Results To reveal close protein relatives of the Hepeviridae family, similarity searching of the GenBank database was carried out using a complete Orthohepevirus A, HEV genotype I (GI) ORF1 protein sequence and individual proteins. The closest non-Hepeviridae homologues to the HEV ORF1 encoded polyprotein were found to be those from the lepidopteran-infecting Alphatetraviridae family members. A consistent relationship to this was found using a phylogenetic approach; the Hepeviridae RdRp clustered with those of the Alphatetraviridae and Benyviridae families. This puts the Hepeviridae ORF1 region within the “Alpha-like” super-group of viruses. In marked contrast, the HEV GI capsid was found to be most closely related to the chicken astrovirus capsid, with phylogenetic trees clustering the Hepeviridae capsid together with those from the Astroviridae family, and surprisingly within the “Picorna-like” supergroup. These results indicate an ancient recombination event has occurred at the junction of the non-structural and structure encoding regions, which led to the emergence of the entire Hepeviridae family. The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses, whilst its non-structural region is related to viruses of the Potyviridae; a large family of plant-infecting viruses with a flexible filamentous rod-shaped virion. Thus, we identified a separate inter-viral family recombination event, again at the non-structural/structural junction, which likely led to the creation of the Astroviridae. Conclusions In summary, we have shown that new viral families have been created though recombination at the junction of the genome that encodes non-structural and structural proteins, and such recombination events are implicated in the genesis of important human pathogens; HEV, astrovirus and rubella virus. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0785-y) contains supplementary material, which is available to authorized users.
Genome Wide Identification and Functional Classification of Tomato (Solanum lycopersicum) Aldehyde Dehydrogenase (ALDH) Gene Superfamily
Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling. Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling.
Characterization of the Neisseria meningitidis Helicase RecG
Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant. Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant.
FUS/TLS acts as an aggregation dependent modifier of polyglutamine disease model mice
FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington’s disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of (HD, but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS+/− double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration. FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington’s disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of (HD, but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS+/− double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration.
Genomic Organization, Phylogenetic and Expression Analysis of the B BOX Gene Family in Tomato
The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family. The B-BOX (BBX) proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT (CO, CO-like and TOC1) motif, which play important roles in regulating plant growth, development and stress response. Nevertheless, no systematic study of BBX genes has undertaken in tomato (Solanum lycopersicum). Here we present the results of a genome-wide analysis of the 29 BBX genes in this important vegetable species. Their structures, conserved domains, phylogenetic relationships, subcellular localizations, and promoter cis-regulatory elements were analyzed; their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail. Tomato BBX genes can be divided into five subfamilies, and twelve of them were found to be segmentally duplicated. Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns. The expression of most BBX genes can be induced by drought, polyethylene glycol-6000 or heat stress. Some BBX genes were induced strongly by phytohormones such as abscisic acid, gibberellic acid, or ethephon. The majority of tomato BBX proteins was predicted to be located in nuclei, and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested (SlBBX5, 7, 15, 17, 20, 22, and 24) were localized in nucleus. Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family.
Interactome transcriptome analysis discovers signatures complementary to GWAS Loci of Type 2 Diabetes
Protein interactions play significant roles in complex diseases. We analyzed peripheral blood mononuclear cells (PBMC) transcriptome using a multi-method strategy. We constructed a tissue-specific interactome (T2Di) and identified 420 molecular signatures associated with T2D-related comorbidity and symptoms, mainly implicated in inflammation, adipogenesis, protein phosphorylation and hormonal secretion. Apart from explaining the residual associations within the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) study, the T2Di signatures were enriched in pathogenic cell type-specific regulatory elements related to fetal development, immunity and expression quantitative trait loci (eQTL). The T2Di revealed a novel locus near a well-established GWAS loci AChE, in which SRRT interacts with JAZF1, a T2D-GWAS gene implicated in pancreatic function. The T2Di also included known anti-diabetic drug targets (e.g. PPARD, MAOB) and identified possible druggable targets (e.g. NCOR2, PDGFR). These T2Di signatures were validated by an independent computational method, and by expression data of pancreatic islet, muscle and liver with some of the signatures (CEBPB, SREBF1, MLST8, SRF, SRRT and SLC12A9) confirmed in PBMC from an independent cohort of 66 T2D and 66 control subjects. By combining prior knowledge and transcriptome analysis, we have constructed an interactome to explain the multi-layered regulatory pathways in T2D. Protein interactions play significant roles in complex diseases. We analyzed peripheral blood mononuclear cells (PBMC) transcriptome using a multi-method strategy. We constructed a tissue-specific interactome (T2Di) and identified 420 molecular signatures associated with T2D-related comorbidity and symptoms, mainly implicated in inflammation, adipogenesis, protein phosphorylation and hormonal secretion. Apart from explaining the residual associations within the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) study, the T2Di signatures were enriched in pathogenic cell type-specific regulatory elements related to fetal development, immunity and expression quantitative trait loci (eQTL). The T2Di revealed a novel locus near a well-established GWAS loci AChE, in which SRRT interacts with JAZF1, a T2D-GWAS gene implicated in pancreatic function. The T2Di also included known anti-diabetic drug targets (e.g. PPARD, MAOB) and identified possible druggable targets (e.g. NCOR2, PDGFR). These T2Di signatures were validated by an independent computational method, and by expression data of pancreatic islet, muscle and liver with some of the signatures (CEBPB, SREBF1, MLST8, SRF, SRRT and SLC12A9) confirmed in PBMC from an independent cohort of 66 T2D and 66 control subjects. By combining prior knowledge and transcriptome analysis, we have constructed an interactome to explain the multi-layered regulatory pathways in T2D.
FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma
Abstract SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F‐box)‐type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW 7α. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation‐resistant SOX9 mutant reveals activation of pro‐metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7‐dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment. Abstract SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F‐box)‐type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW 7α. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation‐resistant SOX9 mutant reveals activation of pro‐metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7‐dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.
Novel Ciliate Genetic Code Variants Including the Reassignment of All Three Stop Codons to Sense Codons in Condylostoma magnum
mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3′ end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state. mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3′ end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state.
Physiological, genomic and transcriptional diversity in responses to boron deficiency in rapeseed genotypes
Highlight A comprehensive examination is made of physiological and transcriptional variations and genetic diversity of rapeseed genotypes differing in their response to boron deficiency, and transcriptomics-assisted QTL-seq analyses are found to expedite the identification of quantitative trait genes in plant species with complex genomes. Highlight A comprehensive examination is made of physiological and transcriptional variations and genetic diversity of rapeseed genotypes differing in their response to boron deficiency, and transcriptomics-assisted QTL-seq analyses are found to expedite the identification of quantitative trait genes in plant species with complex genomes.Allotetraploid rapeseed (Brassica napus L. AnAnCnCn, 2n=4x=38) is highly susceptible to boron (B) deficiency, a widespread limiting factor that causes severe losses in seed yield. The genetic variation in the sensitivity to B deficiency found in rapeseed genotypes emphasizes the complex response architecture. In this research, a B-inefficient genotype, ‘Westar 10’ (‘W10’), responded to B deficiencies during vegetative and reproductive development with an over-accumulation of reactive oxygen species, severe lipid peroxidation, evident plasmolysis, abnormal floral organogenesis, and widespread sterility compared to a B-efficient genotype, ‘Qingyou 10’ (‘QY10’). Whole-genome re-sequencing (WGS) of ‘QY10’ and ‘W10’ revealed a total of 1 605 747 single nucleotide polymorphisms and 218 755 insertions/deletions unevenly distributed across the allotetraploid rapeseed genome (~1130Mb). Digital gene expression (DGE) profiling identified more genes related to B transporters, antioxidant enzymes, and the maintenance of cell walls and membranes with higher transcript levels in the roots of ‘QY10’ than in ‘W10’ under B deficiency. Furthermore, based on WGS and bulked segregant analysis of the doubled haploid (DH) line pools derived from ‘QY10’ and ‘W10’, two significant quantitative trait loci (QTLs) for B efficiency were characterized on chromosome C2, and DGE-assisted QTL-seq analyses then identified a nodulin 26-like intrinsic protein gene and an ATP-binding cassette (ABC) transporter gene as the corresponding candidates regulating B efficiency. This research facilitates a more comprehensive understanding of the differential physiological and transcriptional responses to B deficiency and abundant genetic diversity in rapeseed genotypes, and the DGE-assisted QTL-seq analyses provide novel insights regarding the rapid dissection of quantitative trait genes in plant species with complex genomes. Allotetraploid rapeseed (Brassica napus L. AnAnCnCn, 2n=4x=38) is highly susceptible to boron (B) deficiency, a widespread limiting factor that causes severe losses in seed yield. The genetic variation in the sensitivity to B deficiency found in rapeseed genotypes emphasizes the complex response architecture. In this research, a B-inefficient genotype, ‘Westar 10’ (‘W10’), responded to B deficiencies during vegetative and reproductive development with an over-accumulation of reactive oxygen species, severe lipid peroxidation, evident plasmolysis, abnormal floral organogenesis, and widespread sterility compared to a B-efficient genotype, ‘Qingyou 10’ (‘QY10’). Whole-genome re-sequencing (WGS) of ‘QY10’ and ‘W10’ revealed a total of 1 605 747 single nucleotide polymorphisms and 218 755 insertions/deletions unevenly distributed across the allotetraploid rapeseed genome (~1130Mb). Digital gene expression (DGE) profiling identified more genes related to B transporters, antioxidant enzymes, and the maintenance of cell walls and membranes with higher transcript levels in the roots of ‘QY10’ than in ‘W10’ under B deficiency. Furthermore, based on WGS and bulked segregant analysis of the doubled haploid (DH) line pools derived from ‘QY10’ and ‘W10’, two significant quantitative trait loci (QTLs) for B efficiency were characterized on chromosome C2, and DGE-assisted QTL-seq analyses then identified a nodulin 26-like intrinsic protein gene and an ATP-binding cassette (ABC) transporter gene as the corresponding candidates regulating B efficiency. This research facilitates a more comprehensive understanding of the differential physiological and transcriptional responses to B deficiency and abundant genetic diversity in rapeseed genotypes, and the DGE-assisted QTL-seq analyses provide novel insights regarding the rapid dissection of quantitative trait genes in plant species with complex genomes.
HER2 encoded mir 4728 forms a receptor independent circuit with miR 21 5p through the non canonical poly(A) polymerase PAPD5
We previously reported that the human HER2 gene encodes the intronic microRNA mir-4728, which is overexpressed together with its oncogenic host gene and may act independently of the HER2 receptor. More recently, we also reported that the oncogenic miR-21-5p is regulated by 3′ tailing and trimming by the non-canonical poly(A) polymerase PAPD5 and the ribonuclease PARN. Here we demonstrate a dual function for the HER2 locus in upregulation of miR-21-5p; while HER2 signalling activates transcription of mir-21, miR-4728-3p specifically stabilises miR-21-5p through inhibition of PAPD5. Our results establish a new and unexpected oncogenic role for the HER2 locus that is not currently being targeted by any anti-HER2 therapy. We previously reported that the human HER2 gene encodes the intronic microRNA mir-4728, which is overexpressed together with its oncogenic host gene and may act independently of the HER2 receptor. More recently, we also reported that the oncogenic miR-21-5p is regulated by 3′ tailing and trimming by the non-canonical poly(A) polymerase PAPD5 and the ribonuclease PARN. Here we demonstrate a dual function for the HER2 locus in upregulation of miR-21-5p; while HER2 signalling activates transcription of mir-21, miR-4728-3p specifically stabilises miR-21-5p through inhibition of PAPD5. Our results establish a new and unexpected oncogenic role for the HER2 locus that is not currently being targeted by any anti-HER2 therapy.
Diversity in the Toll Like Receptor Genes of the African Penguin (Spheniscus demersus)
The African penguin, Spheniscus demersus, is listed as Endangered by the IUCN Red List of Threatened Species due to the drastic reduction in population numbers over the last 20 years. To date, the only studies on immunogenetic variation in penguins have been conducted on the major histocompatibility complex (MHC) genes. It was shown in humans that up to half of the genetic variability in immune responses to pathogens are located in non-MHC genes. Toll-like receptors (TLRs) are now increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune region of African penguins similar to that observed in New Zealand robin that has undergone several severe population bottlenecks. Single nucleotide polymorphism (SNP) diversity across TLRs varied between ex situ and in situ penguins with the number of non-synonymous alterations in ex situ populations (n = 14) being reduced in comparison to in situ populations (n = 16). Maintaining adaptive diversity is of vital importance in the assurance populations as these animals may potentially be used in the future for re-introductions. Therefore, this study provides essential data on immune gene diversity in penguins and will assist in providing an additional monitoring tool for African penguin in the wild, as well as to monitor diversity in ex situ populations and to ensure that diversity found in the in situ populations are captured in the assurance populations. The African penguin, Spheniscus demersus, is listed as Endangered by the IUCN Red List of Threatened Species due to the drastic reduction in population numbers over the last 20 years. To date, the only studies on immunogenetic variation in penguins have been conducted on the major histocompatibility complex (MHC) genes. It was shown in humans that up to half of the genetic variability in immune responses to pathogens are located in non-MHC genes. Toll-like receptors (TLRs) are now increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune region of African penguins similar to that observed in New Zealand robin that has undergone several severe population bottlenecks. Single nucleotide polymorphism (SNP) diversity across TLRs varied between ex situ and in situ penguins with the number of non-synonymous alterations in ex situ populations (n = 14) being reduced in comparison to in situ populations (n = 16). Maintaining adaptive diversity is of vital importance in the assurance populations as these animals may potentially be used in the future for re-introductions. Therefore, this study provides essential data on immune gene diversity in penguins and will assist in providing an additional monitoring tool for African penguin in the wild, as well as to monitor diversity in ex situ populations and to ensure that diversity found in the in situ populations are captured in the assurance populations.
Adhesion of the genome sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants
Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7813-0) contains supplementary material, which is available to authorized users. Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7813-0) contains supplementary material, which is available to authorized users.
Comprehensive analysis of lncRNA expression profiles reveals a novel lncRNA signature to discriminate nonequivalent outcomes in patients with ovarian cancer
There is growing evidence of dysregulated long non-coding RNAs (lncRNAs) serving as potential biomarkers for cancer prognosis. However, systematic efforts of searching for an expression-based lncRNA signature for prognosis prediction in ovarian cancer (OvCa) have not been made yet. Here, we performed comprehensive analysis for lncRNA expression profiles and clinical data of 544 OvCa patients from The Cancer Genome Atlas (TCGA), and identified an eight-lncRNA signature with ability to classify patients of the training cohort into high-risk group showing poor outcome and low-risk group showing significantly improved outcome, which was further validated in the validation cohort and entire TCGA cohort. Multivariate Cox regression analysis and stratified analysis demonstrated that the prognostic value of this signature was independent of other clinicopathological factors. Associating the outcome prediction with BRCA1 and/or BRCA2 mutation revealed a superior prognosis performance both in BRCA1/2-mutated and BRCA1/2 wild-type tumors. Finally, a significantly correlation was found between the lncRNA signature and the complete response rate of chemotherapy, suggesting that this eight-lncRNA signature may be a measure to predict chemotherapy response and identify platinum-resistant patients who might benefit from other more efficacious therapies. With further prospective validation, this eight-lncRNA signature may have important implications for outcome prediction and therapy decisions. There is growing evidence of dysregulated long non-coding RNAs (lncRNAs) serving as potential biomarkers for cancer prognosis. However, systematic efforts of searching for an expression-based lncRNA signature for prognosis prediction in ovarian cancer (OvCa) have not been made yet. Here, we performed comprehensive analysis for lncRNA expression profiles and clinical data of 544 OvCa patients from The Cancer Genome Atlas (TCGA), and identified an eight-lncRNA signature with ability to classify patients of the training cohort into high-risk group showing poor outcome and low-risk group showing significantly improved outcome, which was further validated in the validation cohort and entire TCGA cohort. Multivariate Cox regression analysis and stratified analysis demonstrated that the prognostic value of this signature was independent of other clinicopathological factors. Associating the outcome prediction with BRCA1 and/or BRCA2 mutation revealed a superior prognosis performance both in BRCA1/2-mutated and BRCA1/2 wild-type tumors. Finally, a significantly correlation was found between the lncRNA signature and the complete response rate of chemotherapy, suggesting that this eight-lncRNA signature may be a measure to predict chemotherapy response and identify platinum-resistant patients who might benefit from other more efficacious therapies. With further prospective validation, this eight-lncRNA signature may have important implications for outcome prediction and therapy decisions.
Symmetry mismatch reconstruction of genomes and associated proteins within icosahedral viruses using cryo EM
Although near-atomic resolutions have been routinely achieved for structural determination of many icosahedral viral capsids, structures of genomes and associated proteins within the capsids are still less characterized because the genome information is overlapped by the highly symmetric capsid information in the virus particle images. We recently developed a software package for symmetry-mismatch structural reconstruction and determined the structures of the genome and RNA polymerases within an icosahedral virus for the first time. Here, we describe the protocol used for this structural determination, which may facilitate structural biologists in investigating the structures of viral genome and associated proteins. Electronic supplementary material The online version of this article (doi:10.1007/s41048-016-0024-5) contains supplementary material, which is available to authorized users. Although near-atomic resolutions have been routinely achieved for structural determination of many icosahedral viral capsids, structures of genomes and associated proteins within the capsids are still less characterized because the genome information is overlapped by the highly symmetric capsid information in the virus particle images. We recently developed a software package for symmetry-mismatch structural reconstruction and determined the structures of the genome and RNA polymerases within an icosahedral virus for the first time. Here, we describe the protocol used for this structural determination, which may facilitate structural biologists in investigating the structures of viral genome and associated proteins. Electronic supplementary material The online version of this article (doi:10.1007/s41048-016-0024-5) contains supplementary material, which is available to authorized users.
Purification and Characterization of a Novel Cold Shock Protein Like Bacteriocin Synthesized by Bacillus thuringiensis
Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5–7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL, and MBC values of 12.5 μg/mL and 6.25 μg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases. Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5–7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL, and MBC values of 12.5 μg/mL and 6.25 μg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases.
Regulation of transcription by the Arabidopsis UVR8 photoreceptor involves a specific histone modification
The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) specifically mediates photomorphogenic responses to UV-B wavelengths. UVR8 acts by regulating transcription of a set of genes, but the underlying mechanisms are unknown. Previous research indicated that UVR8 can associate with chromatin, but the specificity and functional significance of this interaction are not clear. Here we show, by chromatin immunoprecipitation, that UV-B exposure of Arabidopsis increases acetylation of lysines K9 and/or K14 of histone H3 at UVR8-regulated gene loci in a UVR8-dependent manner. The transcription factors HY5 and/or HYH, which mediate UVR8-regulated transcription, are also required for this chromatin modification, at least for the ELIP1 gene. Furthermore, sequencing of the immunoprecipitated DNA revealed that all UV-B-induced enrichments in H3K9,14diacetylation across the genome are UVR8-dependent, and approximately 40 % of the enriched loci contain known UVR8-regulated genes. In addition, inhibition of histone acetylation by anacardic acid reduces the UV-B induced, UVR8 mediated expression of ELIP1 and CHS. No evidence was obtained in yeast 2-hybrid assays for a direct interaction between either UVR8 or HY5 and several proteins involved in light-regulated histone modification, nor for the involvement of these proteins in UVR8-mediated responses in plants, although functional redundancy between proteins could influence the results. In summary, this study shows that UVR8 regulates a specific chromatin modification associated with transcriptional regulation of a set of UVR8-target genes. Electronic supplementary material The online version of this article (doi:10.1007/s11103-016-0522-3) contains supplementary material, which is available to authorized users. The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) specifically mediates photomorphogenic responses to UV-B wavelengths. UVR8 acts by regulating transcription of a set of genes, but the underlying mechanisms are unknown. Previous research indicated that UVR8 can associate with chromatin, but the specificity and functional significance of this interaction are not clear. Here we show, by chromatin immunoprecipitation, that UV-B exposure of Arabidopsis increases acetylation of lysines K9 and/or K14 of histone H3 at UVR8-regulated gene loci in a UVR8-dependent manner. The transcription factors HY5 and/or HYH, which mediate UVR8-regulated transcription, are also required for this chromatin modification, at least for the ELIP1 gene. Furthermore, sequencing of the immunoprecipitated DNA revealed that all UV-B-induced enrichments in H3K9,14diacetylation across the genome are UVR8-dependent, and approximately 40 % of the enriched loci contain known UVR8-regulated genes. In addition, inhibition of histone acetylation by anacardic acid reduces the UV-B induced, UVR8 mediated expression of ELIP1 and CHS. No evidence was obtained in yeast 2-hybrid assays for a direct interaction between either UVR8 or HY5 and several proteins involved in light-regulated histone modification, nor for the involvement of these proteins in UVR8-mediated responses in plants, although functional redundancy between proteins could influence the results. In summary, this study shows that UVR8 regulates a specific chromatin modification associated with transcriptional regulation of a set of UVR8-target genes. Electronic supplementary material The online version of this article (doi:10.1007/s11103-016-0522-3) contains supplementary material, which is available to authorized users.
Cloning and expression of SgCYP450 4 from Siraitia grosvenorii
CYP450 plays an essential role in the development and growth of the fruits of Siraitia grosvenorii. However, little is known about the SgCYP450-4 gene in S. grosvenorii. Here, based on transcriptome data, a full-length cDNA sequence of SgCYP450-4 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) strategies. SgCYP450-4 is 1677 bp in length (GenBank accession No. AEM42985.1) and contains a complete open reading frame (ORF) of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI) is 8.8, and the protein was predicted to possess cytochrome P450 domains. SgCYP450-4 gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of SgCYP450-4 had the highest level and then decreased over time, which was consistent with the development of fruits of S. Grosvenorii. Hormonal treatment could significantly induce the expression of SgCYP450-4. These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents. CYP450 plays an essential role in the development and growth of the fruits of Siraitia grosvenorii. However, little is known about the SgCYP450-4 gene in S. grosvenorii. Here, based on transcriptome data, a full-length cDNA sequence of SgCYP450-4 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) strategies. SgCYP450-4 is 1677 bp in length (GenBank accession No. AEM42985.1) and contains a complete open reading frame (ORF) of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI) is 8.8, and the protein was predicted to possess cytochrome P450 domains. SgCYP450-4 gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of SgCYP450-4 had the highest level and then decreased over time, which was consistent with the development of fruits of S. Grosvenorii. Hormonal treatment could significantly induce the expression of SgCYP450-4. These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents.
Identification, Expression and IAA Amide Synthetase Activity Analysis of Gretchen Hagen 3 in Papaya Fruit (Carica papaya L.) during Postharvest Process
Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya. Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya.
Microbiological and bioinformatics analysis of primary Sjögren’s syndrome patients with normal salivation§
Background Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. Objective This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. Methods Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. Results Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree. Conclusions While Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation. Background Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. Objective This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. Methods Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. Results Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree. Conclusions While Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation.
Chloroplast sequence of treegourd (Crescentia cujete, Bignoniaceae) to study phylogeography and domestication1
Premise of the study: Crescentia cujete (Bignoniaceae) fruit rinds are traditionally used for storage vessels and handicrafts. We assembled its chloroplast genome and identified single-nucleotide polymorphisms (SNPs). Methods and Results: Using a genome skimming approach, the whole chloroplast of C. cujete was assembled using 3,106,928 sequence reads of 150 bp. The chloroplast is 154,662 bp in length, structurally divided into a large single copy region (84,788 bp), a small single copy region (18,299 bp), and two inverted repeat regions (51,575 bp) with 88 genes annotated. By resequencing the whole chloroplast, we identified 66 SNPs in C. cujete (N = 30) and 68 SNPs in C. amazonica (N = 6). Nucleotide diversity was estimated at 1.1 × 10−3 and 3.5 × 10−3 for C. cujete and C. amazonica, respectively. Conclusions: This broadened C. cujete genetic toolkit will be important to study the origin, domestication, diversity, and phylogeography of treegourds in the Neotropics. Premise of the study: Crescentia cujete (Bignoniaceae) fruit rinds are traditionally used for storage vessels and handicrafts. We assembled its chloroplast genome and identified single-nucleotide polymorphisms (SNPs). Methods and Results: Using a genome skimming approach, the whole chloroplast of C. cujete was assembled using 3,106,928 sequence reads of 150 bp. The chloroplast is 154,662 bp in length, structurally divided into a large single copy region (84,788 bp), a small single copy region (18,299 bp), and two inverted repeat regions (51,575 bp) with 88 genes annotated. By resequencing the whole chloroplast, we identified 66 SNPs in C. cujete (N = 30) and 68 SNPs in C. amazonica (N = 6). Nucleotide diversity was estimated at 1.1 × 10−3 and 3.5 × 10−3 for C. cujete and C. amazonica, respectively. Conclusions: This broadened C. cujete genetic toolkit will be important to study the origin, domestication, diversity, and phylogeography of treegourds in the Neotropics.
Integrating network, sequence and functional features using machine learning approaches towards identification of novel Alzheimer genes
Background Alzheimer’s disease (AD) is a complex progressive neurodegenerative disorder commonly characterized by short term memory loss. Presently no effective therapeutic treatments exist that can completely cure this disease. The cause of Alzheimer’s is still unclear, however one of the other major factors involved in AD pathogenesis are the genetic factors and around 70 % risk of the disease is assumed to be due to the large number of genes involved. Although genetic association studies have revealed a number of potential AD susceptibility genes, there still exists a need for identification of unidentified AD-associated genes and therapeutic targets to have better understanding of the disease-causing mechanisms of Alzheimer’s towards development of effective AD therapeutics. Results In the present study, we have used machine learning approach to identify candidate AD associated genes by integrating topological properties of the genes from the protein-protein interaction networks, sequence features and functional annotations. We also used molecular docking approach and screened already known anti-Alzheimer drugs against the novel predicted probable targets of AD and observed that an investigational drug, AL-108, had high affinity for majority of the possible therapeutic targets. Furthermore, we performed molecular dynamics simulations and MM/GBSA calculations on the docked complexes to validate our preliminary findings. Conclusions To the best of our knowledge, this is the first comprehensive study of its kind for identification of putative Alzheimer-associated genes using machine learning approaches and we propose that such computational studies can improve our understanding on the core etiology of AD which could lead to the development of effective anti-Alzheimer drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3108-1) contains supplementary material, which is available to authorized users. Background Alzheimer’s disease (AD) is a complex progressive neurodegenerative disorder commonly characterized by short term memory loss. Presently no effective therapeutic treatments exist that can completely cure this disease. The cause of Alzheimer’s is still unclear, however one of the other major factors involved in AD pathogenesis are the genetic factors and around 70 % risk of the disease is assumed to be due to the large number of genes involved. Although genetic association studies have revealed a number of potential AD susceptibility genes, there still exists a need for identification of unidentified AD-associated genes and therapeutic targets to have better understanding of the disease-causing mechanisms of Alzheimer’s towards development of effective AD therapeutics. Results In the present study, we have used machine learning approach to identify candidate AD associated genes by integrating topological properties of the genes from the protein-protein interaction networks, sequence features and functional annotations. We also used molecular docking approach and screened already known anti-Alzheimer drugs against the novel predicted probable targets of AD and observed that an investigational drug, AL-108, had high affinity for majority of the possible therapeutic targets. Furthermore, we performed molecular dynamics simulations and MM/GBSA calculations on the docked complexes to validate our preliminary findings. Conclusions To the best of our knowledge, this is the first comprehensive study of its kind for identification of putative Alzheimer-associated genes using machine learning approaches and we propose that such computational studies can improve our understanding on the core etiology of AD which could lead to the development of effective anti-Alzheimer drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3108-1) contains supplementary material, which is available to authorized users.
Characterization of polymorphic microsatellite markers in Pinus armandii (Pinaceae), an endemic conifer species to China1
Premise of the study: Pinus armandii (Pinaceae) is an important conifer tree species in central and southwestern China, and it plays a key role in the local forest ecosystems. To investigate its population genetics and design effective conservation strategies, we characterized 18 polymorphic microsatellite markers for this species. Methods and Results: Eighteen novel polymorphic and 16 monomorphic microsatellite loci of P. armandii were isolated using Illumina MiSeq technology. The number of alleles per locus ranged from two to five. The expected heterozygosity ranged from 0.061 to 0.609 with an average of 0.384, and the observed heterozygosity ranged from 0.063 to 0.947 with an average of 0.436. Seventeen loci could be successfully transferred to five related Pinus species (P. koraiensis, P. griffithii, P. sibirica, P. pumila, and P. bungeana). Conclusions: These novel microsatellites could potentially be used to investigate the population genetics of P. armandii and related species. Premise of the study: Pinus armandii (Pinaceae) is an important conifer tree species in central and southwestern China, and it plays a key role in the local forest ecosystems. To investigate its population genetics and design effective conservation strategies, we characterized 18 polymorphic microsatellite markers for this species. Methods and Results: Eighteen novel polymorphic and 16 monomorphic microsatellite loci of P. armandii were isolated using Illumina MiSeq technology. The number of alleles per locus ranged from two to five. The expected heterozygosity ranged from 0.061 to 0.609 with an average of 0.384, and the observed heterozygosity ranged from 0.063 to 0.947 with an average of 0.436. Seventeen loci could be successfully transferred to five related Pinus species (P. koraiensis, P. griffithii, P. sibirica, P. pumila, and P. bungeana). Conclusions: These novel microsatellites could potentially be used to investigate the population genetics of P. armandii and related species.
Transcriptomic resources and marker validation for diploid and polyploid Veronica (Plantaginaceae) from New Zealand and Europe1
Premise of the study: Polyploidy may generate novel variation, leading to adaptation and species diversification. An excellent natural system to study polyploid evolution in a comparative framework is Veronica (Plantaginaceae), which comprises several parallel, recently evolved polyploid series. Methods: Over 105 million Illumina paired-end sequence reads were generated from cDNA libraries of leaf tissue from eight individuals representing three European and four New Zealand species. Forty-eight simple sequence repeat (SSR) and 48 low-copy nuclear (LCN) markers were developed and validated with Fluidigm microfluidic PCR and Illumina MiSeq amplicon sequencing on 48 different individuals each. Results: Individual Trinity assemblies were similar regarding annotated transcripts (13,009–14,271), mean contig length (635–742 bp), N50 value (916–1133 bp), E90N50 value (1099–1308 bp), contigs with positive BLAST hits (42–63%), and gene ontology terms. Analyses of 29,738 single-nucleotide polymorphisms (8746 phylogenetically informative) mined from these transcriptomes plus two outgroups (Picrorhiza kurrooa and Plantago ovata) showed moderate to high bootstrap support for all branches and reticulation among sampled European Veronica. Discussion: The transcriptome sequences themselves, as well as the validated SSR (40/48) and LCN (11/48) markers derived from them, show inter- and intraspecific genetic variation. These resources will be invaluable for future population genetic, phylogenetic, and functional genetic investigations in polyploid Veronica. Premise of the study: Polyploidy may generate novel variation, leading to adaptation and species diversification. An excellent natural system to study polyploid evolution in a comparative framework is Veronica (Plantaginaceae), which comprises several parallel, recently evolved polyploid series. Methods: Over 105 million Illumina paired-end sequence reads were generated from cDNA libraries of leaf tissue from eight individuals representing three European and four New Zealand species. Forty-eight simple sequence repeat (SSR) and 48 low-copy nuclear (LCN) markers were developed and validated with Fluidigm microfluidic PCR and Illumina MiSeq amplicon sequencing on 48 different individuals each. Results: Individual Trinity assemblies were similar regarding annotated transcripts (13,009–14,271), mean contig length (635–742 bp), N50 value (916–1133 bp), E90N50 value (1099–1308 bp), contigs with positive BLAST hits (42–63%), and gene ontology terms. Analyses of 29,738 single-nucleotide polymorphisms (8746 phylogenetically informative) mined from these transcriptomes plus two outgroups (Picrorhiza kurrooa and Plantago ovata) showed moderate to high bootstrap support for all branches and reticulation among sampled European Veronica. Discussion: The transcriptome sequences themselves, as well as the validated SSR (40/48) and LCN (11/48) markers derived from them, show inter- and intraspecific genetic variation. These resources will be invaluable for future population genetic, phylogenetic, and functional genetic investigations in polyploid Veronica.
Integrating Candida albicans metabolism with biofilm heterogeneity by transcriptome mapping
Candida albicans biofilm formation is an important virulence factor in the pathogenesis of disease, a characteristic which has been shown to be heterogeneous in clinical isolates. Using an unbiased computational approach we investigated the central metabolic pathways driving biofilm heterogeneity. Transcripts from high (HBF) and low (LBF) biofilm forming isolates were analysed by RNA sequencing, with 6312 genes identified to be expressed in these two phenotypes. With a dedicated computational approach we identified and validated a significantly differentially expressed subnetwork of genes associated with these biofilm phenotypes. Our analysis revealed amino acid metabolism, such as arginine, proline, aspartate and glutamate metabolism, were predominantly upregulated in the HBF phenotype. On the contrary, purine, starch and sucrose metabolism was generally upregulated in the LBF phenotype. The aspartate aminotransferase gene AAT1 was found to be a common member of these amino acid pathways and significantly upregulated in the HBF phenotype. Pharmacological inhibition of AAT1 enzyme activity significantly reduced biofilm formation in a dose-dependent manner. Collectively, these findings provide evidence that biofilm phenotype is associated with differential regulation of metabolic pathways. Understanding and targeting such pathways, such as amino acid metabolism, is potentially useful for developing diagnostics and new antifungals to treat biofilm-based infections. Candida albicans biofilm formation is an important virulence factor in the pathogenesis of disease, a characteristic which has been shown to be heterogeneous in clinical isolates. Using an unbiased computational approach we investigated the central metabolic pathways driving biofilm heterogeneity. Transcripts from high (HBF) and low (LBF) biofilm forming isolates were analysed by RNA sequencing, with 6312 genes identified to be expressed in these two phenotypes. With a dedicated computational approach we identified and validated a significantly differentially expressed subnetwork of genes associated with these biofilm phenotypes. Our analysis revealed amino acid metabolism, such as arginine, proline, aspartate and glutamate metabolism, were predominantly upregulated in the HBF phenotype. On the contrary, purine, starch and sucrose metabolism was generally upregulated in the LBF phenotype. The aspartate aminotransferase gene AAT1 was found to be a common member of these amino acid pathways and significantly upregulated in the HBF phenotype. Pharmacological inhibition of AAT1 enzyme activity significantly reduced biofilm formation in a dose-dependent manner. Collectively, these findings provide evidence that biofilm phenotype is associated with differential regulation of metabolic pathways. Understanding and targeting such pathways, such as amino acid metabolism, is potentially useful for developing diagnostics and new antifungals to treat biofilm-based infections.
Identification of internalin A like virulent proteins in Leishmania donovani
Background An active immune surveillance and a range of barriers to infection allow the host to effectively eliminate microbial pathogens. However, pathogens may use diverse strategies to subdue such host defences. For instance, one such mechanism is the use of leucine-rich repeat (LRR) proteins by pathogens (microbial) to cause infection. In this study, we aimed at identifying novel virulence factor(s) in Leishmania donovani, based on the possibility of lateral gene transfers of bacterial virulence factor(s) to L. donovani. Methods Rigorous homology searching protocols including Hidden Markov Model (HMM) and BLASTp based searches were employed to detect remote but significant similarities between L. donovani proteins and bacterial virulence factors. Results We found that some L. donovani proteins are similar to internalin-A (Inl-A) protein of Listeria monocytogenes, a surface LRR protein that helps mediate host cell invasion by interacting with E-cadherin on the cell membrane. However, to date, no such invasion mechanism has been reported in Leishmania donovani, the causative agent of visceral leishmaniasis. Moreover, a comparative LRR motif analysis of L. donovani Inl-A-like proteins against the Inl-A protein of L. monocytogenes revealed existence of characteristic consensus LRR regions, suggesting a reliable evolutionary relationship between them. Further, through rigorous three dimensional (3D) modeling of L. donovani Inl-A-like proteins and subsequent molecular docking studies we suggest the probability of human E-cadherin binding with the L. donovani Inl-A-like proteins. Conclusions We have identified three potential candidates (UniProt ID: E9B7L9, E9BMT7 and E9BUL5) of Inl-A-like LRR containing proteins in L. donovani with the help of systematic whole genome sequence analysis. Thus, herein we propose the existence of a novel class of Inl-A-like virulence factor proteins in L. donovani and other Leishmania species based on sequence similarity, phylogenetic analysis and molecular modelling studies in L. donovani. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1842-5) contains supplementary material, which is available to authorized users. Background An active immune surveillance and a range of barriers to infection allow the host to effectively eliminate microbial pathogens. However, pathogens may use diverse strategies to subdue such host defences. For instance, one such mechanism is the use of leucine-rich repeat (LRR) proteins by pathogens (microbial) to cause infection. In this study, we aimed at identifying novel virulence factor(s) in Leishmania donovani, based on the possibility of lateral gene transfers of bacterial virulence factor(s) to L. donovani. Methods Rigorous homology searching protocols including Hidden Markov Model (HMM) and BLASTp based searches were employed to detect remote but significant similarities between L. donovani proteins and bacterial virulence factors. Results We found that some L. donovani proteins are similar to internalin-A (Inl-A) protein of Listeria monocytogenes, a surface LRR protein that helps mediate host cell invasion by interacting with E-cadherin on the cell membrane. However, to date, no such invasion mechanism has been reported in Leishmania donovani, the causative agent of visceral leishmaniasis. Moreover, a comparative LRR motif analysis of L. donovani Inl-A-like proteins against the Inl-A protein of L. monocytogenes revealed existence of characteristic consensus LRR regions, suggesting a reliable evolutionary relationship between them. Further, through rigorous three dimensional (3D) modeling of L. donovani Inl-A-like proteins and subsequent molecular docking studies we suggest the probability of human E-cadherin binding with the L. donovani Inl-A-like proteins. Conclusions We have identified three potential candidates (UniProt ID: E9B7L9, E9BMT7 and E9BUL5) of Inl-A-like LRR containing proteins in L. donovani with the help of systematic whole genome sequence analysis. Thus, herein we propose the existence of a novel class of Inl-A-like virulence factor proteins in L. donovani and other Leishmania species based on sequence similarity, phylogenetic analysis and molecular modelling studies in L. donovani. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1842-5) contains supplementary material, which is available to authorized users.
High throughput genomic sequencing of bioaerosols in broiler chicken production facilities
Summary Chronic inhalation exposure to agricultural dust promotes the development of chronic respiratory diseases among poultry workers. Poultry dust is composed of dander, chicken feed, litter bedding and microbes. However, the microbial composition and abundance has not been fully elucidated. Genomic DNA was extracted from settled dust and personal inhalable dust collected while performing litter sampling or mortality collection tasks. DNA libraries were sequenced using a paired‐end sequencing‐by‐synthesis approach on an Illumina HiSeq 2500. Sequencing data showed that poultry dust is predominantly composed of bacteria (64–67%) with a small quantity of avian, human and feed DNA (< 2% of total reads). Staphylococcus sp. AL1, Salinicoccus carnicancri and Lactobacillus crispatus were the most abundant bacterial species in personal exposure samples of inhalable dust. Settled dust had a moderate relative abundance of these species as well as Staphylococcus lentus and Lactobacillus salivarius. There was a statistical difference between the microbial composition of aerosolized and settled dust. Unlike settled dust composition, aerosolized dust composition had little variance between samples. These data provide an extensive analysis of the microbial composition and relative abundance in personal inhalable poultry dust and settled poultry dust. Summary Chronic inhalation exposure to agricultural dust promotes the development of chronic respiratory diseases among poultry workers. Poultry dust is composed of dander, chicken feed, litter bedding and microbes. However, the microbial composition and abundance has not been fully elucidated. Genomic DNA was extracted from settled dust and personal inhalable dust collected while performing litter sampling or mortality collection tasks. DNA libraries were sequenced using a paired‐end sequencing‐by‐synthesis approach on an Illumina HiSeq 2500. Sequencing data showed that poultry dust is predominantly composed of bacteria (64–67%) with a small quantity of avian, human and feed DNA (< 2% of total reads). Staphylococcus sp. AL1, Salinicoccus carnicancri and Lactobacillus crispatus were the most abundant bacterial species in personal exposure samples of inhalable dust. Settled dust had a moderate relative abundance of these species as well as Staphylococcus lentus and Lactobacillus salivarius. There was a statistical difference between the microbial composition of aerosolized and settled dust. Unlike settled dust composition, aerosolized dust composition had little variance between samples. These data provide an extensive analysis of the microbial composition and relative abundance in personal inhalable poultry dust and settled poultry dust.
Fine Mapping of a Dravet Syndrome Modifier Locus on Mouse Chromosome 5 and Candidate Gene Analysis by RNA Seq
Author Summary Epilepsy is a neurological disorder affecting approximately 3 million Americans and 1% of the worldwide population. Approximately 70% of patients diagnosed with epilepsy have a genetic basis for their disease. The same genetic mutation can result in epilepsy with varying clinical severity in some individuals. This suggests that additional factors modify the effect of the primary mutation, resulting in the differences observed. Mouse models of epilepsy also exhibit variable severity depending on the inbred strain background. This is a useful model system that enables us to determine other genetic factors that influence phenotype presentation. This study investigates a mouse model of Dravet syndrome. Similar to Dravet syndrome patients, the mice experience a variety of seizure types and have increased risk for premature death. Interestingly, the Dravet syndrome phenotype is completely masked on one mouse strain background. Through a series of genetic and pharmacological techniques we were able to identify a gene that is likely responsible for the strain-dependent phenotype difference of Dravet mice. Identifying genes that modify diseases can help predict patient outcomes and suggest new therapies for the treatment of epilepsy. Author Summary Epilepsy is a neurological disorder affecting approximately 3 million Americans and 1% of the worldwide population. Approximately 70% of patients diagnosed with epilepsy have a genetic basis for their disease. The same genetic mutation can result in epilepsy with varying clinical severity in some individuals. This suggests that additional factors modify the effect of the primary mutation, resulting in the differences observed. Mouse models of epilepsy also exhibit variable severity depending on the inbred strain background. This is a useful model system that enables us to determine other genetic factors that influence phenotype presentation. This study investigates a mouse model of Dravet syndrome. Similar to Dravet syndrome patients, the mice experience a variety of seizure types and have increased risk for premature death. Interestingly, the Dravet syndrome phenotype is completely masked on one mouse strain background. Through a series of genetic and pharmacological techniques we were able to identify a gene that is likely responsible for the strain-dependent phenotype difference of Dravet mice. Identifying genes that modify diseases can help predict patient outcomes and suggest new therapies for the treatment of epilepsy.A substantial number of mutations have been identified in voltage-gated sodium channel genes that result in various forms of human epilepsy. SCN1A mutations result in a spectrum of severity ranging from mild febrile seizures to Dravet syndrome, an infant-onset epileptic encephalopathy. Dravet syndrome patients experience multiple seizures types that are often refractory to treatment, developmental delays, and elevated risk for SUDEP. The same sodium channel mutation can produce epilepsy phenotypes of varying clinical severity. This suggests that other factors, including genetic, modify the primary mutation and change disease severity. Mouse models provide a useful tool in studying the genetic basis of epilepsy. The mouse strain background can alter phenotype severity, supporting a contribution of genetic modifiers in epilepsy. The Scn1a+/- mouse model has a strain-dependent epilepsy phenotype. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have a normal phenotype and lifespan, while [129xC57BL/6J]F1-Scn1a+/- mice experience spontaneous seizures, hyperthermia-induced seizures and high rates of premature death. We hypothesize the phenotypic differences are due to strain-specific genetic modifiers that influence expressivity of the Scn1a+/- phenotype. Low resolution mapping of Scn1a+/- identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival. One locus of interest, Dsm1 located on chromosome 5, was fine mapped to a 9 Mb region using interval specific congenics. RNA-Seq was then utilized to identify candidate modifier genes within this narrowed region. Three genes with significant total gene expression differences between 129S6/SvEvTac and [129xC57BL/6J]F1 were identified, including the GABAA receptor subunit, Gabra2. Further analysis of Gabra2 demonstrated allele-specific expression. Pharmological manipulation by clobazam, a common anticonvulsant with preferential affinity for the GABRA2 receptor, revealed dose-dependent protection against hyperthermia-induced seizures in Scn1a+/- mice. These findings support Gabra2 as a genetic modifier of the Scn1a+/- mouse model of Dravet syndrome. A substantial number of mutations have been identified in voltage-gated sodium channel genes that result in various forms of human epilepsy. SCN1A mutations result in a spectrum of severity ranging from mild febrile seizures to Dravet syndrome, an infant-onset epileptic encephalopathy. Dravet syndrome patients experience multiple seizures types that are often refractory to treatment, developmental delays, and elevated risk for SUDEP. The same sodium channel mutation can produce epilepsy phenotypes of varying clinical severity. This suggests that other factors, including genetic, modify the primary mutation and change disease severity. Mouse models provide a useful tool in studying the genetic basis of epilepsy. The mouse strain background can alter phenotype severity, supporting a contribution of genetic modifiers in epilepsy. The Scn1a+/- mouse model has a strain-dependent epilepsy phenotype. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have a normal phenotype and lifespan, while [129xC57BL/6J]F1-Scn1a+/- mice experience spontaneous seizures, hyperthermia-induced seizures and high rates of premature death. We hypothesize the phenotypic differences are due to strain-specific genetic modifiers that influence expressivity of the Scn1a+/- phenotype. Low resolution mapping of Scn1a+/- identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival. One locus of interest, Dsm1 located on chromosome 5, was fine mapped to a 9 Mb region using interval specific congenics. RNA-Seq was then utilized to identify candidate modifier genes within this narrowed region. Three genes with significant total gene expression differences between 129S6/SvEvTac and [129xC57BL/6J]F1 were identified, including the GABAA receptor subunit, Gabra2. Further analysis of Gabra2 demonstrated allele-specific expression. Pharmological manipulation by clobazam, a common anticonvulsant with preferential affinity for the GABRA2 receptor, revealed dose-dependent protection against hyperthermia-induced seizures in Scn1a+/- mice. These findings support Gabra2 as a genetic modifier of the Scn1a+/- mouse model of Dravet syndrome.
Genomic copy number variation association study in Caucasian patients with nonsyndromic cryptorchidism
Background Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population. Methods Genome wide genotyping were performed in 559 cases and 1772 controls (Group 1) using Illumina HumanHap550 v1, HumanHap550 v3 or Human610-Quad platforms and in 353 cases and 1149 controls (Group 2) using the Illumina Human OmniExpress 12v1 or Human OmniExpress 12v1-1. Signal intensity data including log R ratio (LRR) and B allele frequency (BAF) for each single nucleotide polymorphism (SNP) were used for CNV detection using PennCNV software. After sample quality control, gene- and CNV-based association tests were performed using cleaned data from Group 1 (493 cases and 1586 controls) and Group 2 (307 cases and 1102 controls) using ParseCNV software. Meta-analysis was performed using gene-based test results as input to identify significant genes, and CNVs in or around significant genes were identified in CNV-based association test results. Called CNVs passing quality control and signal intensity visualization examination were considered for validation using TaqMan CNV assays and QuantStudio® 3D Digital PCR System. Results The meta-analysis identified 373 genome wide significant (p < 5X10−4) genes/loci including 49 genes/loci with deletions and 324 with duplications. Among them, 17 genes with deletion and 1 gene with duplication were identified in CNV-based association results in both Group 1 and Group 2. Only 2 genes (NUCB2 and UPF2) containing deletions passed CNV quality control in both groups and signal intensity visualization examination, but laboratory validation failed to verify these deletions. Conclusions Our data do not support that structural variation is a major cause of nonsyndromic cryptorchidism. Electronic supplementary material The online version of this article (doi:10.1186/s12894-016-0180-4) contains supplementary material, which is available to authorized users. Background Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population. Methods Genome wide genotyping were performed in 559 cases and 1772 controls (Group 1) using Illumina HumanHap550 v1, HumanHap550 v3 or Human610-Quad platforms and in 353 cases and 1149 controls (Group 2) using the Illumina Human OmniExpress 12v1 or Human OmniExpress 12v1-1. Signal intensity data including log R ratio (LRR) and B allele frequency (BAF) for each single nucleotide polymorphism (SNP) were used for CNV detection using PennCNV software. After sample quality control, gene- and CNV-based association tests were performed using cleaned data from Group 1 (493 cases and 1586 controls) and Group 2 (307 cases and 1102 controls) using ParseCNV software. Meta-analysis was performed using gene-based test results as input to identify significant genes, and CNVs in or around significant genes were identified in CNV-based association test results. Called CNVs passing quality control and signal intensity visualization examination were considered for validation using TaqMan CNV assays and QuantStudio® 3D Digital PCR System. Results The meta-analysis identified 373 genome wide significant (p < 5X10−4) genes/loci including 49 genes/loci with deletions and 324 with duplications. Among them, 17 genes with deletion and 1 gene with duplication were identified in CNV-based association results in both Group 1 and Group 2. Only 2 genes (NUCB2 and UPF2) containing deletions passed CNV quality control in both groups and signal intensity visualization examination, but laboratory validation failed to verify these deletions. Conclusions Our data do not support that structural variation is a major cause of nonsyndromic cryptorchidism. Electronic supplementary material The online version of this article (doi:10.1186/s12894-016-0180-4) contains supplementary material, which is available to authorized users.
Heterozygous mutations in HSD17B4 cause juvenile peroxisomal D bifunctional protein deficiency
Objective: To determine the genetic cause of slowly progressive cerebellar ataxia, sensorineural deafness, and hypergonadotropic hypogonadism in 5 patients from 3 different families. Methods: The patients comprised 2 sib pairs and 1 sporadic patient. Clinical assessment included history, physical examination, and brain MRI. Linkage analysis was performed separately on the 2 sets of sib pairs using single nucleotide polymorphism microarrays, followed by analysis of the intersection of the regions. Exome sequencing was performed on 1 affected patient with variant filtering and prioritization undertaken using these intersected regions. Results: Using a combination of sequencing technologies, we identified compound heterozygous mutations in HSD17B4 in all 5 affected patients. In all 3 families, peroxisomal D-bifunctional protein (DBP) deficiency was caused by compound heterozygosity for 1 nonsense/deletion mutation and 1 missense mutation. Conclusions: We describe 5 patients with juvenile DBP deficiency from 3 different families, bringing the total number of reported patients to 14, from 8 families. This report broadens and consolidates the phenotype associated with juvenile DBP deficiency. Objective: To determine the genetic cause of slowly progressive cerebellar ataxia, sensorineural deafness, and hypergonadotropic hypogonadism in 5 patients from 3 different families. Methods: The patients comprised 2 sib pairs and 1 sporadic patient. Clinical assessment included history, physical examination, and brain MRI. Linkage analysis was performed separately on the 2 sets of sib pairs using single nucleotide polymorphism microarrays, followed by analysis of the intersection of the regions. Exome sequencing was performed on 1 affected patient with variant filtering and prioritization undertaken using these intersected regions. Results: Using a combination of sequencing technologies, we identified compound heterozygous mutations in HSD17B4 in all 5 affected patients. In all 3 families, peroxisomal D-bifunctional protein (DBP) deficiency was caused by compound heterozygosity for 1 nonsense/deletion mutation and 1 missense mutation. Conclusions: We describe 5 patients with juvenile DBP deficiency from 3 different families, bringing the total number of reported patients to 14, from 8 families. This report broadens and consolidates the phenotype associated with juvenile DBP deficiency.
Viral Genome Sequencing Proves Nosocomial Transmission of Fatal Varicella
We report the first use of whole viral genome sequencing to identify nosocomial transmission of varicella-zoster virus with fatal outcome. The index case patient, nursed in source isolation, developed disseminated zoster with rash present for 1 day before being transferred to the intensive care unit (ICU). Two patients who had received renal transplants while inpatients in an adjacent ward developed chickenpox and 1 died; neither patient had direct contact with the index patient. We report the first use of whole viral genome sequencing to identify nosocomial transmission of varicella-zoster virus with fatal outcome. The index case patient, nursed in source isolation, developed disseminated zoster with rash present for 1 day before being transferred to the intensive care unit (ICU). Two patients who had received renal transplants while inpatients in an adjacent ward developed chickenpox and 1 died; neither patient had direct contact with the index patient.
Overexpression of Nictaba Like Lectin Genes from Glycine max Confers Tolerance toward Pseudomonas syringae Infection, Aphid Infestation and Salt Stress in Transgenic Arabidopsis Plants
Plants have evolved a sophisticated immune system that allows them to recognize invading pathogens by specialized receptors. Carbohydrate-binding proteins or lectins are part of this immune system and especially the lectins that reside in the nucleocytoplasmic compartment are known to be implicated in biotic and abiotic stress responses. The class of Nictaba-like lectins (NLL) groups all proteins with homology to the tobacco (Nicotiana tabacum) lectin, known as a stress-inducible lectin. Here we focus on two Nictaba homologs from soybean (Glycine max), referred to as GmNLL1 and GmNLL2. Confocal laser scanning microscopy of fusion constructs with the green fluorescent protein either transiently expressed in Nicotiana benthamiana leaves or stably transformed in tobacco BY-2 suspension cells revealed a nucleocytoplasmic localization for the GmNLLs under study. RT-qPCR analysis of the transcript levels for the Nictaba-like lectins in soybean demonstrated that the genes are expressed in several tissues throughout the development of the plant. Furthermore, it was shown that salt treatment, Phytophthora sojae infection and Aphis glycines infestation trigger the expression of particular NLL genes. Stress experiments with Arabidopsis lines overexpressing the NLLs from soybean yielded an enhanced tolerance of the plant toward bacterial infection (Pseudomonas syringae), insect infestation (Myzus persicae) and salinity. Our data showed a better performance of the transgenic lines compared to wild type plants, indicating that the NLLs from soybean are implicated in the stress response. These data can help to further elucidate the physiological importance of the Nictaba-like lectins from soybean, which can ultimately lead to the design of crop plants with a better tolerance to changing environmental conditions. Plants have evolved a sophisticated immune system that allows them to recognize invading pathogens by specialized receptors. Carbohydrate-binding proteins or lectins are part of this immune system and especially the lectins that reside in the nucleocytoplasmic compartment are known to be implicated in biotic and abiotic stress responses. The class of Nictaba-like lectins (NLL) groups all proteins with homology to the tobacco (Nicotiana tabacum) lectin, known as a stress-inducible lectin. Here we focus on two Nictaba homologs from soybean (Glycine max), referred to as GmNLL1 and GmNLL2. Confocal laser scanning microscopy of fusion constructs with the green fluorescent protein either transiently expressed in Nicotiana benthamiana leaves or stably transformed in tobacco BY-2 suspension cells revealed a nucleocytoplasmic localization for the GmNLLs under study. RT-qPCR analysis of the transcript levels for the Nictaba-like lectins in soybean demonstrated that the genes are expressed in several tissues throughout the development of the plant. Furthermore, it was shown that salt treatment, Phytophthora sojae infection and Aphis glycines infestation trigger the expression of particular NLL genes. Stress experiments with Arabidopsis lines overexpressing the NLLs from soybean yielded an enhanced tolerance of the plant toward bacterial infection (Pseudomonas syringae), insect infestation (Myzus persicae) and salinity. Our data showed a better performance of the transgenic lines compared to wild type plants, indicating that the NLLs from soybean are implicated in the stress response. These data can help to further elucidate the physiological importance of the Nictaba-like lectins from soybean, which can ultimately lead to the design of crop plants with a better tolerance to changing environmental conditions.
Ligand bound Structures and Site directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE*
GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential. GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.
Contrasting Influences of Geographic Range and Distribution of Populations on Patterns of Genetic Diversity in Two Sympatric Pilbara Acacias
The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species. The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species.
Improving sensitivity of linear regression based cell type specific differential expression deconvolution with per gene vs. global significance threshold
Background The goal of many human disease-oriented studies is to detect molecular mechanisms different between healthy controls and patients. Yet, commonly used gene expression measurements from blood samples suffer from variability of cell composition. This variability hinders the detection of differentially expressed genes and is often ignored. Combined with cell counts, heterogeneous gene expression may provide deeper insights into the gene expression differences on the cell type-specific level. Published computational methods use linear regression to estimate cell type-specific differential expression, and a global cutoff to judge significance, such as False Discovery Rate (FDR). Yet, they do not consider many artifacts hidden in high-dimensional gene expression data that may negatively affect linear regression. In this paper we quantify the parameter space affecting the performance of linear regression (sensitivity of cell type-specific differential expression detection) on a per-gene basis. Results We evaluated the effect of sample sizes, cell type-specific proportion variability, and mean squared error on sensitivity of cell type-specific differential expression detection using linear regression. Each parameter affected variability of cell type-specific expression estimates and, subsequently, the sensitivity of differential expression detection. We provide the R package, LRCDE, which performs linear regression-based cell type-specific differential expression (deconvolution) detection on a gene-by-gene basis. Accounting for variability around cell type-specific gene expression estimates, it computes per-gene t-statistics of differential detection, p-values, t-statistic-based sensitivity, group-specific mean squared error, and several gene-specific diagnostic metrics. Conclusions The sensitivity of linear regression-based cell type-specific differential expression detection differed for each gene as a function of mean squared error, per group sample sizes, and variability of the proportions of target cell (cell type being analyzed). We demonstrate that LRCDE, which uses Welch’s t-test to compare per-gene cell type-specific gene expression estimates, is more sensitive in detecting cell type-specific differential expression at α < 0.05 missed by the global false discovery rate threshold FDR < 0.3. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1226-z) contains supplementary material, which is available to authorized users. Background The goal of many human disease-oriented studies is to detect molecular mechanisms different between healthy controls and patients. Yet, commonly used gene expression measurements from blood samples suffer from variability of cell composition. This variability hinders the detection of differentially expressed genes and is often ignored. Combined with cell counts, heterogeneous gene expression may provide deeper insights into the gene expression differences on the cell type-specific level. Published computational methods use linear regression to estimate cell type-specific differential expression, and a global cutoff to judge significance, such as False Discovery Rate (FDR). Yet, they do not consider many artifacts hidden in high-dimensional gene expression data that may negatively affect linear regression. In this paper we quantify the parameter space affecting the performance of linear regression (sensitivity of cell type-specific differential expression detection) on a per-gene basis. Results We evaluated the effect of sample sizes, cell type-specific proportion variability, and mean squared error on sensitivity of cell type-specific differential expression detection using linear regression. Each parameter affected variability of cell type-specific expression estimates and, subsequently, the sensitivity of differential expression detection. We provide the R package, LRCDE, which performs linear regression-based cell type-specific differential expression (deconvolution) detection on a gene-by-gene basis. Accounting for variability around cell type-specific gene expression estimates, it computes per-gene t-statistics of differential detection, p-values, t-statistic-based sensitivity, group-specific mean squared error, and several gene-specific diagnostic metrics. Conclusions The sensitivity of linear regression-based cell type-specific differential expression detection differed for each gene as a function of mean squared error, per group sample sizes, and variability of the proportions of target cell (cell type being analyzed). We demonstrate that LRCDE, which uses Welch’s t-test to compare per-gene cell type-specific gene expression estimates, is more sensitive in detecting cell type-specific differential expression at α < 0.05 missed by the global false discovery rate threshold FDR < 0.3. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1226-z) contains supplementary material, which is available to authorized users.
Draft Genome Sequence of Extremely Drug Resistant Pseudomonas aeruginosa (ST357) Strain CMC_VB_PA_B22862 Isolated from a Community Acquired Bloodstream Infection
Extremely drug-resistant Pseudomonas aeruginosa strains causing severe infections have become a serious concern across the world. Here, we report draft genome sequence of P. aeruginosa with an extremely drug-resistant profile isolated from a patient with community-acquired bloodstream infection in India. Extremely drug-resistant Pseudomonas aeruginosa strains causing severe infections have become a serious concern across the world. Here, we report draft genome sequence of P. aeruginosa with an extremely drug-resistant profile isolated from a patient with community-acquired bloodstream infection in India.
Structures and stabilization of kinetoplastid specific split rRNAs revealed by comparing leishmanial and human ribosomes
Leishmania donovani is a protozoan parasite that can cause fatal infections in humans. Here the authors present a high resolution cryoEM structure of the L. donovani 80S ribosome and compare it to its human counterpart to provide insight into the basis for drug selectivity towards this eukaryotic parasite. Leishmania donovani is a protozoan parasite that can cause fatal infections in humans. Here the authors present a high resolution cryoEM structure of the L. donovani 80S ribosome and compare it to its human counterpart to provide insight into the basis for drug selectivity towards this eukaryotic parasite.The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome. The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.
Draft Genome Sequence of Leifsonia xyli subsp. xyli Strain gdw1
Here, we report the draft genome sequence of Leifsonia xyli subsp. xyli strain gdw1, isolated from the stem of Badila sugarcane located at the Guangdong Key Laboratory for Crops Genetic Improvement (Guanzhou, China), that causes ratoon stunting disease of sugarcane. The de novo genome of Leifsonia xyli subsp. xyli was assembled with 48 scaffolds and a G+C content of 67.68%, and contained 2.6 Mb bp and 2,838 coding sequences. Here, we report the draft genome sequence of Leifsonia xyli subsp. xyli strain gdw1, isolated from the stem of Badila sugarcane located at the Guangdong Key Laboratory for Crops Genetic Improvement (Guanzhou, China), that causes ratoon stunting disease of sugarcane. The de novo genome of Leifsonia xyli subsp. xyli was assembled with 48 scaffolds and a G+C content of 67.68%, and contained 2.6 Mb bp and 2,838 coding sequences.
Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell
Background A defining feature of eukaryotic cells is the presence of various distinct membrane-bound compartments with different metabolic roles. Material exchange between most compartments occurs via a sophisticated vesicle trafficking system. This intricate cellular architecture of eukaryotes appears to have emerged suddenly, about 2 billion years ago, from much less complex ancestors. How the eukaryotic cell acquired its internal complexity is poorly understood, partly because no prokaryotic precursors have been found for many key factors involved in compartmentalization. One exception is the Cdc48 protein family, which consists of several distinct classical ATPases associated with various cellular activities (AAA+) proteins with two consecutive AAA domains. Results Here, we have classified the Cdc48 family through iterative use of hidden Markov models and tree building. We found only one type, Cdc48, in prokaryotes, although a set of eight diverged members that function at distinct subcellular compartments were retrieved from eukaryotes and were probably present in the last eukaryotic common ancestor (LECA). Pronounced changes in sequence and domain structure during the radiation into the LECA set are delineated. Moreover, our analysis brings to light lineage-specific losses and duplications that often reflect important biological changes. Remarkably, we also found evidence for internal duplications within the LECA set that probably occurred during the rise of the eukaryotic cell. Conclusions Our analysis corroborates the idea that the diversification of the Cdc48 family is closely intertwined with the development of the compartments of the eukaryotic cell. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0790-1) contains supplementary material, which is available to authorized users. Background A defining feature of eukaryotic cells is the presence of various distinct membrane-bound compartments with different metabolic roles. Material exchange between most compartments occurs via a sophisticated vesicle trafficking system. This intricate cellular architecture of eukaryotes appears to have emerged suddenly, about 2 billion years ago, from much less complex ancestors. How the eukaryotic cell acquired its internal complexity is poorly understood, partly because no prokaryotic precursors have been found for many key factors involved in compartmentalization. One exception is the Cdc48 protein family, which consists of several distinct classical ATPases associated with various cellular activities (AAA+) proteins with two consecutive AAA domains. Results Here, we have classified the Cdc48 family through iterative use of hidden Markov models and tree building. We found only one type, Cdc48, in prokaryotes, although a set of eight diverged members that function at distinct subcellular compartments were retrieved from eukaryotes and were probably present in the last eukaryotic common ancestor (LECA). Pronounced changes in sequence and domain structure during the radiation into the LECA set are delineated. Moreover, our analysis brings to light lineage-specific losses and duplications that often reflect important biological changes. Remarkably, we also found evidence for internal duplications within the LECA set that probably occurred during the rise of the eukaryotic cell. Conclusions Our analysis corroborates the idea that the diversification of the Cdc48 family is closely intertwined with the development of the compartments of the eukaryotic cell. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0790-1) contains supplementary material, which is available to authorized users.
Klf5 regulates muscle differentiation by directly targeting muscle specific genes in cooperation with MyoD in mice
Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2. DOI: http://dx.doi.org/10.7554/eLife.17462.001 Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2. DOI: http://dx.doi.org/10.7554/eLife.17462.001
High Quality Draft Genome Sequence of an Endophytic Pseudomonas viridiflava Strain with Herbicidal Properties against Its Host, the Weed Lepidium draba L.
Here, we report the draft genome sequence of Pseudomonas viridiflava strain CDRTc14 a pectinolytic bacterium showing herbicidal activity, isolated from the root of Lepidium draba L. growing as a weed in an Austrian vineyard. The availability of this genome sequence allows us to investigate the genetic basis of plant–microbe interactions. Here, we report the draft genome sequence of Pseudomonas viridiflava strain CDRTc14 a pectinolytic bacterium showing herbicidal activity, isolated from the root of Lepidium draba L. growing as a weed in an Austrian vineyard. The availability of this genome sequence allows us to investigate the genetic basis of plant–microbe interactions.
Genetic diversity and population differentiation of small giant clam Tridacna maxima in Comoros islands assessed by microsatellite markers
Small giant clam, Tridacna maxima, widely distributed from French Polynesia to East Africa, has faced population declines due to over-exploitation. Comoros islands are an important biogeographic region due to potential richness of marine species, but no relevant information is available. In order to facilitate devising effective conservation management plan for T. maxima, nine microsatellite markers were used to survey genetic diversity and population differentiation of 72 specimens collected from three Comoros islands, Grande Comore, Moheli and Anjouan. A total of 51 alleles were detected ranged from 2 to 8 per locus. Observed and expected heterozygosity varied from 0.260 to 0.790 and from 0.542 to 0.830, respectively. All populations have high genetic diversity, especially the population in Moheli, a protected area, has higher genetic diversity than the others. Significant heterozygote deficiencies were recorded, and null alleles were probably the main factor leading to these deficits. F ST value indicated medium genetic differentiation among the populations. Although significant, AMOVA revealed 48.9 % of genetic variation within individuals and only a small variation of 8.9 % was found between populations. Gene flow was high (Nm = 12.40) between Grande Comore and Moheli, while lower (Nm = 1.80) between Grande Comore and Anjouan, explaining geographic barriers to genetic exchanges might exist in these two islands. Global gene flow analysis (Nm = 5.50) showed that larval dispersal is enough to move between the islands. The high genetic diversity and medium population differentiation revealed in the present study offer useful information on genetic conservation of small giant clams. Small giant clam, Tridacna maxima, widely distributed from French Polynesia to East Africa, has faced population declines due to over-exploitation. Comoros islands are an important biogeographic region due to potential richness of marine species, but no relevant information is available. In order to facilitate devising effective conservation management plan for T. maxima, nine microsatellite markers were used to survey genetic diversity and population differentiation of 72 specimens collected from three Comoros islands, Grande Comore, Moheli and Anjouan. A total of 51 alleles were detected ranged from 2 to 8 per locus. Observed and expected heterozygosity varied from 0.260 to 0.790 and from 0.542 to 0.830, respectively. All populations have high genetic diversity, especially the population in Moheli, a protected area, has higher genetic diversity than the others. Significant heterozygote deficiencies were recorded, and null alleles were probably the main factor leading to these deficits. F ST value indicated medium genetic differentiation among the populations. Although significant, AMOVA revealed 48.9 % of genetic variation within individuals and only a small variation of 8.9 % was found between populations. Gene flow was high (Nm = 12.40) between Grande Comore and Moheli, while lower (Nm = 1.80) between Grande Comore and Anjouan, explaining geographic barriers to genetic exchanges might exist in these two islands. Global gene flow analysis (Nm = 5.50) showed that larval dispersal is enough to move between the islands. The high genetic diversity and medium population differentiation revealed in the present study offer useful information on genetic conservation of small giant clams.
Complete Genome Sequence of Spiroplasma helicoides TABS 2T (DSM 22551), a Bacterium Isolated from a Horsefly (Tabanus abactor)
Spiroplasma helicoides TABS-2T (DSM 22551) was isolated from the gut of a horsefly (Tabanus abactor) collected near Ardmore, Oklahoma, USA, in 1987. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species. Spiroplasma helicoides TABS-2T (DSM 22551) was isolated from the gut of a horsefly (Tabanus abactor) collected near Ardmore, Oklahoma, USA, in 1987. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species.
Wild Carrot Differentiation in Europe and Selection at DcAOX1 Gene?
By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity. By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.
Targeted inhibition of the COP9 signalosome for treatment of cancer
Dysregulation of protein degradation by the ubiquitin-proteasome system is a feature commonly associated with cancer. Here, the authors develop an orally available small molecule that inhibits CSN5, the proteolytic subunit of the COP9 signalosome, and blocks tumour growth in a xenograft model. Dysregulation of protein degradation by the ubiquitin-proteasome system is a feature commonly associated with cancer. Here, the authors develop an orally available small molecule that inhibits CSN5, the proteolytic subunit of the COP9 signalosome, and blocks tumour growth in a xenograft model.The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin–proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy. The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin–proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.
Production and verification of a 2nd generation clonal group of Japanese flounder, Paralichthys olivaceus
Clonal fishes are useful tools in biology and aquaculture studies due to their isogenicity. In Japanese flounder (Paralichthys olivaceus), a group of homozygous clones was created by inducing meiogynogenesis in eggs from a mitogynogenetic homozygous diploid. As the clones reached sexual maturity, meiogynogenesis was again induced in order to produce a 2nd generation clonal group of Japanese flounder. After 3 months, there were 611 healthy, surviving individuals. Twenty-four microsatellite markers, that covered all the linkage groups of Japanese flounder, were used to identify the homozygosity of the 2nd generation clones; no heterozygous locus was detected. This indicates that the production of a 2nd generation clonal group of Japanese flounder was successful. Restriction-site DNA associated sequencing at the genomic level also confirmed the homozygosity and clonality of the 2nd generation clonal group. Furthermore, these 2nd generation clones had a small coefficient of variation for body shape indices at 210 days of age and showed a high degree of similarity in body characteristics among individuals. The successful production of 2nd generation clones has laid the foundation for the large-scale production of clonal Japanese flounder. Clonal fishes are useful tools in biology and aquaculture studies due to their isogenicity. In Japanese flounder (Paralichthys olivaceus), a group of homozygous clones was created by inducing meiogynogenesis in eggs from a mitogynogenetic homozygous diploid. As the clones reached sexual maturity, meiogynogenesis was again induced in order to produce a 2nd generation clonal group of Japanese flounder. After 3 months, there were 611 healthy, surviving individuals. Twenty-four microsatellite markers, that covered all the linkage groups of Japanese flounder, were used to identify the homozygosity of the 2nd generation clones; no heterozygous locus was detected. This indicates that the production of a 2nd generation clonal group of Japanese flounder was successful. Restriction-site DNA associated sequencing at the genomic level also confirmed the homozygosity and clonality of the 2nd generation clonal group. Furthermore, these 2nd generation clones had a small coefficient of variation for body shape indices at 210 days of age and showed a high degree of similarity in body characteristics among individuals. The successful production of 2nd generation clones has laid the foundation for the large-scale production of clonal Japanese flounder.
Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling
Pepper pericarp microbiota plays an important role in the pepper peeling process for the production of white pepper. We collected pepper samples at different peeling time points from Hainan Province, China, and used a metagenomic approach to identify changes in the pericarp microbiota based on functional gene analysis. UniFrac distance-based principal coordinates analysis revealed significant changes in the pericarp microbiota structure during peeling, which were attributed to increases in bacteria from the genera Selenomonas and Prevotella. We identified 28 core operational taxonomic units at each time point, mainly belonging to Selenomonas, Prevotella, Megasphaera, Anaerovibrio, and Clostridium genera. The results were confirmed by quantitative polymerase chain reaction. At the functional level, we observed significant increases in microbial features related to acetyl xylan esterase and pectinesterase for pericarp degradation during peeling. These findings offer a new insight into biodegradation for pepper peeling and will promote the development of the white pepper industry. Pepper pericarp microbiota plays an important role in the pepper peeling process for the production of white pepper. We collected pepper samples at different peeling time points from Hainan Province, China, and used a metagenomic approach to identify changes in the pericarp microbiota based on functional gene analysis. UniFrac distance-based principal coordinates analysis revealed significant changes in the pericarp microbiota structure during peeling, which were attributed to increases in bacteria from the genera Selenomonas and Prevotella. We identified 28 core operational taxonomic units at each time point, mainly belonging to Selenomonas, Prevotella, Megasphaera, Anaerovibrio, and Clostridium genera. The results were confirmed by quantitative polymerase chain reaction. At the functional level, we observed significant increases in microbial features related to acetyl xylan esterase and pectinesterase for pericarp degradation during peeling. These findings offer a new insight into biodegradation for pepper peeling and will promote the development of the white pepper industry.
Draft Genome Sequence of Streptomyces sp. TP A0874, a Catechoserine Producer
We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis. We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis.
Draft genome sequence of Streptomyces sp. TP A0867, an alchivemycin producer
Streptomyces sp. TP-A0867 (=NBRC 109436) produces structurally complex polyketides designated alchivemycins A and B. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation, and analysis of the genome sequence. The 9.9 Mb genome of Streptomyces sp. TP-A0867 encodes 8,385 putative ORFs, of which 7,232 were assigned with COG categories. We successfully identified a hybrid polyketide synthase (PKS)/ nonribosomal peptide synthetase (NRPS) gene cluster that could be responsible for alchivemycin biosynthesis, and propose the biosynthetic pathway. The alchivemycin biosynthetic gene cluster is also present in Streptomyces rapamycinicus NRRL 5491T, Streptomyces hygroscopicus subsp. hygroscopicus NBRC 16556, and Streptomyces ascomycinicus NBRC 13981T, which are taxonomically highly close to strain TP-A0867. This study shows a representative example that distribution of secondary metabolite genes is correlated with evolution within the genus Streptomyces. Streptomyces sp. TP-A0867 (=NBRC 109436) produces structurally complex polyketides designated alchivemycins A and B. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation, and analysis of the genome sequence. The 9.9 Mb genome of Streptomyces sp. TP-A0867 encodes 8,385 putative ORFs, of which 7,232 were assigned with COG categories. We successfully identified a hybrid polyketide synthase (PKS)/ nonribosomal peptide synthetase (NRPS) gene cluster that could be responsible for alchivemycin biosynthesis, and propose the biosynthetic pathway. The alchivemycin biosynthetic gene cluster is also present in Streptomyces rapamycinicus NRRL 5491T, Streptomyces hygroscopicus subsp. hygroscopicus NBRC 16556, and Streptomyces ascomycinicus NBRC 13981T, which are taxonomically highly close to strain TP-A0867. This study shows a representative example that distribution of secondary metabolite genes is correlated with evolution within the genus Streptomyces.
Differentially expressed circulating LncRNAs and mRNA identified by microarray analysis in obese patients
Circulating long non-coding RNAs (lncRNAs) serve as valuable biomarkers in a number of human diseases. However, lncRNA biomarkers have yet to be identified in obesity. We aim to characterize circulating lncRNA expression in obese and non-obese human subjects. First, we assessed the genome-wide circulating lncRNA expression profiles in blood from 3 obese and 3 non-obese human subjects. We found a significant decrease in circulating levels of three lncRNAs (lncRNA-p5549, lncRNA-p21015 and lncRNA-p19461) in obese human subjects only. Next, using RT-PCR we measured the expression levels of these three lncRNAs in 33 obese and 33 non-obese human subjects and found similar differences. Moreover, we found a negative correlation between circulating levels of these three lncRNAs and body mass index (BMI), waist circumference, waist to hip ratio and fasting insulin. There was also a significant negative correlation between expression of lncRNA-p19461 and homeostasis model assessment-estimated insulin resistance. Finally, we tested the circulating levels of these three lncRNAs in 8 obese human subjects after a 12-week diet-induced weight loss program. We found that only lncRNA-p19461 expression level significantly increased. In summary, circulating lncRNAs are deregulated in obesity. Weight loss–induced changes in this profile support this observation and suggest a potential mechanistic relevance. Circulating long non-coding RNAs (lncRNAs) serve as valuable biomarkers in a number of human diseases. However, lncRNA biomarkers have yet to be identified in obesity. We aim to characterize circulating lncRNA expression in obese and non-obese human subjects. First, we assessed the genome-wide circulating lncRNA expression profiles in blood from 3 obese and 3 non-obese human subjects. We found a significant decrease in circulating levels of three lncRNAs (lncRNA-p5549, lncRNA-p21015 and lncRNA-p19461) in obese human subjects only. Next, using RT-PCR we measured the expression levels of these three lncRNAs in 33 obese and 33 non-obese human subjects and found similar differences. Moreover, we found a negative correlation between circulating levels of these three lncRNAs and body mass index (BMI), waist circumference, waist to hip ratio and fasting insulin. There was also a significant negative correlation between expression of lncRNA-p19461 and homeostasis model assessment-estimated insulin resistance. Finally, we tested the circulating levels of these three lncRNAs in 8 obese human subjects after a 12-week diet-induced weight loss program. We found that only lncRNA-p19461 expression level significantly increased. In summary, circulating lncRNAs are deregulated in obesity. Weight loss–induced changes in this profile support this observation and suggest a potential mechanistic relevance.
Differential Gene Expression in Rhododendron fortunei Roots Colonized by an Ericoid Mycorrhizal Fungus and Increased Nitrogen Absorption and Plant Growth
Ericoid mycorrhizal (ERM) fungi are specifically symbiotic with plants in the family Ericaceae. Little is known thus far about their symbiotic establishment and subsequent nitrogen (N) uptake at the molecular level. The present study devised a system for establishing a symbiotic relationship between Rhododendron fortunei Lindl. and an ERM fungus (Oidiodendron maius var. maius strain Om19), quantified seedling growth and N uptake, and compared transcriptome profiling between colonized and uncolonized roots using RNA-Seq. The Om19 colonization induced 16,892 genes that were differentially expressed in plant roots, of which 14,364 were upregulated and 2,528 were downregulated. These genes included those homologous to ATP-binding cassette transporters, calcium/calmodulin-dependent kinases, and symbiosis receptor-like kinases. N metabolism was particularly active in Om19-colonized roots, and 51 genes were upregulated, such as nitrate transporters, nitrate reductase, nitrite reductase, ammonium transporters, glutamine synthetase, and glutamate synthase. Transcriptome analysis also identified a series of genes involving endocytosis, Fc-gamma R-mediated phagocytosis, glycerophospholipid metabolism, and Gonadotropin-releasing hormone (GnRH) signal pathway that have not been reported previously. Their roles in the symbiosis require further investigation. The Om19 colonization significantly increased N uptake and seedling growth. Total N content and dry weight of colonized seedlings were 36.6 and 46.6% greater than control seedlings. This is the first transcriptome analysis of a species from the family Ericaceae colonized by an ERM fungus. The findings from this study will shed light on the mechanisms underlying symbiotic relationships of ericaceous species with ERM fungi and the symbiosis-resultant N uptake and plant growth. Ericoid mycorrhizal (ERM) fungi are specifically symbiotic with plants in the family Ericaceae. Little is known thus far about their symbiotic establishment and subsequent nitrogen (N) uptake at the molecular level. The present study devised a system for establishing a symbiotic relationship between Rhododendron fortunei Lindl. and an ERM fungus (Oidiodendron maius var. maius strain Om19), quantified seedling growth and N uptake, and compared transcriptome profiling between colonized and uncolonized roots using RNA-Seq. The Om19 colonization induced 16,892 genes that were differentially expressed in plant roots, of which 14,364 were upregulated and 2,528 were downregulated. These genes included those homologous to ATP-binding cassette transporters, calcium/calmodulin-dependent kinases, and symbiosis receptor-like kinases. N metabolism was particularly active in Om19-colonized roots, and 51 genes were upregulated, such as nitrate transporters, nitrate reductase, nitrite reductase, ammonium transporters, glutamine synthetase, and glutamate synthase. Transcriptome analysis also identified a series of genes involving endocytosis, Fc-gamma R-mediated phagocytosis, glycerophospholipid metabolism, and Gonadotropin-releasing hormone (GnRH) signal pathway that have not been reported previously. Their roles in the symbiosis require further investigation. The Om19 colonization significantly increased N uptake and seedling growth. Total N content and dry weight of colonized seedlings were 36.6 and 46.6% greater than control seedlings. This is the first transcriptome analysis of a species from the family Ericaceae colonized by an ERM fungus. The findings from this study will shed light on the mechanisms underlying symbiotic relationships of ericaceous species with ERM fungi and the symbiosis-resultant N uptake and plant growth.
The Chloroplast Genome of Utricularia reniformis Sheds Light on the Evolution of the ndh Gene Complex of Terrestrial Carnivorous Plants from the Lentibulariaceae Family
Lentibulariaceae is the richest family of carnivorous plants spanning three genera including Pinguicula, Genlisea, and Utricularia. Utricularia is globally distributed, and, unlike Pinguicula and Genlisea, has both aquatic and terrestrial forms. In this study we present the analysis of the chloroplast (cp) genome of the terrestrial Utricularia reniformis. U. reniformis has a standard cp genome of 139,725bp, encoding a gene repertoire similar to essentially all photosynthetic organisms. However, an exclusive combination of losses and pseudogenization of the plastid NAD(P)H-dehydrogenase (ndh) gene complex were observed. Comparisons among aquatic and terrestrial forms of Pinguicula, Genlisea, and Utricularia indicate that, whereas the aquatic forms retained functional copies of the eleven ndh genes, these have been lost or truncated in terrestrial forms, suggesting that the ndh function may be dispensable in terrestrial Lentibulariaceae. Phylogenetic scenarios of the ndh gene loss and recovery among Pinguicula, Genlisea, and Utricularia to the ancestral Lentibulariaceae cladeare proposed. Interestingly, RNAseq analysis evidenced that U. reniformis cp genes are transcribed, including the truncated ndh genes, suggesting that these are not completely inactivated. In addition, potential novel RNA-editing sites were identified in at least six U. reniformis cp genes, while none were identified in the truncated ndh genes. Moreover, phylogenomic analyses support that Lentibulariaceae is monophyletic, belonging to the higher core Lamiales clade, corroborating the hypothesis that the first Utricularia lineage emerged in terrestrial habitats and then evolved to epiphytic and aquatic forms. Furthermore, several truncated cp genes were found interspersed with U. reniformis mitochondrial and nuclear genome scaffolds, indicating that as observed in other smaller plant genomes, such as Arabidopsis thaliana, and the related and carnivorous Genlisea nigrocaulis and G. hispidula, the endosymbiotic gene transfer may also shape the U. reniformis genome in a similar fashion. Overall the comparative analysis of the U. reniformis cp genome provides new insight into the ndh genes and cp genome evolution of carnivorous plants from Lentibulariaceae family. Lentibulariaceae is the richest family of carnivorous plants spanning three genera including Pinguicula, Genlisea, and Utricularia. Utricularia is globally distributed, and, unlike Pinguicula and Genlisea, has both aquatic and terrestrial forms. In this study we present the analysis of the chloroplast (cp) genome of the terrestrial Utricularia reniformis. U. reniformis has a standard cp genome of 139,725bp, encoding a gene repertoire similar to essentially all photosynthetic organisms. However, an exclusive combination of losses and pseudogenization of the plastid NAD(P)H-dehydrogenase (ndh) gene complex were observed. Comparisons among aquatic and terrestrial forms of Pinguicula, Genlisea, and Utricularia indicate that, whereas the aquatic forms retained functional copies of the eleven ndh genes, these have been lost or truncated in terrestrial forms, suggesting that the ndh function may be dispensable in terrestrial Lentibulariaceae. Phylogenetic scenarios of the ndh gene loss and recovery among Pinguicula, Genlisea, and Utricularia to the ancestral Lentibulariaceae cladeare proposed. Interestingly, RNAseq analysis evidenced that U. reniformis cp genes are transcribed, including the truncated ndh genes, suggesting that these are not completely inactivated. In addition, potential novel RNA-editing sites were identified in at least six U. reniformis cp genes, while none were identified in the truncated ndh genes. Moreover, phylogenomic analyses support that Lentibulariaceae is monophyletic, belonging to the higher core Lamiales clade, corroborating the hypothesis that the first Utricularia lineage emerged in terrestrial habitats and then evolved to epiphytic and aquatic forms. Furthermore, several truncated cp genes were found interspersed with U. reniformis mitochondrial and nuclear genome scaffolds, indicating that as observed in other smaller plant genomes, such as Arabidopsis thaliana, and the related and carnivorous Genlisea nigrocaulis and G. hispidula, the endosymbiotic gene transfer may also shape the U. reniformis genome in a similar fashion. Overall the comparative analysis of the U. reniformis cp genome provides new insight into the ndh genes and cp genome evolution of carnivorous plants from Lentibulariaceae family.
TRPM8 genetic variations associated with COPD risk in the Chinese Han population
TRPM8 plays a key role in COPD. The development of pulmonary hypertension (PH) in COPD adversely affects survival and exercise capacity. Thus, the aim of this study was to evaluate the possible association between single nucleotide polymorphisms (SNPs) in TRPM8 and COPD or PH in COPD among the Chinese Han population. A total of 513 COPD patients and 506 controls were enrolled in the study. Six tag SNPs (tSNPs) were genotyped. The relationship between COPD or PH in COPD and the six tSNPs was evaluated using the χ2 test and genetic model analysis. In the rs9789398 polymorphism, the T/C genotype was associated with an increased risk for COPD (P=0.005). Under the assumption of models of inheritance, there was an association between the rs9789398 polymorphism and COPD. In the rs9789675 polymorphism, the G/A genotype was associated with an increased risk for COPD (P=0.021). Furthermore, by the χ2 test, we found that the minor allele “A” of rs9789675 (odds ratio [OR] =0.63, 95% confidence interval [CI], 0.42–0.97, P=0.034) and the minor allele “C” of rs9789398 (OR =1.59, 95% CI, 1.03–2.44, P=0.034) were associated with a decreased risk of PH in COPD in allele models. In genetic models, the genotypes “GA” and “AA” of rs9789675 were associated with a decreased risk of PH in COPD. The genotypes “TC” and “CC” of rs9789398 were associated with a decreased risk of PH in COPD. Moreover, “CG” of rs1004478 was significantly associated with a decreased risk of PH in COPD. There was a significant association between the five SNPs (rs2362290, rs9789675, rs9789398, rs1003540, and rs104478) in the TRPM8 gene and the risk of PH in COPD. Our findings indicated that rs9789398 in the TRPM8 gene was significantly associated with the risk of COPD in the Chinese Han population. Moreover, rs9789675, rs9789398, and rs1004478 were significantly associated with the risk of PH in COPD. This study provides a novel insight into COPD and PH in the development of COPD. TRPM8 plays a key role in COPD. The development of pulmonary hypertension (PH) in COPD adversely affects survival and exercise capacity. Thus, the aim of this study was to evaluate the possible association between single nucleotide polymorphisms (SNPs) in TRPM8 and COPD or PH in COPD among the Chinese Han population. A total of 513 COPD patients and 506 controls were enrolled in the study. Six tag SNPs (tSNPs) were genotyped. The relationship between COPD or PH in COPD and the six tSNPs was evaluated using the χ2 test and genetic model analysis. In the rs9789398 polymorphism, the T/C genotype was associated with an increased risk for COPD (P=0.005). Under the assumption of models of inheritance, there was an association between the rs9789398 polymorphism and COPD. In the rs9789675 polymorphism, the G/A genotype was associated with an increased risk for COPD (P=0.021). Furthermore, by the χ2 test, we found that the minor allele “A” of rs9789675 (odds ratio [OR] =0.63, 95% confidence interval [CI], 0.42–0.97, P=0.034) and the minor allele “C” of rs9789398 (OR =1.59, 95% CI, 1.03–2.44, P=0.034) were associated with a decreased risk of PH in COPD in allele models. In genetic models, the genotypes “GA” and “AA” of rs9789675 were associated with a decreased risk of PH in COPD. The genotypes “TC” and “CC” of rs9789398 were associated with a decreased risk of PH in COPD. Moreover, “CG” of rs1004478 was significantly associated with a decreased risk of PH in COPD. There was a significant association between the five SNPs (rs2362290, rs9789675, rs9789398, rs1003540, and rs104478) in the TRPM8 gene and the risk of PH in COPD. Our findings indicated that rs9789398 in the TRPM8 gene was significantly associated with the risk of COPD in the Chinese Han population. Moreover, rs9789675, rs9789398, and rs1004478 were significantly associated with the risk of PH in COPD. This study provides a novel insight into COPD and PH in the development of COPD.
Responses of Bovine Innate Immunity to Mycobacterium avium subsp. paratuberculosis Infection Revealed by Changes in Gene Expression and Levels of MicroRNA
Paratuberculosis in cattle is a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which is endemic worldwide. In dairy herds, it is responsible for huge economic losses. However, current diagnostic methods do not detect subclinical infection making control of the disease difficult. The identification of MAP infected animals during the sub-clinical phase of infection would play a key role in preventing the dissemination of the pathogen and in reducing transmission. Gene expression and circulating microRNA (miRNA) signatures have been proposed as biomarkers of disease both in the human and veterinary medicine. In this paper, gene expression and related miRNA levels were investigated in cows positive for MAP, by ELISA and culture, in order to identify potential biomarkers to improve diagnosis of MAP infection. Three groups, each of 5 animals, were used to compare the results of gene expression from positive, exposed and negative cows. Overall 258 differentially expressed genes were identified between unexposed, exposed, but ELISA negative and positive groups which were involved in biological functions related to inflammatory response, lipid metabolism and small molecule biochemistry. Differentially expressed miRNA was also found among the three groups: 7 miRNAs were at a lower level and 2 at a higher level in positive animals vs unexposed animals, while 5 and 3 miRNAs were respectively reduced and increased in the exposed group compared to the unexposed group. Among the differentially expressed miRNAs 6 have been previously described as immune-response related and two were novel miRNAs. Analysis of the miRNA levels showed correlation with expression of their target genes, known to be involved in the immune process. This study suggests that miRNA expression is affected by MAP infection and play a key role in tuning the host response to infection. The miRNA and gene expression profiles may be biomarkers of infection and potential diagnostic of MAP infection earlier than the current ELISA based diagnostic tests. Paratuberculosis in cattle is a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which is endemic worldwide. In dairy herds, it is responsible for huge economic losses. However, current diagnostic methods do not detect subclinical infection making control of the disease difficult. The identification of MAP infected animals during the sub-clinical phase of infection would play a key role in preventing the dissemination of the pathogen and in reducing transmission. Gene expression and circulating microRNA (miRNA) signatures have been proposed as biomarkers of disease both in the human and veterinary medicine. In this paper, gene expression and related miRNA levels were investigated in cows positive for MAP, by ELISA and culture, in order to identify potential biomarkers to improve diagnosis of MAP infection. Three groups, each of 5 animals, were used to compare the results of gene expression from positive, exposed and negative cows. Overall 258 differentially expressed genes were identified between unexposed, exposed, but ELISA negative and positive groups which were involved in biological functions related to inflammatory response, lipid metabolism and small molecule biochemistry. Differentially expressed miRNA was also found among the three groups: 7 miRNAs were at a lower level and 2 at a higher level in positive animals vs unexposed animals, while 5 and 3 miRNAs were respectively reduced and increased in the exposed group compared to the unexposed group. Among the differentially expressed miRNAs 6 have been previously described as immune-response related and two were novel miRNAs. Analysis of the miRNA levels showed correlation with expression of their target genes, known to be involved in the immune process. This study suggests that miRNA expression is affected by MAP infection and play a key role in tuning the host response to infection. The miRNA and gene expression profiles may be biomarkers of infection and potential diagnostic of MAP infection earlier than the current ELISA based diagnostic tests.
Assessing Marine Microbial Induced Corrosion at Santa Catalina Island, California
High iron and eutrophic conditions are reported as environmental factors leading to accelerated low-water corrosion, an enhanced form of near-shore microbial induced corrosion. To explore this hypothesis, we deployed flow-through colonization systems in laboratory-based aquarium tanks under a continuous flow of surface seawater from Santa Catalina Island, CA, USA, for periods of 2 and 6 months. Substrates consisted of mild steel – a major constituent of maritime infrastructure – and the naturally occurring iron sulfide mineral pyrite. Four conditions were tested: free-venting “high-flux” conditions; a “stagnant” condition; an “active” flow-through condition with seawater slowly pumped over the substrates; and an “enrichment” condition where the slow pumping of seawater was supplemented with nutrient rich medium. Electron microscopy analyses of the 2-month high flux incubations document coating of substrates with “twisted stalks,” resembling iron oxyhydroxide bioprecipitates made by marine neutrophilic Fe-oxidizing bacteria (FeOB). Six-month incubations exhibit increased biofilm and substrate corrosion in the active flow and nutrient enriched conditions relative to the stagnant condition. A scarcity of twisted stalks was observed for all 6 month slow-flow conditions compared to the high-flux condition, which may be attributable to oxygen concentrations in the slow-flux conditions being prohibitively low for sustained growth of stalk-producing bacteria. All substrates developed microbial communities reflective of the original seawater input, as based on 16S rRNA gene sequencing. Deltaproteobacteria sequences increased in relative abundance in the active flow and nutrient enrichment conditions, whereas Gammaproteobacteria sequences were relatively more abundant in the stagnant condition. These results indicate that (i) high-flux incubations with higher oxygen availability favor the development of biofilms with twisted stalks resembling those of marine neutrophilic FeOB and (ii) long-term nutrient stimulation results in substrate corrosion and biofilms with different bacterial community composition and structure relative to stagnant and non-nutritionally enhanced incubations. Similar microbial succession scenarios, involving increases in nutritional input leading to the proliferation of anaerobic iron and sulfur-cycling guilds, may occur at the nearby Port of Los Angeles and cause potential damage to maritime port infrastructure. High iron and eutrophic conditions are reported as environmental factors leading to accelerated low-water corrosion, an enhanced form of near-shore microbial induced corrosion. To explore this hypothesis, we deployed flow-through colonization systems in laboratory-based aquarium tanks under a continuous flow of surface seawater from Santa Catalina Island, CA, USA, for periods of 2 and 6 months. Substrates consisted of mild steel – a major constituent of maritime infrastructure – and the naturally occurring iron sulfide mineral pyrite. Four conditions were tested: free-venting “high-flux” conditions; a “stagnant” condition; an “active” flow-through condition with seawater slowly pumped over the substrates; and an “enrichment” condition where the slow pumping of seawater was supplemented with nutrient rich medium. Electron microscopy analyses of the 2-month high flux incubations document coating of substrates with “twisted stalks,” resembling iron oxyhydroxide bioprecipitates made by marine neutrophilic Fe-oxidizing bacteria (FeOB). Six-month incubations exhibit increased biofilm and substrate corrosion in the active flow and nutrient enriched conditions relative to the stagnant condition. A scarcity of twisted stalks was observed for all 6 month slow-flow conditions compared to the high-flux condition, which may be attributable to oxygen concentrations in the slow-flux conditions being prohibitively low for sustained growth of stalk-producing bacteria. All substrates developed microbial communities reflective of the original seawater input, as based on 16S rRNA gene sequencing. Deltaproteobacteria sequences increased in relative abundance in the active flow and nutrient enrichment conditions, whereas Gammaproteobacteria sequences were relatively more abundant in the stagnant condition. These results indicate that (i) high-flux incubations with higher oxygen availability favor the development of biofilms with twisted stalks resembling those of marine neutrophilic FeOB and (ii) long-term nutrient stimulation results in substrate corrosion and biofilms with different bacterial community composition and structure relative to stagnant and non-nutritionally enhanced incubations. Similar microbial succession scenarios, involving increases in nutritional input leading to the proliferation of anaerobic iron and sulfur-cycling guilds, may occur at the nearby Port of Los Angeles and cause potential damage to maritime port infrastructure.
Morphological Characterization and Gene Expression Profiling during Bud Development in a Tropical Perennial, Litchi chinensis Sonn.
Tropical evergreen perennials undergo recurrent flush growth, and their terminal buds alternate between growth and dormancy. In sharp contrast to the intensive studies on bud development in temperate deciduous trees, there is little information about bud development regulation in tropical trees. In this study, litchi (Litchi chinensis Sonn.) was used as a model tropical perennial for morphological characterization and transcriptomic analysis of bud development. Litchi buds are naked with apical meristem embraced by rudimentary leaves, which are brown at dormant stage (Stage I). They swell and turn greenish as buds break (Stage II), and as growth accelerates, the rudimentary leaves elongate and open exposing the inner leaf primodia. With the outgrowth of the needle-like leaflets, bud growth reaches a maximum (Stage III). When leaflets expand, bud growth cease with the abortion of the rudimentary leaves at upper positions (Stage IV). Then buds turn brown and reenter dormant status. Budbreak occurs again when new leaves become hard green. Buds at four stages (Stage I to IV) were collected for respiration measurements and in-depth RNA sequencing. Respiration rate was the lowest at Stage I and highest at Stage II, decreasing toward growth cessation. RNA sequencing obtained over 5 Gb data from each of the bud samples and de novo assembly generated a total of 59,999 unigenes, 40,119 of which were annotated. Pair-wise comparison of gene expression between stages, gene profiling across stages, GO/KEGG enrichment analysis, and the expression patterns of 17 major genes highlighted by principal component (PC) analysis displayed significant changes in stress resistance, hormone signal pathways, circadian rhythm, photosynthesis, cell division, carbohydrate metabolism, programmed cell death during bud development, which might be under epigenetic control involving chromatin methylation. The qPCR results of 8 selected unigenes with high PC scores agreed with the RPKM values obtained from RNA-seq. Three Short Vegetative Phase (SVP) genes, namely LcSVP1, LcSVP2, and LcSVP3 displayed different expression patterns, suggesting their differential roles in bud development regulation. The study brought an understanding about biological processes associated with the phase transitions, molecular regulation of bud development, as well as cyclic bud growth as a strategy to survive tropical conditions. Tropical evergreen perennials undergo recurrent flush growth, and their terminal buds alternate between growth and dormancy. In sharp contrast to the intensive studies on bud development in temperate deciduous trees, there is little information about bud development regulation in tropical trees. In this study, litchi (Litchi chinensis Sonn.) was used as a model tropical perennial for morphological characterization and transcriptomic analysis of bud development. Litchi buds are naked with apical meristem embraced by rudimentary leaves, which are brown at dormant stage (Stage I). They swell and turn greenish as buds break (Stage II), and as growth accelerates, the rudimentary leaves elongate and open exposing the inner leaf primodia. With the outgrowth of the needle-like leaflets, bud growth reaches a maximum (Stage III). When leaflets expand, bud growth cease with the abortion of the rudimentary leaves at upper positions (Stage IV). Then buds turn brown and reenter dormant status. Budbreak occurs again when new leaves become hard green. Buds at four stages (Stage I to IV) were collected for respiration measurements and in-depth RNA sequencing. Respiration rate was the lowest at Stage I and highest at Stage II, decreasing toward growth cessation. RNA sequencing obtained over 5 Gb data from each of the bud samples and de novo assembly generated a total of 59,999 unigenes, 40,119 of which were annotated. Pair-wise comparison of gene expression between stages, gene profiling across stages, GO/KEGG enrichment analysis, and the expression patterns of 17 major genes highlighted by principal component (PC) analysis displayed significant changes in stress resistance, hormone signal pathways, circadian rhythm, photosynthesis, cell division, carbohydrate metabolism, programmed cell death during bud development, which might be under epigenetic control involving chromatin methylation. The qPCR results of 8 selected unigenes with high PC scores agreed with the RPKM values obtained from RNA-seq. Three Short Vegetative Phase (SVP) genes, namely LcSVP1, LcSVP2, and LcSVP3 displayed different expression patterns, suggesting their differential roles in bud development regulation. The study brought an understanding about biological processes associated with the phase transitions, molecular regulation of bud development, as well as cyclic bud growth as a strategy to survive tropical conditions.
Isolation and identification of EST SSR markers in Chunia bucklandioides (Hamamelidaceae)1
Premise of the study: Chunia bucklandioides (Hamamelidaceae), endemic to Hainan, China, is listed as threatened in the IUCN Red List and is now only found on Mt. Diaoluo and Mt. Jianfeng. Thus, microsatellite markers were developed for future conservation genetic studies of this species. Methods and Results: A total of 115 primers were designed on the basis of the transcriptome data of C. bucklandioides. Of them, 59 successfully amplified in C. bucklandioides and polymorphisms were detected in 11; the number of alleles per locus varied from two to five, the observed heterozygosity ranged from 0.000 to 0.941, and the expected heterozygosity ranged from 0.000 to 0.699. A total of 13 primers amplified in Mytilaria laosensis, and five primers amplified in Exbucklandia tonkinensis and E. populnea. Conclusions: The markers screened here provide a basis to assess genetic structure and further establish conservation strategies for C. bucklandioides. Premise of the study: Chunia bucklandioides (Hamamelidaceae), endemic to Hainan, China, is listed as threatened in the IUCN Red List and is now only found on Mt. Diaoluo and Mt. Jianfeng. Thus, microsatellite markers were developed for future conservation genetic studies of this species. Methods and Results: A total of 115 primers were designed on the basis of the transcriptome data of C. bucklandioides. Of them, 59 successfully amplified in C. bucklandioides and polymorphisms were detected in 11; the number of alleles per locus varied from two to five, the observed heterozygosity ranged from 0.000 to 0.941, and the expected heterozygosity ranged from 0.000 to 0.699. A total of 13 primers amplified in Mytilaria laosensis, and five primers amplified in Exbucklandia tonkinensis and E. populnea. Conclusions: The markers screened here provide a basis to assess genetic structure and further establish conservation strategies for C. bucklandioides.
Molecular Characterization of Salmonella Serovars Anatum and Ealing Associated with Two Historical Outbreaks, Linked to Contaminated Powdered Infant Formula
Powdered infant formula (PIF) is not intended to be produced as a sterile product unless explicitly stated and on occasion may become contaminated during production with pathogens such as Salmonella enterica. This retrospective study focused on two historically reported salmonellosis outbreaks associated with PIF from the United Kingdom and France, in 1985 and 1996/1997. In this paper, the molecular characterization of the two outbreaks associated Salmonella serovars Anatum and Ealing is reported. Initially the isolates were analyzed using pulsed-field gel electrophoresis (PFGE), which revealed the clonal nature of the two outbreaks. Following from this two representative isolates, one from each serovar was selected for whole genome sequencing (WGS), wherein analysis focused on the Salmonella pathogenicity islands. Furthermore, the ability of these isolates to survive the host intercellular environment was determined using an ex vivo gentamicin protection assay. Results suggest a high level of genetic diversity that may have contributed to survival and virulence of isolates from these outbreaks. Powdered infant formula (PIF) is not intended to be produced as a sterile product unless explicitly stated and on occasion may become contaminated during production with pathogens such as Salmonella enterica. This retrospective study focused on two historically reported salmonellosis outbreaks associated with PIF from the United Kingdom and France, in 1985 and 1996/1997. In this paper, the molecular characterization of the two outbreaks associated Salmonella serovars Anatum and Ealing is reported. Initially the isolates were analyzed using pulsed-field gel electrophoresis (PFGE), which revealed the clonal nature of the two outbreaks. Following from this two representative isolates, one from each serovar was selected for whole genome sequencing (WGS), wherein analysis focused on the Salmonella pathogenicity islands. Furthermore, the ability of these isolates to survive the host intercellular environment was determined using an ex vivo gentamicin protection assay. Results suggest a high level of genetic diversity that may have contributed to survival and virulence of isolates from these outbreaks.
Prokaryotic Responses to Ammonium and Organic Carbon Reveal Alternative CO2 Fixation Pathways and Importance of Alkaline Phosphatase in the Mesopelagic North Atlantic
To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates—assumed to be related to autotrophic metabolisms—were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention. To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates—assumed to be related to autotrophic metabolisms—were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.
Cooperation of Nutlin 3a and a Wip1 inhibitor to induce p53 activity
Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists. Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists.
CD4 Transgenic Zebrafish Reveal Tissue Resident Th2 and Regulatory T Cell–like Populations and Diverse Mononuclear Phagocytes
CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b–expressing Th2-like cells, which do not coexpress il-4/13a. Additionally, we identify a contrasting population of regulatory T cell–like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity. CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b–expressing Th2-like cells, which do not coexpress il-4/13a. Additionally, we identify a contrasting population of regulatory T cell–like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.
Helicobacter pylori Infection Aggravates Diet induced Insulin Resistance in Association With Gut Microbiota of Mice
Emerging evidence suggests that Helicobacter pylori infection is associated with insulin resistance (IR) yet the underlying mechanisms are still obscure. The vital role of gut microbiota in triggering IR has been increasingly reported, however, no study has explored the correlation of gut microbiota and H. pylori-associated IR. Using H. pylori-infected mice model fed different diet structures, we demonstrated that H. pylori infection significantly aggravated high-fat diet (HFD)-induced metabolic disorders at the early stage, the extent of which was close to the effect of long-term HFD. Interestingly, we observed dynamic alterations in gut microbiota that were consistent with the changes in the metabolic phenotype induced by H. pylori and HFD. There may be an interaction among H. pylori, diet and gut microbiota, which dysregulates the host metabolic homeostasis, and treatment of H. pylori may be beneficial to the patients with impaired glucose tolerance in addition to diet control. Emerging evidence suggests that Helicobacter pylori infection is associated with insulin resistance (IR) yet the underlying mechanisms are still obscure. The vital role of gut microbiota in triggering IR has been increasingly reported, however, no study has explored the correlation of gut microbiota and H. pylori-associated IR. Using H. pylori-infected mice model fed different diet structures, we demonstrated that H. pylori infection significantly aggravated high-fat diet (HFD)-induced metabolic disorders at the early stage, the extent of which was close to the effect of long-term HFD. Interestingly, we observed dynamic alterations in gut microbiota that were consistent with the changes in the metabolic phenotype induced by H. pylori and HFD. There may be an interaction among H. pylori, diet and gut microbiota, which dysregulates the host metabolic homeostasis, and treatment of H. pylori may be beneficial to the patients with impaired glucose tolerance in addition to diet control.
Draft Genomic Analysis of an Avian Multidrug Resistant Morganella morganii Isolate Carrying qnrD1
Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen. Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen.
Insertion of a knockout first cassette in Ampd1 gene leads to neonatal death by disruption of neighboring genes expression
AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. To understand the physiological function of AMPD1, we obtained a strain of Ampd1 mutant mice from KOMP repository, which was generated by a knockout-first strategy. An elevated AMP level and almost complete lack of IMP was detected in the skeletal muscle of E18.5 Ampd1tm1a/tm1a mice. However, Ampd1tm1a/tm1a mice died in 2 days postnatally, which was contradicting to previous reports. After removal of the knockout-first cassette and critical exon, mice homozygous for the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1tm1a/tm1a mice, but normal in the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1tm1a/tm1a mice was consistent with the Man1a2-deficient mice. Our results indicated the knockout-first cassette may cause off-target effect by influence the expression of neighboring genes. This study, together with other reports, strongly suggests that removal of targeting cassette by site-specific recombinases is very important for the accurate phenotypic interpretation on mice generated by target mutations. AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. To understand the physiological function of AMPD1, we obtained a strain of Ampd1 mutant mice from KOMP repository, which was generated by a knockout-first strategy. An elevated AMP level and almost complete lack of IMP was detected in the skeletal muscle of E18.5 Ampd1tm1a/tm1a mice. However, Ampd1tm1a/tm1a mice died in 2 days postnatally, which was contradicting to previous reports. After removal of the knockout-first cassette and critical exon, mice homozygous for the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1tm1a/tm1a mice, but normal in the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1tm1a/tm1a mice was consistent with the Man1a2-deficient mice. Our results indicated the knockout-first cassette may cause off-target effect by influence the expression of neighboring genes. This study, together with other reports, strongly suggests that removal of targeting cassette by site-specific recombinases is very important for the accurate phenotypic interpretation on mice generated by target mutations.
Past climate changes, population dynamics and the origin of Bison in Europe
Background Climatic and environmental fluctuations as well as anthropogenic pressure have led to the extinction of much of Europe’s megafauna. The European bison or wisent (Bison bonasus), one of the last wild European large mammals, narrowly escaped extinction at the onset of the 20th century owing to hunting and habitat fragmentation. Little is known, however, about its origin, evolutionary history and population dynamics during the Pleistocene. Results Through ancient DNA analysis we show that the emblematic European bison has experienced several waves of population expansion, contraction, and extinction during the last 50,000 years in Europe, culminating in a major reduction of genetic diversity during the Holocene. Fifty-seven complete and partial ancient mitogenomes from throughout Europe, the Caucasus, and Siberia reveal that three populations of wisent (Bison bonasus) and steppe bison (B. priscus) alternately occupied Western Europe, correlating with climate-induced environmental changes. The Late Pleistocene European steppe bison originated from northern Eurasia, whereas the modern wisent population emerged from a refuge in the southern Caucasus after the last glacial maximum. A population overlap during a transition period is reflected in ca. 36,000-year-old paintings in the French Chauvet cave. Bayesian analyses of these complete ancient mitogenomes yielded new dates of the various branching events during the evolution of Bison and its radiation with Bos, which lead us to propose that the genetic affiliation between the wisent and cattle mitogenomes result from incomplete lineage sorting rather than post-speciation gene flow. Conclusion The paleogenetic analysis of bison remains from the last 50,000 years reveals the influence of climate changes on the dynamics of the various bison populations in Europe, only one of which survived into the Holocene, where it experienced severe reductions in its genetic diversity. The time depth and geographical scope of this study enables us to propose temperate Western Europe as a suitable biotope for the wisent compatible with its reintroduction. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0317-7) contains supplementary material, which is available to authorized users. Background Climatic and environmental fluctuations as well as anthropogenic pressure have led to the extinction of much of Europe’s megafauna. The European bison or wisent (Bison bonasus), one of the last wild European large mammals, narrowly escaped extinction at the onset of the 20th century owing to hunting and habitat fragmentation. Little is known, however, about its origin, evolutionary history and population dynamics during the Pleistocene. Results Through ancient DNA analysis we show that the emblematic European bison has experienced several waves of population expansion, contraction, and extinction during the last 50,000 years in Europe, culminating in a major reduction of genetic diversity during the Holocene. Fifty-seven complete and partial ancient mitogenomes from throughout Europe, the Caucasus, and Siberia reveal that three populations of wisent (Bison bonasus) and steppe bison (B. priscus) alternately occupied Western Europe, correlating with climate-induced environmental changes. The Late Pleistocene European steppe bison originated from northern Eurasia, whereas the modern wisent population emerged from a refuge in the southern Caucasus after the last glacial maximum. A population overlap during a transition period is reflected in ca. 36,000-year-old paintings in the French Chauvet cave. Bayesian analyses of these complete ancient mitogenomes yielded new dates of the various branching events during the evolution of Bison and its radiation with Bos, which lead us to propose that the genetic affiliation between the wisent and cattle mitogenomes result from incomplete lineage sorting rather than post-speciation gene flow. Conclusion The paleogenetic analysis of bison remains from the last 50,000 years reveals the influence of climate changes on the dynamics of the various bison populations in Europe, only one of which survived into the Holocene, where it experienced severe reductions in its genetic diversity. The time depth and geographical scope of this study enables us to propose temperate Western Europe as a suitable biotope for the wisent compatible with its reintroduction. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0317-7) contains supplementary material, which is available to authorized users.
Intra individual changes in DNA methylation not mediated by cell type composition are correlated with aging during childhood
Background Several studies have reported age-associated changes in DNA methylation in the first few years of life and in adult populations, but the extent of such changes during childhood is less well studied. The goals of this study were to investigate to what degree intra-individual changes in DNA methylation are associated with aging during childhood and dissect the methylation changes directly associated with aging from the effect mediated through variation in cell-type composition (CTC). Results We performed reduced representation bisulfite sequencing (RRBS) in peripheral whole-blood samples collected at 2, 10, and 16 years of age. We identified age-associated longitudinal changes in DNA methylation at 346 CpGs in 178 genes. Analyses separating the effect mediated by CTC variability across age identified 26 CpGs located in 12 genes that associated directly with age. Hence, the CTC changes across age appear to act as a mediator of the observed DNA methylation associated with age. The results were replicated using EpiTYPER in a second sample set selected from the same cohort. Gene ontology analyses revealed enrichment of transcriptional regulation and developmental processes. Further, comparisons of the mean DNA methylation differences between the time points reveal greater differences between 2 to 10 years and 10 to 16 years, suggesting that the identified age-associated DNA methylation patterns manifests in early childhood. Conclusions This study reveals insights into the epigenetic dynamics associated with aging early in life. Such information could ultimately provide clues and point towards molecular pathways that are susceptible to aging-related disease-associated epigenetic dysregulation. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0277-3) contains supplementary material, which is available to authorized users. Background Several studies have reported age-associated changes in DNA methylation in the first few years of life and in adult populations, but the extent of such changes during childhood is less well studied. The goals of this study were to investigate to what degree intra-individual changes in DNA methylation are associated with aging during childhood and dissect the methylation changes directly associated with aging from the effect mediated through variation in cell-type composition (CTC). Results We performed reduced representation bisulfite sequencing (RRBS) in peripheral whole-blood samples collected at 2, 10, and 16 years of age. We identified age-associated longitudinal changes in DNA methylation at 346 CpGs in 178 genes. Analyses separating the effect mediated by CTC variability across age identified 26 CpGs located in 12 genes that associated directly with age. Hence, the CTC changes across age appear to act as a mediator of the observed DNA methylation associated with age. The results were replicated using EpiTYPER in a second sample set selected from the same cohort. Gene ontology analyses revealed enrichment of transcriptional regulation and developmental processes. Further, comparisons of the mean DNA methylation differences between the time points reveal greater differences between 2 to 10 years and 10 to 16 years, suggesting that the identified age-associated DNA methylation patterns manifests in early childhood. Conclusions This study reveals insights into the epigenetic dynamics associated with aging early in life. Such information could ultimately provide clues and point towards molecular pathways that are susceptible to aging-related disease-associated epigenetic dysregulation. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0277-3) contains supplementary material, which is available to authorized users.
Immune genes and divergent antimicrobial peptides in flies of the subgenus Drosophila
Background Drosophila is an important model for studying the evolution of animal immunity, due to the powerful genetic tools developed for D. melanogaster. However, Drosophila is an incredibly speciose lineage with a wide range of ecologies, natural histories, and diverse natural enemies. Surprisingly little functional work has been done on immune systems of species other than D. melanogaster. In this study, we examine the evolution of immune genes in the speciose subgenus Drosophila, which diverged from the subgenus Sophophora (that includes D. melanogaster) approximately 25–40 Mya. We focus on D. neotestacea, a woodland species used to study interactions between insects and parasitic nematodes, and combine recent transcriptomic data with infection experiments to elucidate aspects of host immunity. Results We found that the vast majority of genes involved in the D. melanogaster immune response are conserved in D. neotestacea, with a few interesting exceptions, particularly in antimicrobial peptides (AMPs); until recently, AMPs were not thought to evolve rapidly in Drosophila. Unexpectedly, we found a distinct diptericin in subgenus Drosophila flies that appears to have evolved under diversifying (positive) selection. We also describe the presence of the AMP drosocin, which was previously thought to be restricted to the subgenus Sophophora, in the subgenus Drosophila. We challenged two subgenus Drosophila species, D. neotestacea and D. virilis with bacterial and fungal pathogens and quantified AMP expression. Conclusions While diptericin in D. virilis was induced by exposure to gram-negative bacteria, it was not induced in D. neotestacea, showing that conservation of immune genes does not necessarily imply conservation of the realized immune response. Our study lends support to the idea that invertebrate AMPs evolve rapidly, and that Drosophila harbor a diverse repertoire of AMPs with potentially important functional consequences. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0805-y) contains supplementary material, which is available to authorized users. Background Drosophila is an important model for studying the evolution of animal immunity, due to the powerful genetic tools developed for D. melanogaster. However, Drosophila is an incredibly speciose lineage with a wide range of ecologies, natural histories, and diverse natural enemies. Surprisingly little functional work has been done on immune systems of species other than D. melanogaster. In this study, we examine the evolution of immune genes in the speciose subgenus Drosophila, which diverged from the subgenus Sophophora (that includes D. melanogaster) approximately 25–40 Mya. We focus on D. neotestacea, a woodland species used to study interactions between insects and parasitic nematodes, and combine recent transcriptomic data with infection experiments to elucidate aspects of host immunity. Results We found that the vast majority of genes involved in the D. melanogaster immune response are conserved in D. neotestacea, with a few interesting exceptions, particularly in antimicrobial peptides (AMPs); until recently, AMPs were not thought to evolve rapidly in Drosophila. Unexpectedly, we found a distinct diptericin in subgenus Drosophila flies that appears to have evolved under diversifying (positive) selection. We also describe the presence of the AMP drosocin, which was previously thought to be restricted to the subgenus Sophophora, in the subgenus Drosophila. We challenged two subgenus Drosophila species, D. neotestacea and D. virilis with bacterial and fungal pathogens and quantified AMP expression. Conclusions While diptericin in D. virilis was induced by exposure to gram-negative bacteria, it was not induced in D. neotestacea, showing that conservation of immune genes does not necessarily imply conservation of the realized immune response. Our study lends support to the idea that invertebrate AMPs evolve rapidly, and that Drosophila harbor a diverse repertoire of AMPs with potentially important functional consequences. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0805-y) contains supplementary material, which is available to authorized users.
Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species specific genomic properties and numerous putative antibiotic resistance determinants
Background Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. Results The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. Conclusion Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3152-x) contains supplementary material, which is available to authorized users. Background Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. Results The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. Conclusion Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3152-x) contains supplementary material, which is available to authorized users.
Nuclear receptor nhr 48 is required for pathogenicity of the second stage (J2) of the plant parasite Meloidogyne incognita
Nuclear receptors (NRs) are a diverse class of transcription factors, which are involved in regulating a large number of physiological events in metazoans. However, the function of NRs is poorly understood in plant-parasitic nematodes. Here, members of the NR1J+K group of NRs in nematodes, including the free-living and plant parasites, were examined and phylogenetically analyzed. We found that the number of members of the NR1J+K group in plant-parasitic nematodes was less than that in the free-living nematodes, suggesting this reduction of NR1J+K group members in plant parasites maybe arose during the separation of the free-living and intermediately plant parasitic nematodes (Bursaphelenchus xylophilus). Interestingly, the DNA-binding domain (DBD) and ligand-binding domain (LBD) of NR1J+K members were separated into two gene locations in the plant parasites. Knockdown of Meloidogyne incognita WBMinc13296, the ortholog of Caenorhabditis elegans nhr-48 DBD, reduced infectivity, delayed development, and decreased reproductivity. J2 of M. incognita subjected to silencing of WBMinc13295, the orthologs of B. xylophilus nhr-48 LBD, exhibited developmental lag within the host and reduced reproductivity. This study provides new insights into the function of NRs and suggests that NRs are potential targets for developing effective strategies for biological control of plant-parasitic nematodes. Nuclear receptors (NRs) are a diverse class of transcription factors, which are involved in regulating a large number of physiological events in metazoans. However, the function of NRs is poorly understood in plant-parasitic nematodes. Here, members of the NR1J+K group of NRs in nematodes, including the free-living and plant parasites, were examined and phylogenetically analyzed. We found that the number of members of the NR1J+K group in plant-parasitic nematodes was less than that in the free-living nematodes, suggesting this reduction of NR1J+K group members in plant parasites maybe arose during the separation of the free-living and intermediately plant parasitic nematodes (Bursaphelenchus xylophilus). Interestingly, the DNA-binding domain (DBD) and ligand-binding domain (LBD) of NR1J+K members were separated into two gene locations in the plant parasites. Knockdown of Meloidogyne incognita WBMinc13296, the ortholog of Caenorhabditis elegans nhr-48 DBD, reduced infectivity, delayed development, and decreased reproductivity. J2 of M. incognita subjected to silencing of WBMinc13295, the orthologs of B. xylophilus nhr-48 LBD, exhibited developmental lag within the host and reduced reproductivity. This study provides new insights into the function of NRs and suggests that NRs are potential targets for developing effective strategies for biological control of plant-parasitic nematodes.
Early cave art and ancient DNA record the origin of European bison
The ancestry of the European bison (wisent) remains a mystery. Here, Cooper and colleagues examine ancient DNA from fossil remains of extinct bison, and reveal the wisent originated through the hybridization of the extinct Steppe bison and ancestors of modern cattle. The ancestry of the European bison (wisent) remains a mystery. Here, Cooper and colleagues examine ancient DNA from fossil remains of extinct bison, and reveal the wisent originated through the hybridization of the extinct Steppe bison and ancestors of modern cattle.The two living species of bison (European and American) are among the few terrestrial megafauna to have survived the late Pleistocene extinctions. Despite the extensive bovid fossil record in Eurasia, the evolutionary history of the European bison (or wisent, Bison bonasus) before the Holocene (<11.7 thousand years ago (kya)) remains a mystery. We use complete ancient mitochondrial genomes and genome-wide nuclear DNA surveys to reveal that the wisent is the product of hybridization between the extinct steppe bison (Bison priscus) and ancestors of modern cattle (aurochs, Bos primigenius) before 120 kya, and contains up to 10% aurochs genomic ancestry. Although undetected within the fossil record, ancestors of the wisent have alternated ecological dominance with steppe bison in association with major environmental shifts since at least 55 kya. Early cave artists recorded distinct morphological forms consistent with these replacement events, around the Last Glacial Maximum (LGM, ∼21–18 kya). The two living species of bison (European and American) are among the few terrestrial megafauna to have survived the late Pleistocene extinctions. Despite the extensive bovid fossil record in Eurasia, the evolutionary history of the European bison (or wisent, Bison bonasus) before the Holocene (<11.7 thousand years ago (kya)) remains a mystery. We use complete ancient mitochondrial genomes and genome-wide nuclear DNA surveys to reveal that the wisent is the product of hybridization between the extinct steppe bison (Bison priscus) and ancestors of modern cattle (aurochs, Bos primigenius) before 120 kya, and contains up to 10% aurochs genomic ancestry. Although undetected within the fossil record, ancestors of the wisent have alternated ecological dominance with steppe bison in association with major environmental shifts since at least 55 kya. Early cave artists recorded distinct morphological forms consistent with these replacement events, around the Last Glacial Maximum (LGM, ∼21–18 kya).
Distinct transcriptome responses to water limitation in isohydric and anisohydric grapevine cultivars
Background Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades. Results Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar. Conclusions Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3136-x) contains supplementary material, which is available to authorized users. Background Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades. Results Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar. Conclusions Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3136-x) contains supplementary material, which is available to authorized users.
Gene expression profile of whole blood cells differs in pregnant women with positive screening and negative diagnosis for gestational diabetes
Objective To evaluate the gene expression profile of whole blood cells in pregnant women without diabetes (with positive screening and negative diagnosis for gestational diabetes mellitus (GDM)) compared with pregnant women with negative screening for GDM. Research design and methods Pregnant women were recruited in the Diabetes Perinatal Research Centre—Botucatu Medical School-UNESP and Botucatuense Mercy Hospital (UNIMED). Distributed into 2 groups: control (n=8), women with negative screening and non-diabetic (ND, n=13), with positive screening and negative diagnosis of GDM. A peripheral blood sample was collected for glucose, glycated hemoglobin, and microarray gene expression analyses. Results The evaluation of gene expression profiles showed significant differences between the control group and the ND group, with 22 differentially expressed gene sequences. Gene networks and interaction tables were generated to evaluate the biological processes associated with differentially expressed genes of interest. Conclusions In the group with positive screening, there is an apparent regulatory balance between the functions of the differentially expressed genes related to the pathogenesis of diabetes and a compensatory attempt to mitigate the possible etiology. These results support the ‘two-step Carpenter-Coustan’ strategy because pregnant women with negative screening do not need to continue on diagnostic investigation of gestational diabetes, thus reducing the cost of healthcare and the medicalization of pregnancy. Although not diabetic, they do have risk factors, and thus attention to these genes is important when considering disease evolution because this pregnant women are a step toward developing diabetes compared with women without these risk factors. Objective To evaluate the gene expression profile of whole blood cells in pregnant women without diabetes (with positive screening and negative diagnosis for gestational diabetes mellitus (GDM)) compared with pregnant women with negative screening for GDM. Research design and methods Pregnant women were recruited in the Diabetes Perinatal Research Centre—Botucatu Medical School-UNESP and Botucatuense Mercy Hospital (UNIMED). Distributed into 2 groups: control (n=8), women with negative screening and non-diabetic (ND, n=13), with positive screening and negative diagnosis of GDM. A peripheral blood sample was collected for glucose, glycated hemoglobin, and microarray gene expression analyses. Results The evaluation of gene expression profiles showed significant differences between the control group and the ND group, with 22 differentially expressed gene sequences. Gene networks and interaction tables were generated to evaluate the biological processes associated with differentially expressed genes of interest. Conclusions In the group with positive screening, there is an apparent regulatory balance between the functions of the differentially expressed genes related to the pathogenesis of diabetes and a compensatory attempt to mitigate the possible etiology. These results support the ‘two-step Carpenter-Coustan’ strategy because pregnant women with negative screening do not need to continue on diagnostic investigation of gestational diabetes, thus reducing the cost of healthcare and the medicalization of pregnancy. Although not diabetic, they do have risk factors, and thus attention to these genes is important when considering disease evolution because this pregnant women are a step toward developing diabetes compared with women without these risk factors.
Physical activity delays hippocampal neurodegeneration and rescues memory deficits in an Alzheimer disease mouse model
The evidence for a protective role of physical activity on the risk and progression of Alzheimer's disease (AD) has been growing in the last years. Here we studied the influence of a prolonged physical and cognitive stimulation on neurodegeneration, with special emphasis on hippocampal neuron loss and associated behavioral impairment in the Tg4-42 mouse model of AD. Tg4-42 mice overexpress Aβ4-42 without any mutations, and develop an age-dependent hippocampal neuron loss associated with a severe memory decline. We demonstrate that long-term voluntary exercise diminishes CA1 neuron loss and completely rescues spatial memory deficits in different experimental settings. This was accompanied by changes in the gene expression profile of Tg4-42 mice. Deep sequencing analysis revealed an upregulation of chaperones involved in endoplasmatic reticulum protein processing, which might be intimately linked to the beneficial effects seen upon long-term exercise. We believe that we provide evidence for the first time that enhanced physical activity counteracts neuron loss and behavioral deficits in a transgenic AD mouse model. The present findings underscore the relevance of increased physical activity as a potential strategy in the prevention of dementia. The evidence for a protective role of physical activity on the risk and progression of Alzheimer's disease (AD) has been growing in the last years. Here we studied the influence of a prolonged physical and cognitive stimulation on neurodegeneration, with special emphasis on hippocampal neuron loss and associated behavioral impairment in the Tg4-42 mouse model of AD. Tg4-42 mice overexpress Aβ4-42 without any mutations, and develop an age-dependent hippocampal neuron loss associated with a severe memory decline. We demonstrate that long-term voluntary exercise diminishes CA1 neuron loss and completely rescues spatial memory deficits in different experimental settings. This was accompanied by changes in the gene expression profile of Tg4-42 mice. Deep sequencing analysis revealed an upregulation of chaperones involved in endoplasmatic reticulum protein processing, which might be intimately linked to the beneficial effects seen upon long-term exercise. We believe that we provide evidence for the first time that enhanced physical activity counteracts neuron loss and behavioral deficits in a transgenic AD mouse model. The present findings underscore the relevance of increased physical activity as a potential strategy in the prevention of dementia.
Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3
A gene encoding a thermotolerant lipase with broad pH was isolated from an Antarctic Pseudomonas strain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10–70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni2+ decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature. A gene encoding a thermotolerant lipase with broad pH was isolated from an Antarctic Pseudomonas strain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10–70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni2+ decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature.
Taxonomic and Functional Diversity of Soil and Hypolithic Microbial Communities in Miers Valley, McMurdo Dry Valleys, Antarctica
The McMurdo Dry Valleys of Antarctica are an extreme polar desert. Mineral soils support subsurface microbial communities and translucent rocks support development of hypolithic communities on ventral surfaces in soil contact. Despite significant research attention, relatively little is known about taxonomic and functional diversity or their inter-relationships. Here we report a combined diversity and functional interrogation for soil and hypoliths of the Miers Valley in the McMurdo Dry Valleys of Antarctica. The study employed 16S rRNA fingerprinting and high throughput sequencing combined with the GeoChip functional microarray. The soil community was revealed as a highly diverse reservoir of bacterial diversity dominated by actinobacteria. Hypolithic communities were less diverse and dominated by cyanobacteria. Major differences in putative functionality were that soil communities displayed greater diversity in stress tolerance and recalcitrant substrate utilization pathways, whilst hypolithic communities supported greater diversity of nutrient limitation adaptation pathways. A relatively high level of functional redundancy in both soil and hypoliths may indicate adaptation of these communities to fluctuating environmental conditions. The McMurdo Dry Valleys of Antarctica are an extreme polar desert. Mineral soils support subsurface microbial communities and translucent rocks support development of hypolithic communities on ventral surfaces in soil contact. Despite significant research attention, relatively little is known about taxonomic and functional diversity or their inter-relationships. Here we report a combined diversity and functional interrogation for soil and hypoliths of the Miers Valley in the McMurdo Dry Valleys of Antarctica. The study employed 16S rRNA fingerprinting and high throughput sequencing combined with the GeoChip functional microarray. The soil community was revealed as a highly diverse reservoir of bacterial diversity dominated by actinobacteria. Hypolithic communities were less diverse and dominated by cyanobacteria. Major differences in putative functionality were that soil communities displayed greater diversity in stress tolerance and recalcitrant substrate utilization pathways, whilst hypolithic communities supported greater diversity of nutrient limitation adaptation pathways. A relatively high level of functional redundancy in both soil and hypoliths may indicate adaptation of these communities to fluctuating environmental conditions.