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Epigenetic Silencing of the Key Antioxidant Enzyme Catalase in Karyotypically Abnormal Human Pluripotent Stem Cells
Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells, however, the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase, a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues.
A comparative phylogeographic study reveals discordant evolutionary histories of alpine ground beetles (Coleoptera, Carabidae)
Taiwan, an island with three major mountain ranges, provides an ideal topography to study mountain–island effect on organisms that would be diversified in the isolation areas. Glaciations, however, might drive these organisms to lower elevations, causing gene flow among previously isolated populations. Two hypotheses have been proposed to depict the possible refugia for alpine organisms during glaciations. Nunatak hypothesis suggests that alpine species might have stayed in situ in high mountain areas during glaciations. Massif de refuge, on the other hand, proposes that alpine species might have migrated to lower ice‐free areas. By sampling five sympatric carabid species of Nebria and Leistus, and using two mitochondrial genes and two nuclear genes, we evaluated the mountain–island effect on alpine carabids and tested the two proposed hypotheses with comparative phylogeographic method. Results from the phylogenetic relationships, network analysis, lineage calibration, and genetic structure indicate that the deep divergence among populations in all L. smetanai, N. formosana, and N. niitakana was subjected to long‐term isolation, a phenomenon in agreement with the nunatak hypothesis. However, genetic admixture among populations of N. uenoiana and some populations of L. nokoensis complex suggests that gene flow occurred during glaciations, as a massif de refuge depicts. The speciation event in N. niitakana is estimated to have occurred before 1.89 million years ago (Mya), while differentiation among isolated populations in N. niitakana, N. formosana, L. smetanai, and L. nokoensis complex might have taken place during 0.65–1.65 Mya. While each of the alpine carabids arriving in Taiwan during different glaciation events acquired its evolutionary history, all of them had confronted the existing mountain ranges.
Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer
The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.
Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein
Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions.
Genetic and cellular studies highlight that A Disintegrin and Metalloproteinase 19 is a protective biomarker in human prostate cancer
Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Current treatments include surgery, androgen ablation and radiation. Introduction of more targeted therapies in prostate cancer, based on a detailed knowledge of the signalling pathways, aims to reduce side effects, leading to better clinical outcomes for the patient. ADAM19 (A Disintegrin And Metalloproteinase 19) is a transmembrane and soluble protein which can regulate cell phenotype through cell adhesion and proteolysis. ADAM19 has been positively associated with numerous diseases, but has not been shown to be a tumor suppressor in the pathogenesis of any human cancers. Our group sought to investigate the role of ADAM19 in human prostate cancer. ADAM19 mRNA and protein levels were assessed in well characterised human prostate cancer cohorts. ADAM19 expression was assessed in normal prostate epithelial cells (RWPE-1) and prostate cancer cells (LNCaP, PC3) using western blotting and immunocytochemistry. Proliferation assays were conducted in LNCaP cells in which ADAM19 was over-expressed. In vitro scratch assays were performed in PC3 cells over-expressing ADAM19. Immunohistochemical studies highlighted that ADAM19 protein levels were elevated in normal prostate tissue compared to prostate cancer biopsies. Results from the clinical cohorts demonstrated that high levels of ADAM19 in microarrays are positively associated with lower stage (p = 0.02591) and reduced relapse (p = 0.00277) of human prostate cancer. In vitro, ADAM19 expression was higher in RWPE-1 cells compared to LNCaP cells. In addition, human ADAM19 over-expression reduced LNCaP cell proliferation and PC3 cell migration. Taken together, our immunohistochemical and microarray results and cellular studies have shown for the first time that ADAM19 is a protective factor for human prostate cancer. Further, this study suggests that upregulation of ADAM19 expression could be of therapeutic potential in human prostate cancer. The online version of this article (doi:10.1186/s12885-016-2178-4) contains supplementary material, which is available to authorized users.
Diffusion and self assembly of C60 molecules on monolayer graphyne sheets
The motion of a fullerene (C60) on 5 different types of graphyne is studied by all-atom molecular dynamics simulations and compared with former studies on the motion of C60 on graphene. The motion shows a diffusive behavior which consists of either a continuous motion or discrete movements between trapping sites depending on the type of the graphyne sheet. For graphyne-4 and graphyne-5, fullerenes could detach from the surface of the graphyne sheet at room temperature which was not reported for similar cases on graphene sheets. Collective motion of a group of fullerenes interacting with a graphyne studied and it is shown that fullerenes exhibit stable assemblies. Depending on the type of graphyne, these assemblies can have either single or double layers. The mobility of the assembled structures is also dependent on the type of the graphyne sheet. The observed properties of the motion suggests novel applications for the complexes of fullerene and monolayer graphynes.
Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study
Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco’s Modified Eagle’s Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.
Prevalence and factors associated with violence and abuse of older adults in Mexico’s 2012 National Health and Nutrition Survey
Factors associated with violence and the abuse of older adults are understudied and its prevalence in Mexico has not been reported. The aim of this study was to identify the prevalence and factors associated with violence and abuse of older adults in Mexico. We used Mexico’s 2012 National Health and Nutrition Survey, which included a sample of 8,894 individuals who are 60 years or older and who self-reported a negative health event related to robbery, aggression or violence in the previous 12 months. We used chi-squared test and Fisher’s exact test to analyze the variables related to violence. Adjusted estimates were completed with multiple logistic regression models for complex surveys. The prevalence of violence was 1.7 % for both men and women. In 95 % of the cases, the aggression was from an unknown party. Verbal aggressions were the most prevalent (60 %). Among men, physical aggression was more common. Violence frequently occurred in the home (37.6 %); however, men were primarily assaulted in public places (42.4 %), in comparison to women (30.7 %). There were also differences in the risk factors for violence. Among men, risk was associated with younger age (60–64 years), higher education (secondary school or above) and higher socioeconomic status. Among women, risk was associated with depression, not being the head of the family, and region of the country. Violence against older adults presents differently for men and women, which means it is necessary to increase knowledge about the dynamics of the social determinants of violence, particularly in regards to the role of education among men. The relatively low prevalence found in this study may reflect the difficulty and fear of discussing the topic of violence. This may occur because of cultural factors, as well as by the perception of helplessness perpetuated by the scarce access to social programs that ensure protection and problem solving with regards to the complex social determinants of individual and family violence that this population group endures.
TCF4 Targeting miR 124 is Differentially Expressed amongst Dendritic Cell Subsets
Dendritic cells (DCs) are professional antigen-presenting cells that sample their environment and present antigens to naïve T lymphocytes for the subsequent antigen-specific immune responses. DCs exist in a range of distinct subpopulations including plasmacytoid DCs (pDCs) and classical DCs (cDCs), with the latter consisting of the cDC1 and cDC2 lineages. Although the roles of DC-specific transcription factors across the DC subsets have become understood, the posttranscriptional mechanisms that regulate DC development are yet to be elucidated. MicroRNAs (miRNAs) are pivotal posttranscriptional regulators of gene expression in a myriad of biological processes, but their contribution to the immune system is just beginning to surface. In this study, our in-house probe collection was screened to identify miRNAs possibly involved in DC development and function by targeting the transcripts of relevant mouse transcription factors. Examination of DC subsets from the culture of mouse bone marrow with Flt3 ligand identified high expression of miR-124 which was able to target the transcript of TCF4, a transcription factor critical for the development and homeostasis of pDCs. Further expression profiling of mouse DC subsets isolated from in vitro culture as well as via ex vivo purification demonstrated that miR-124 was outstandingly expressed in CD24+ cDC1 cells compared to in pDCs and CD172α+ cDC2 cells. These results imply that miR-124 is likely involved in the processes of DC subset development by posttranscriptional regulation of a transcription factor(s).
Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector
A specialized quantum area detector for electron diffraction studies makes it possible to solve the structure of small organic compound nanocrystals in non-cryo conditions by direct methods. Until recently, structure determination by transmission electron microscopy of beam-sensitive three-dimensional nanocrystals required electron diffraction tomography data collection at liquid-nitrogen temperature, in order to reduce radiation damage. Here it is shown that the novel Timepix detector combines a high dynamic range with a very high signal-to-noise ratio and single-electron sensitivity, enabling ab initio phasing of beam-sensitive organic compounds. Low-dose electron diffraction data (∼0.013 e− Å−2 s−1) were collected at room temperature with the rotation method. It was ascertained that the data were of sufficient quality for structure solution using direct methods using software developed for X-ray crystallography (XDS, SHELX) and for electron crystallography (ADT3D/PETS, SIR2014).
Comparison of ablation defect on MR imaging with computer simulation estimated treatment zone following irreversible electroporation of patient prostate
To determine whether patient specific numerical simulations of irreversible electroporation (IRE) of the prostate correlates with the treatment effect seen on follow-up MR imaging. Computer models were created using intra-operative US images, post-treatment follow-up MR images and clinical data from six patients receiving IRE. Isoelectric contours drawn using simulation results were compared with MR imaging to estimate the energy threshold separating treated and untreated tissue. Simulation estimates of injury to the neurovascular bundle and rectum were compared with clinical follow-up and patient reported outcomes. At the electric field strength of 700 V/cm, simulation estimated electric field distribution was not different from the ablation defect seen on follow-up MR imaging (p = 0.43). Simulation predicted cross sectional area of treatment (mean 532.33 ± 142.32 mm2) corresponded well with the treatment zone seen on MR imaging (mean 540.16 ± 237.13 mm2). Simulation results did not suggest injury to the rectum or neurovascular bundle, matching clinical follow-up at 3 months. Computer simulation estimated zone of irreversible electroporation in the prostate at 700 V/cm was comparable to measurements made on follow-up MR imaging. Numerical simulation may aid treatment planning for irreversible electroporation of the prostate in patients.
An omics investigation into chronic widespread musculoskeletal pain reveals epiandrosterone sulfate as a potential biomarker
An “omics” investigation of chronic widespread pain in 2 population samples reveals a genetic variant in CYP3A5 as underlying steroid hormone abnormalities seen in CWP. Chronic widespread musculoskeletal pain (CWP) is common, having a population prevalence of 10%. This study aimed to define the biological basis of the CWP/body mass association by using a systems biology approach. Adult female twins (n = 2444) from the TwinsUK registry who had extensive clinical, anthropometric, and “omic” data were included. Nontargeted metabolomics screening including 324 metabolites was carried out for CWP and body composition using dual-energy X-ray absorptiometry. The biological basis of these associations was explored through a genome-wide association study and replicated in an independent population sample (Cooperative Health Research in the Region of Augsburg [KORA] study, n = 2483). A causal role for the genetic variants identified was sought in CWP using a Mendelian randomisation study design. Fat mass/height2 was the body composition variable most strongly associated with CWP (TwinsUK: P = 2.4 × 10−15 and KORA: P = 1.59 × 10−10). Of 324 metabolites examined, epiandrosterone sulfate (EAS) was highly associated with both CWP (P = 1.05 × 10−09 in TwinsUK and P = 3.70 × 10−06 in KORA) and fat mass/height2. Genome-wide association study of EAS identified imputed single nucleotide polymorphism rs1581492 at 7q22.1 to be strikingly associated with EAS levels (P ≤ 2.49 × 10−78), and this result was replicated in KORA (P = 2.12 × 10−9). Mendelian randomization by rs1581492 genotype showed that EAS is unlikely to be causally related to CWP. Using an agnostic omics approach to focus on the association of CWP with body mass index, we have confirmed a steroid hormone association and identified a genetic variant upstream of the CYP genes, which likely controls this response. This study suggests that steroid hormone abnormalities result from pain rather than causing it, and EAS may provide a biomarker that identifies subgroups at risk of CWP.
Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation
Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids. Light scattering experiments showed formation of lower order associated species around 50–70 °C followed by rapid cooperativity to β-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous β-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in β-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in β-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to “broom-like” fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation.
Diversity of Bacterial Communities on Four Frequently Used Surfaces in a Large Brazilian Teaching Hospital
Frequently used hand-touch surfaces in hospital settings have been implicated as a vehicle of microbial transmission. In this study, we aimed to investigate the overall bacterial population on four frequently used surfaces using a culture-independent Illumina massively parallel sequencing approach of the 16S rRNA genes. Surface samples were collected from four sites, namely elevator buttons (EB), bank machine keyboard buttons (BMKB), restroom surfaces, and the employee biometric time clock system (EBTCS), in a large public and teaching hospital in São Paulo. Taxonomical composition revealed the abundance of Firmicutes phyla, followed by Actinobacteria and Proteobacteria, with a total of 926 bacterial families and 2832 bacterial genera. Moreover, our analysis revealed the presence of some potential pathogenic bacterial genera, including Salmonella enterica, Klebsiella pneumoniae, and Staphylococcus aureus. The presence of these pathogens in frequently used surfaces enhances the risk of exposure to any susceptible individuals. Some of the factors that may contribute to the richness of bacterial diversity on these surfaces are poor personal hygiene and ineffective routine schedules of cleaning, sanitizing, and disinfecting. Strict standards of infection control in hospitals and increased public education about hand hygiene are recommended to decrease the risk of transmission in hospitals among patients.
Bacterial Community Succession in Pine Wood Decomposition
Though bacteria and fungi are common inhabitants of decaying wood, little is known about the relationship between bacterial and fungal community dynamics during natural wood decay. Based on previous studies involving inoculated wood blocks, strong fungal selection on bacteria abundance and community composition was expected to occur during natural wood decay. Here, we focused on bacterial and fungal community compositions in pine wood samples collected from dead trees in different stages of decomposition. We showed that bacterial communities undergo less drastic changes than fungal communities during wood decay. Furthermore, we found that bacterial community assembly was a stochastic process at initial stage of wood decay and became more deterministic in later stages, likely due to environmental factors. Moreover, composition of bacterial communities did not respond to the changes in the major fungal species present in the wood but rather to the stage of decay reflected by the wood density. We concluded that the shifts in the bacterial communities were a result of the changes in wood properties during decomposition and largely independent of the composition of the wood-decaying fungal communities.
Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model
Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl) under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs. Summary: Live imaging in the medaka, a popular fish model for human bone research, shows that osteoclast inhibition by bisphosphonates triggers an efficient repair of bone defects by redistributed osteoblasts.
Detecting Selection on Temporal and Spatial Scales: A Genomic Time Series Assessment of Selective Responses to Devil Facial Tumor Disease
Detecting loci under selection is an important task in evolutionary biology. In conservation genetics detecting selection is key to investigating adaptation to the spread of infectious disease. Loci under selection can be detected on a spatial scale, accounting for differences in demographic history among populations, or on a temporal scale, tracing changes in allele frequencies over time. Here we use these two approaches to investigate selective responses to the spread of an infectious cancer—devil facial tumor disease (DFTD)—that since 1996 has ravaged the Tasmanian devil (Sarcophilus harrisii). Using time-series ‘restriction site associated DNA’ (RAD) markers from populations pre- and post DFTD arrival, and DFTD free populations, we infer loci under selection due to DFTD and investigate signatures of selection that are incongruent among methods, populations, and times. The lack of congruence among populations influenced by DFTD with respect to inferred loci under selection, and the direction of that selection, fail to implicate a consistent selective role for DFTD. Instead genetic drift is more likely driving the observed allele frequency changes over time. Our study illustrates the importance of applying methods with different performance optima e.g. accounting for population structure and background selection, and assessing congruence of the results.
Standardized orthotopic xenografts in zebrafish reveal glioma cell line specific characteristics and tumor cell heterogeneity
Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening. Summary: This zebrafish xenotransplant model of glioblastoma enables in vivo imaging of tumor cells and rapid screening for anti-glioma agents. It provides standardization of a model that is easily replicated across laboratories.
SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane
Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells. SPARC is an extracellular matrix protein that is present at high levels in many metastatic cancers where it promotes tumor invasion into neighboring tissues. The mechanism linking a surplus of SPARC to cell invasion, however, is not clear due to the challenge of examining SPARCs function in complex tumor environments. We have used anchor cell invasion in C. elegans development to understand how an excess of SPARC promotes invasion in a native tissue setting. Anchor cell invasion allows experimental examination and visualization of the interactions between an invasive cell, neighboring tissues, and the basement membrane, a sheet-like extracellular matrix that surrounds tissues. We find that increased SPARC expression potently enhances the ability of weakly invasive anchor cells to breach the basement membrane. Our data indicate that SPARC functions normally to transport the basement membrane component type IV collagen between tissues to precisely regulate its deposition into basement membranes. Collagen molecules are covalently cross-linked and provide basement membranes their barrier properties. Our results indicate that overexpression of SPARC interferes with collagen trafficking and significantly decreases collagen incorporation into basement membranes, potentially weakening this barrier and allowing it to be more easily breached by invasive cells.
Performance Monitoring in Medication Naïve Children with Tourette Syndrome
Background: Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder and its impact on cognitive development needs further study. Evidence from neuropsychological, neuroimaging and electrophysiological studies suggests that the decline in tic severity and the ability to suppress tics relate to the development of self-regulatory functions in late childhood and adolescence. Hence, tasks measuring performance monitoring might provide insight into the regulation of tics in children with TS. Method: Twenty-five children with TS, including 14 with comorbid Attention-deficit/ hyperactivity disorder (ADHD), 39 children with ADHD and 35 typically developing children aged 8–12 years were tested with a modified Eriksen-Flanker task during a 34-channel electroencephalography (EEG) recording. Task performance, as well as stimulus-locked and response-locked event-related potentials (ERP) were analyzed and compared across groups. Results: Participants did not differ in their behavioral performance. Children with TS showed higher amplitudes of an early P3 component of the stimulus-locked ERPs in ensemble averages and in separate trial outcomes, suggesting heightened orienting and/or attention during stimulus evaluation. In response-locked averages, children with TS had a slightly higher positive complex before the motor response, likely also reflecting a late P3. Groups did not differ in post-response components, particularly in the error-related negativity (ERN) and error-related positivity (Pe). Conclusions: These findings suggest that children with TS may employ additional attentional resources as a compensatory mechanism to maintain equal behavioral performance.
Human Adipose Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4 Integrin Expression
Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.
A secretomic view of woody and nonwoody lignocellulose degradation by Pleurotus ostreatus
Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures. Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends. Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results. The online version of this article (doi:10.1186/s13068-016-0462-9) contains supplementary material, which is available to authorized users.
Quantitative map of multiple auditory cortical regions with a stereotaxic fine scale atlas of the mouse brain
Optical imaging studies have recently revealed the presence of multiple auditory cortical regions in the mouse brain. We have previously demonstrated, using flavoprotein fluorescence imaging, at least six regions in the mouse auditory cortex, including the anterior auditory field (AAF), primary auditory cortex (AI), the secondary auditory field (AII), dorsoanterior field (DA), dorsomedial field (DM), and dorsoposterior field (DP). While multiple regions in the visual cortex and somatosensory cortex have been annotated and consolidated in recent brain atlases, the multiple auditory cortical regions have not yet been presented from a coronal view. In the current study, we obtained regional coordinates of the six auditory cortical regions of the C57BL/6 mouse brain and illustrated these regions on template coronal brain slices. These results should reinforce the existing mouse brain atlases and support future studies in the auditory cortex.
Identification of Loci Modulating the Cardiovascular and Skeletal Phenotypes of Marfan Syndrome in Mice
Marfan syndrome (MFS) is an autosomal dominant disease of the connective tissue, affecting mostly the skeletal, ocular and cardiovascular systems, caused by mutations in the FBN1 gene. The existence of modifier genes has been postulated based on the wide clinical variability of manifestations in patients, even among those with the same FBN1 mutation. Although isogenic mouse models of the disease were fundamental in dissecting the molecular mechanism of pathogenesis, they do not address the effect of genetic background on the disease phenotype. Here, we use a new mouse model, mgΔloxPneo, which presents different phenotype severity dependent on the genetic backgrounds, to identify genes involved in modulating MFS phenotype. F2 heterozygotes showed wide phenotypic variability, with no correlations between phenotypic severities of the different affected systems, indicating that each has its specific set of modifier genes. Individual analysis of the phenotypes, with SNP microarrays, identified two suggestive QTL each to the cardiovascular and skeletal, and one significant QTL to the skeletal phenotype. Epistatic interactions between the QTL account for 47.4% and 53.5% of variation in the skeletal and cardiovascular phenotypes, respectively. This is the first study that maps modifier loci for MFS, showing the complex genetic architecture underlying the disease.
Pituitary adenylate cyclase activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP specific receptor
Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.
Phylogenetic Analysis of a ‘Jewel Orchid’ Genus Goodyera (Orchidaceae) Based on DNA Sequence Data from Nuclear and Plastid Regions
A molecular phylogeny of Asiatic species of Goodyera (Orchidaceae, Cranichideae, Goodyerinae) based on the nuclear ribosomal internal transcribed spacer (ITS) region and two chloroplast loci (matK and trnL-F) was presented. Thirty-five species represented by 132 samples of Goodyera were analyzed, along with other 27 genera/48 species, using Pterostylis longifolia and Chloraea gaudichaudii as outgroups. Bayesian inference, maximum parsimony and maximum likelihood methods were used to reveal the intrageneric relationships of Goodyera and its intergeneric relationships to related genera. The results indicate that: 1) Goodyera is not monophyletic; 2) Goodyera could be divided into four sections, viz., Goodyera, Otosepalum, Reticulum and a new section; 3) sect. Reticulum can be further divided into two subsections, viz., Reticulum and Foliosum, whereas sect. Goodyera can in turn be divided into subsections Goodyera and a new subsection.
Combining Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism
Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.
Genome Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells
In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality. In protein synthesis, ribosome assembles to mRNA to initiate translation, followed by the process of elongation to read the codons along the mRNA molecule for polypeptide chain production. It is known that slowing down the elongation speed at certain regions of mRNA is critical for the correct folding of numerous proteins—the so-called “pause-to-fold”. However, it has been an open question to evaluate elongation speed under cellular physiological conditions in genome-wide scale. Here, we used three types of next-generation sequencing approaches to experimentally and computationally address this question. With a new relative measure of elongation velocity index (EVI), we successfully distinguished slow-translating genes. Their protein products are more stable than the background genes. We found that different cell types tended to have distinct slow-translating codons, which might be relevant to the cell/tissue specific tRNA composition. Such elongation deceleration is potentially disease-relevant: cancer cells tend to slow down numerous cancer-favorable genes, and vice versa. Furthermore, we justified that translation initiation and elongation are evolutionarily synergistic as no gene with both high initiation efficiency and low elongation speed was observed: that would cause a traffic jam of ribosomes that should be maximally avoided per evolution.
Blood Transcriptomic Markers in Patients with Late Onset Major Depressive Disorder
We investigated transcriptomic markers of late-onset major depressive disorder (LOD; onset age of first depressive episode ≥ 50 years) from the genes expressed in blood cells and identified state-dependent transcriptomic markers in these patients. We assessed the genes expressed in blood cells by microarray and found that the expression levels of 3,066 probes were state-dependently changed in the blood cells of patients with LOD. To select potential candidates from those probes, we assessed the genes expressed in the blood of an animal model of depression, ovariectomized female mice exposed to chronic ultra-mild stress, by microarray and cross-matched the differentially expressed genes between the patients and the model mice. We identified 14 differentially expressed genes that were similarly changed in both patients and the model mice. By assessing statistical significance using real-time quantitative PCR (RT-qPCR), the following 4 genes were selected as candidates: cell death-inducing DFFA-like effector c (CIDEC), ribonuclease 1 (RNASE1), solute carrier family 36 member-1 (SLC36A1), and serine/threonine/tyrosine interacting-like 1 (STYXL1). The discriminating ability of these 4 candidate genes was evaluated in an independent cohort that was validated. Among them, CIDEC showed the greatest discriminant validity (sensitivity 91.3% and specificity 87.5%). Thus, these 4 biomarkers should be helpful for properly diagnosing LOD.
Transmembrane Domain Lengths Serve as Signatures of Organismal Complexity and Viral Transport Mechanisms
It is known that membrane proteins are important in various secretory pathways, with a possible role of their transmembrane domains (TMDs) as sorting determinant factors. One key aspect of TMDs associated with various “checkposts” (i.e. organelles) of intracellular trafficking is their length. To explore possible linkages in organisms with varying “complexity” and differences in TMD lengths of membrane proteins associated with different organelles (such as Endoplasmic Reticulum, Golgi, Endosomes, Nucleus, Plasma Membrane), we analyzed ~70000 membrane protein sequences in over 300 genomes of fungi, plants, non-mammalian vertebrates and mammals. We report that as we move from simpler to complex organisms, variation in organellar TMD lengths decreases, especially compared to their respective plasma membranes, with increasing organismal complexity. This suggests an evolutionary pressure in modulating length of TMDs of membrane proteins with increasing complexity of communication between sub-cellular compartments. We also report functional applications of our findings by discovering remarkable distinctions in TMD lengths of membrane proteins associated with different intracellular transport pathways. Finally, we show that TMD lengths extracted from viral proteins can serve as somewhat weak indicators of viral replication sites in plant cells but very strong indicators of different entry pathways employed by animal viruses.
Genetic polymorphisms in circadian negative feedback regulation genes predict overall survival and response to chemotherapy in gastric cancer patients
Circadian negative feedback loop (CNFL) genes play important roles in cancer development and progression. To evaluate the effects of single nucleotide polymorphisms (SNPs) in CNFL genes on the survival of GC patients, 13 functional SNPs from 5 CNFL genes were genotyped in a cohort of 1030 resected GC patients (704 in the training set, 326 in the validation set) to explore the association of SNPs with overall survival (OS). Among the 13 SNPs, three SNPs (rs1056560 in CRY1, rs3027178 in PER1 and rs228729 in PER3) were significantly associated with OS of GC in the training set, and verified in the validation set and pooled analysis. Furthermore, a dose-dependent cumulative effect of these SNPs on GC survival was observed, and survival tree analysis showed higher order interactions between these SNPs. In addition, protective effect conferred by adjuvant chemotherapy (ACT) on GC was observed in patients with variant alleles (TG/GG) of rs1056560, but not in those with homozygous wild (TT) genotype. Functional assay suggested rs1056560 genotypes significantly affect CRY1 expression in cancer cells. Our study presents that SNPs in the CNFL genes may be associated with GC prognosis, and provides the guidance in selecting potential GC patients most likely responsive to ACT.
Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation
The atypical cadherin Fat2 binds the WAVE regulatory complex (WRC) and acts with receptor tyrosine phosphatase Dlar through the WRC to control collective cell migration during Drosophila oogenesis. Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.
Newly Isolated Paenibacillus tyrfis sp. nov., from Malaysian Tropical Peat Swamp Soil with Broad Spectrum Antimicrobial Activity
Emergence of antimicrobial resistance coupled with the slowdown in discovery of new antimicrobial compounds points to serious consequences for human health. Therefore, scientists are looking for new antimicrobial compounds from unique and understudied ecosystems such as tropical peat swamp forests. Over the course of isolating antimicrobial producing bacteria from North Selangor tropical peat swamp forest, Malaysia, a Gram variable, rod shaped, endospore forming, facultative anaerobic novel strain MSt1T that exerts potent and broad spectrum antimicrobial activity was isolated. Phylogenetic analysis using 16S rRNA gene sequences showed that strain MSt1T belonged to the genus Paenibacillus with the highest similarity to Paenibacillus elgii SD17T (99.5%). Whole genome comparison between strain MSt1T with its closely related species using average nucleotide identity (ANI) revealed that similarity between strain MSt1T with P. elgii B69 (93.45%) and Paenibacillus ehimensis A2 (90.42%) was below the recommended threshold of 95%. Further analysis using in silico pairwise DDH also showed that similarity between strain MSt1T with P. elgii B69 (55.4%) and P. ehimensis A2 (43.7%) was below the recommended threshold of 70%. Strain MSt1T contained meso-diaminopilemic acid in the cell wall and MK-7 as the major menaquinone. The major fatty acids of strain MSt1T were anteiso-C15:0 (48.2%) and C16:0 (29.0%) whereas the polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unknown lipid, two unknown glycolipids, and one unknown phospholipid. Total DNA G+C content of strain MSt1T was 51.5 mol%. The extract from strain MSt1T exerted strong antimicrobial activity against Escherichia coli ATCC 25922 (MIC = 1.5 μg/mL), MRSA ATCC 700699 (MIC = 25 μg/mL) and Candida albicans IMR (MIC = 12.5 μg/mL). Partially purified active fraction exerted a strong effect against E. coli ATCC 25922 resulting in cell rupture when viewed with SEM. Based on distinctive taxonomic differences between strain MSt1T when compared to its closely related type species, we propose that strain MSt1T represents a novel species within the genus of Paenibacillus, for which the name Paenibacillus tyrfis sp. nov. (= DSM 100708T = MCCC 1K01247T) is proposed.
Whole transcriptome profiling reveals the RNA content of motor axons
Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
Genome wide binding studies reveal DNA binding specificity mechanisms and functional interplay amongst Forkhead transcription factors
Transcription factors belonging to the same transcription factor families contain very similar DNA binding domains and hence have the potential to bind to related DNA sequences. However, subtle differences in binding specificities can be detected in vitro with the potential to direct specific responses in vivo. Here, we have examined the binding properties of three Forkhead (FOX) transcription factors, FOXK2, FOXO3 and FOXJ3 in vivo. Extensive overlap in chromatin binding is observed, although underlying differential DNA binding specificity can dictate the recruitment of FOXK2 and FOXJ3 to chromatin. However, functionally, FOXO3-dependent gene regulation is generally mediated not through uniquely bound regions but through regions occupied by both FOXK2 and FOXO3 where both factors play a regulatory role. Our data point to a model whereby FOX transcription factors control gene expression through dynamically binding and generating partial occupancy of the same site rather than mutually exclusive binding derived by stable binding of individual FOX proteins.
MECP2 missense mutations outside the canonical MBD and TRD domains in males with intellectual disability
Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein highly expressed in neurons that is involved in transcriptional modulation and chromatin remodeling. Mutations in MECP2 in females are associated with Rett syndrome, a neurological disorder characterized by a normal neonatal period, followed by the arrest of development and regression of acquired skills. Although it was initially thought that MECP2 pathogenic mutations in males were not compatible with life, starting from 1999 about 60 male patients have been identified and their phenotype varies from severe neonatal encephalopathy to mild intellectual disability. Targeted Next Generation Sequencing of a panel of intellectual disability related genes was performed on two unrelated male patients, and two missense variants in MECP2 were identified (p.Gly185Val and p.Arg167Trp). These variants lie outside the canonical MBD and TRD domains, where the pathogenicity of missense variants is more difficult to establish. In both families, variants were found in all affected siblings and were inherited from the asymptomatic mother, showing skewed X-chromosome inactivation. We report here the first missense variant located in AT-hook domain 1 and we underline the importance of MECP2 substitutions outside the canonical MeCP2 domains in X-linked intellectual disability.
PARP 2 domain requirements for DNA damage dependent activation and localization to sites of DNA damage
Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs.
Monoamine Oxidase A Occupancy by Moclobemide and Phenelzine: Implications for the Development of Monoamine Oxidase Inhibitors
Monoamine oxidase inhibitors (MAOIs) are being developed for major depressive disorder, Alzheimer’s, and Parkinson’s Disease. Newer MAOIs have minimal sensitivity to tyramine, but a key limitation for optimizing their development is that standards for in vivo monoamine oxidase-A (MAO-A) occupancy in humans are not well established. The objectives were to determine the dose-occupancy relationship of moclobemide and the occupancy of phenelzine at typical clinical dosing. Major depressive episode (MDE) subjects underwent [11C]harmine positron emission tomography scanning prior to and following 6 weeks of treatment with moclobemide or phenelzine. Mean brain MAO-A occupancies were 74.23±8.32% for moclobemide at 300–600mg daily (n = 11), 83.75±5.52% for moclobemide at 900–1200mg daily (n = 9), and 86.82±6.89% for phenelzine at 45–60mg daily (n = 4). The regional dose-occupancy relationship of moclobemide fit a hyperbolic function [F(x) = a(x/[b + x]); F(1,18) = 5.57 to 13.32, p = 0.002 to 0.03, mean ‘a’: 88.62±2.38%, mean ‘b’: 69.88±4.36 mg]. Multivariate analyses of variance showed significantly greater occupancy of phenelzine (45–60mg) and higher-dose moclobemide (900–1200mg) compared to lower-dose moclobemide [300–600mg; F(7,16) = 3.94, p = 0.01]. These findings suggest that for first-line MDE treatment, daily moclobemide doses of 300–600mg correspond to a MAO-A occupancy of 74%, whereas for treatment-resistant MDE, either phenelzine or higher doses of moclobemide correspond to a MAO-A occupancy of at least 84%. Therefore, novel MAO inhibitor development should aim for similar thresholds. The findings provide a rationale in treatment algorithm design to raise moclobemide doses to inhibit more MAO-A sites, but suggest switching from high-dose moclobemide to phenelzine is best justified by binding to additional targets.
The spliceosome associated protein Nrl1 suppresses homologous recombination dependent R loop formation in fission yeast
The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.
Radiographic Comparison between Cervical Spine Lateral and Whole Spine Lateral Standing Radiographs
Study Design Retrospective radiologic study. Objective The sagittal alignment of the cervical spine can be evaluated using either a lateral cervical radiograph or a whole-spine lateral view on which the cervical spine is included. To our knowledge, however, no report has compared the two. The purpose of this work is to identify the difference in radiographic parameters between the cervical spine lateral view and the whole-spine lateral view. Methods We retrospectively analyzed 59 adult patients suffering from neck pain with cervical spine lateral radiographs and whole-spine lateral radiographs from November 2007 to December 2011. The radiographs were measured using standard techniques to obtain the following parameters from the two different radiographs: occipital–C2 angle, C2–C7 angle, C7–sternal angle, sternal slope, T1 slope, C2 central offset distance, the distance between C2 and C7 plumb lines, C4 anteroposterior (AP) diameter, the ratio of C2 central off distance to C4 AP diameter, the ratio of plumb lines' distance to C4 AP diameter. Results We found that the occipital–C2 angle, sternal slope, and C4 AP diameter were similar, but the C2–C7 angle, C7–sternal angle, T1 slope, C2 central offset distance, distance between C2 and C7 plumb lines, ratio of C2 central off distance to C4 AP diameter, and ratio of plumb lines' distance to C4 AP diameter were different. However, the error of measurement was greater than the small angular and linear differences between the two views. Conclusions Most numerical values of the measured radiographic parameters appear to be different between the two views. However, the two views are comparable because the numerical differences were smaller than the errors of measurement.
Nuclear factor 90 uses an ADAR2 like binding mode to recognize specific bases in dsRNA
Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs.
The first crystal structures of RNA–PNA duplexes and a PNA PNA duplex containing mismatches—toward anti sense therapy against TREDs
PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA–PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA–PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA–RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson–Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA.
Structure of the hypusinylated eukaryotic translation factor eIF 5A bound to the ribosome
During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.
Human nonsense mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.
Unfolding the HIV 1 reverse transcriptase RNase H domain – how to lose a molecular tug of war
Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66′ homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66′ subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH′ domain unfolding behavior of the p66/p66′ homodimer. This study demonstrates the feasibility of directly targeting RT maturation with therapeutics.
Transcription coupled DNA supercoiling dictates the chromosomal arrangement of bacterial genes
Over the recent decade, the central importance of DNA supercoiling in chromosome organization and global gene regulation of bacteria became more and more visible. With a regulon comprising more than 2000 genes in Escherichia coli, DNA supercoiling is among the most influential regulators of gene expression found in bacteria so far. However, the mechanism creating thousands of diverse temporal gene expression patterns coordinated by DNA supercoiling remains unclear. In this study we show that a specific chromosomal arrangement of genes modulates the local levels of DNA supercoiling at gene promoters via transcription-coupled DNA supercoiling (TCDS) in the model organism E. coli. Our findings provide a consistent explanation for the strong positive coupling of temporal gene expression patterns of neighboring genes. Using comparative genomics we are furthermore able to provide evidence that TCDS is a driving force for the evolution of chromosomal gene arrangement patterns in other Enterobacteriaceae. With the currently available data of promoter supercoiling sensitivity we prove that the same principle is applicable also for the evolutionary distant gram-positive pathogenic bacterium Streptococcus pneumoniae. Moreover, our findings are fully consistent with recent investigations concerning the regulatory impact of TCDS on gene pairs in eukaryots underpinning the broad applicability of our analysis.
Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster
Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.
Network of protein interactions within the Drosophila inner kinetochore
The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.
A New Thermophilic Nitrilase from an Antarctic Hyperthermophilic Microorganism
Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85°C. Its optimal activity occurred at 85°C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85°C. In addition, this nitrilase is highly versatile demonstrating activity toward different substrates, such as benzonitrile (60 mM, aromatic nitrile) and butyronitrile (60 mM, aliphatic nitrile), with a specific activity of 3286.7 U mg−1 of protein and 4008.2 U mg−1 of protein, respectively. Moreover the enzyme NitM24D13 also presents cyanidase activity. The apparent Michaelis–Menten constant (Km) and Vmáx of this Nitrilase for benzonitrile were 0.3 mM and 333.3 μM min−1, respectively, and the specificity constant (kcat/Km) for benzonitrile was 2.05 × 105 s−1 M−1.
Evidence for the Presence of Non Celiac Gluten Sensitivity in Patients with Functional Gastrointestinal Symptoms: Results from a Multicenter Randomized Double Blind Placebo Controlled Gluten Challenge
Non-celiac gluten sensitivity (NCGS) is characterized by the onset of symptoms after eating gluten-containing food. We aimed to single out NCGS subjects among subjects with functional gastrointestinal symptoms. Patients were enrolled in a multicenter double-blind placebo-controlled trial with crossover. Symptoms and quality of life were evaluated by means of 10-cm VAS and SF36. Iron parameters, transaminases and C reactive protein (CRP) were evaluated. After a three-week-long gluten-free diet (GFD), responsive patients were randomly assigned to gluten intake (5.6 g/day) or placebo for seven days, followed by crossover. The primary endpoint was the worsening of symptoms (VAS increase ≥3 cm) during gluten ingestion compared to placebo. One hundred and forty patients were enrolled and 134 (17 males, mean age 39.1 ± 11.7 years, BMI 22.4 ± 3.8) completed the first period. A total of 101 subjects (10 males, mean age 39.3 ± 11.0 years, BMI 22.3 ± 4.0) reported a symptomatic improvement (VAS score 2.3 ± 1.2 vs. 6.5 ± 2.2 before and after GFD, p = 0.001). 98 patients underwent the gluten challenge and 28 (all females, mean age 38.9 ± 12.7 years, BMI 22.0 ± 2.9) reported a symptomatic relapse and deterioration of quality of life. No parameters were found to be statistically associated with positivity to the challenge. However, 14 patients responded to the placebo ingestion. Taking into account this finding, about 14% of patients responding to gluten withdrawal showed a symptomatic relapse during the gluten challenge. This group is suspected to have NCGS.
The Structural Correlates of Statistical Information Processing during Speech Perception
The processing of continuous and complex auditory signals such as speech relies on the ability to use statistical cues (e.g. transitional probabilities). In this study, participants heard short auditory sequences composed either of Italian syllables or bird songs and completed a regularity-rating task. Behaviorally, participants were better at differentiating between levels of regularity in the syllable sequences than in the bird song sequences. Inter-individual differences in sensitivity to regularity for speech stimuli were correlated with variations in surface-based cortical thickness (CT). These correlations were found in several cortical areas including regions previously associated with statistical structure processing (e.g. bilateral superior temporal sulcus, left precentral sulcus and inferior frontal gyrus), as well other regions (e.g. left insula, bilateral superior frontal gyrus/sulcus and supramarginal gyrus). In all regions, this correlation was positive suggesting that thicker cortex is related to higher sensitivity to variations in the statistical structure of auditory sequences. Overall, these results suggest that inter-individual differences in CT within a distributed network of cortical regions involved in statistical structure processing, attention and memory is predictive of the ability to detect structural structure in auditory speech sequences.
Clinical and epidemiological features of the 2014 large scale dengue outbreak in Guangzhou city, China
Dengue virus is transmitted by mosquito around the tropical and sub-tropical regions. There was a large-scale dengue epidemic in Guangdong province, China during 2014 and around fifty thousands dengue fever cases, including six deaths, have been reported. In this study, we aimed to understand the clinical characteristics of hospitalized patients with laboratory-confirmed dengue virus (DENV) infection and determined the origin of the virus from the outbreak. We have summarized the data from 138 hospitalized patients who were laboratory confirmed for dengue infection in Guangzhou city. Patients were classified as either non-severe dengue fever or severe dengue fever according to the guidelines from the WHO. Viral serotypes were determined by real time RT-PCR. Genetic sequences of the envelope and non-structural genes were amplified and analyzed from the serum samples of eleven patients. Co-circulation of dengue serotype 1 and 2 were identified from the outbreak. Patients infected by serotype 1 or 2 showed similar clinical features. Patients with severe dengue fever showed prolonged hospitalization and significant impairment of organ functions. Four samples from serotype 1 and five samples from serotype 2 were closely related respectively and clustered with Guangzhou isolates from previous years. The remaining isolates of serotype 1 were related to viruses found in Malaysia, India, Bangladesh and Singapore. The phylogenetic grouping of Guangdong isolates suggests that dengue is no longer an imported disease in China. Analysis of the isolates obtained in this study together with the size of the outbreak are suggestive of endemic circulation in Guangdong province. The online version of this article (doi:10.1186/s12879-016-1379-4) contains supplementary material, which is available to authorized users.
Integrative Taxonomic Approach for Describing a New Cryptic Species of Bush Frog (Raorchestes: Anura: Rhacophoridae) from the Western Ghats, India
A new cryptic species of bush frog Raorchestes honnametti sp. nov. is described from the south-eastern part of the Western Ghats, India. This newly described species belongs to the Charius clade and is morphologically similar to other clade members—R. charius and R. griet. Therefore, an integrative taxonomic approach based on molecular and bioacoustic analysis along with morphology was used to delimit the new species. Raorchestes honnametti sp. nov., is currently known only from Biligiri Rangaswamy Temple Tiger Reserve, a part of Biligiri Rangaswamy horst mountain range (a mountain formed due movement of two faults) formed during the Late Quaternary period (1.8–2.58 Ma). Discovery of cryptic species from a highly speciose and well-studied genus Raorchestes hints at the possible existence of several more cryptic species in this genus. We discuss the possible reasons for crypsis and emphasize the need for continued systematic surveys of amphibians across the Western Ghats.
Murine mesothelin: characterization, expression, and inhibition of tumor growth in a murine model of pancreatic cancer
Mesothelin has attracted much interest as a tumor specific antigen; it has been reported to promote tumor development and to be a good target for cancer treatment. Most studies to date have used human mesothelin in immunocompromised mice. Since these models do not allow for study of the natural immune response to mesothelin expressing tumors, we have undertaken the characterization of mouse mesothelin so the effects of this protein can be assessed in immunocompetent mouse strains. We analyzed mouse mesothelin expression, tissue distribution, shedding and biochemistry. In addition we constructed stable mesothelin overexpressing lines of the pancreatic cancer line Panc02 by two methods and tested them for growth and tumorigencity in vitro and in vivo. We show here that mouse mesothelin is similar to human mesothelin in biochemical characteristics, tumor expression and tissue distribution, suggesting the mouse may be a suitable model for study of mesothelin. Stable overexpression of mesothelin in a pancreatic cancer cell line did not increase cell proliferation or anchorage-independent growth in vitro, suggesting that mesothelin is not necessarily a tumor progression factor. Surprisingly overexpression of mesothelin inhibited tumor formation in vivo in immunocompetent mice. The mouse may be a good model for studying mesothelin in the context of an intact immune response. Mesothelin is not necessarily a tumor progression factor, and indeed mesothelin overexpression inhibited tumor growth in immunocompetent mice.
Phenotyping of type 2 diabetes mellitus at onset on the basis of fasting incretin tone: Results of a two‐step cluster analysis
According to some authors, in type 2 diabetes there is a reduced postprandial action of glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP). However, little is known about the role of fasting incretins in glucose homeostasis. Our aim was to evaluate, through a two‐step cluster analysis, the possibility of phenotyping patients with type 2 diabetes at onset on the basis of fasting GLP‐1, GIP and ghrelin. A total of 96 patients with type 2 diabetes within 6 months of onset (mean age 62.40 ± 6.36 years) were cross‐sectionally studied. Clinical, anthropometric and metabolic parameters were evaluated. At fasting the following were carried out: assay of GLP‐1, GIP, ghrelin, insulin, C‐peptide, glucagon and a panel of adipocytokines (visfatin, resistin, leptin, soluble leptin receptor and adiponectin). The analysis resulted in two clusters: cluster 1 (63 patients) had significantly lower levels of GLP‐1 (4.93 ± 0.98 vs 7.81 ± 1.98 pmol/L; P < 0.001), GIP (12.73 ± 9.44 vs 23.88 ± 28.56 pmol/L; P < 0.001) and ghrelin (26.54 ± 2.94 vs 39.47 ± 9.84 pmol/L; P < 0.001) compared with cluster 2 (33 patients). Between the two clusters, no differences in age, duration of disease, sex, clinical‐anthropometric parameters, insulin sensitivity and adipocytokines were highlighted. However, cluster 1 was associated with significantly higher levels of glycated hemoglobin (7.4 ± 0.61 vs 6.68 ± 0.57%, P = 0.007), glucagon (232.02 ± 37.27 vs 183.33 ± 97.29 ng/L; P = 0.001), fasting glucose (7.85 ± 1.60 vs 6.93 ± 1.01 mmol/L; P = 0.003) and significantly lower levels of C‐peptide (0.12 ± 0.11 vs 0.20 ± 0.20 nmol/L; P = 0.017). The present study suggests that fasting incretins play an important role in the pathophysiology of type 2 diabetes, which requires to further investigation.
Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove
HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 Toxoplasma gondii is a parasite that can infect most warm-blooded animals and cause a disease called toxoplasmosis. In humans, toxoplasmosis generally does not cause any noticeable symptoms, but it can cause serious problems in pregnant women and individuals with weakened immune systems. T. gondii is one of many parasites that hide within human cells in an attempt to avoid detection by the immune system. However, proteins called Human Leukocyte Antigens, or HLAs, can reveal hidden parasites by carrying small sections of them from the inside the infected cell to the cell’s surface. The immune system can then recognize the fragments as foreign and attack the parasite. HLAs typically pick up parasite fragments of a certain length, which enables the immune system to recognize that what is being displayed is a piece of parasite. By purifying HLAs from cells that have been infected by T. gondii, McMurtrey et al. have now learned more about which fragments of the parasite are displayed to the immune system. This analysis revealed that the parasite somehow manipulates the HLAs to carry parasite fragments that are considerably longer than can be explained with our current knowledge of how HLAs work. By using a technique called X-ray crystallography, McMurtrey et al. also show that the structure of the HLA assumes a previously unseen configuration when interacting with fragments of T. gondii. In the future, it will be important to understand how infected cells give rise to unusual structural configurations of HLAs and to unravel how these structures affect the immune system’s ability to fight infections. DOI: http://dx.doi.org/10.7554/eLife.12556.002
Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution
The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 Cells sense and respond to the world around them using signaling “circuits” made of proteins and other molecules, and when an important cell circuit breaks, diseases like cancer may arise. Much like with electrical circuits, a given set of molecular components can be used to build different signaling circuits that behave in different ways. However, unlike for electrical circuits we generally do not have design manuals that allow us to work out how a signaling circuit behaves based on the components it includes. Doing this would involve identifying all the molecular parts of a circuit, using them to build every possible circuit, and carefully measuring the associated behavior. A group of proteins called the Ras-superfamily GTPases are important controllers of cell behavior. To investigate the behavior of Ras GTPase signaling circuits, Coyle and Lim built up different circuits from their components and “watched” their behavior with a microscope. Analyzing these behaviors provided the information needed to produce a ‘design manual’ for programming Ras circuits. Coyle and Lim found that the makeup of a Ras signaling circuit strongly affects the timing, duration, shape and size of its output. This means that different cells can use the same core components in different ways to build circuits customized to their specific needs. Nonetheless, this versatility comes with a trade-off: the circuits are fragile, and can break in many different ways to cause disease. In the future Coyle and Lim aim to build other types of important cellular signaling circuits from their component parts. Only by building these systems, turning them on and watching them run can we begin to understand how they actually perform and what they are capable of. DOI: http://dx.doi.org/10.7554/eLife.12435.002
An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor
Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 Plants and other living organisms can survive stress and starvation by digesting and recycling parts of their own cells. This process is known as autophagy and it involves engulfing cellular material inside spherical structures called autophagosomes, before delivering it to sites in the cell where digestive enzymes can break the material down. A form of autophagy, known as selective autophagy, can specifically degrade toxic substances such as disease-causing microbes. Selective autophagy works through proteins called autophagy cargo receptors that define which molecules are targeted for degradation. However, it was not clear whether autophagy protects plants from infections, or how much disease-causing microbes interfere with this process for their own benefit. The microbe that causes late blight of potatoes (called Phytophthora infestans) is infamous for triggering widespread famines in Ireland in the 19th century. This disease-causing microbe continues to pose a serious threat to food security today, and parasitizes plant tissues by releasing proteins called effectors that enter the plant’s cells to subvert the plant’s physiology and counteract its defenses. Dagdas, Belhaj et al. now report that an effector from P. infestans, called PexRD54, can bind to autophagy-related protein from potato, called ATG8CL, and stimulate the formation of autophagosomes. Further experiments revealed that the PexRD54 effector could outcompete a plant autophagy cargo receptor that would otherwise bind to ATG8CL. This plant cargo receptor contributes to the plant’s defences, and by preventing it from interacting with ATG8CL, PexRD54 makes the plant more susceptible to infection by P. infestans. These findings show that the PexRD54 effector has evolved to interact with an autophagy-related protein to counteract the plant’s defences. Dagdas, Belhaj et al. suggest that PexRD54 might do this by activating autophagy to selectively eliminate some of the molecules that the plant use to defend itself. Furthermore, P. infestans might also benefit from the nutrients that are released when cellular material is broken down via autophagy. Future work could test these two hypotheses and explore whether other effectors from disease-causing microbes work in a similar way. DOI: http://dx.doi.org/10.7554/eLife.10856.002
Flowering Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)
Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.
Reappraisal of Supraorbital Sensory Nerve Conduction Recordings: Orthodromic and Antidromic Techniques
To establish a supraorbital nerve sensory conduction recording method and assess its usefulness. Thirty-one healthy subjects without a history of trauma or neurological disease were recruited. For the orthodromic procedure, the recording electrode was attached immediately superior to the supraorbital notch. The stimulation electrode was placed on points along the hairline which evoked the largest sensory nerve action potentials (SNAPs). The antidromic sensory response was recorded after switching the recording and stimulating electrodes. The measured parameters were onset latency, peak latency, and baseline to peak amplitude of the SNAPs. The electrophysiological parameters of the bilateral supraorbital nerves were compared. We also recruited two patients who had sensory deficits on one side of their foreheads because of laceration injuries. The parameters of orthodromically recorded SNAPs were as follows: onset latency 1.21±0.22 ms (range, 0.9–1.6 ms), peak latency 1.54±0.23 ms (range, 1.2–2.2 ms), and baseline to peak amplitude 4.16±1.92 µV (range, 1.4–10 µV). Those of antidromically recorded SNAPs were onset latency 1.31±0.27 ms (range, 0.8–1.7 ms), peak latency 1.62±0.29 ms (range, 1.3–2.2 ms), and baseline to peak amplitude 4.00±1.89 µV (range, 1.5–9.0 µV). There was no statistical difference in onset latency, peak latency, or baseline to peak amplitude between the responses obtained using the orthodromic and antidromic methods, and the parameters also revealed no statistical difference between the supraorbital nerves on both sides. We have successfully recorded supraorbital SNAPs. This conduction technique could be quite useful in evaluating patients with supraorbital nerve lesions.
A New Miocene Divergent Lineage of Old World Racer Snake from India
A distinctive early Miocene-divergent lineage of Old world racer snakes is described as a new genus and species based on three specimens collected from the western Indian state of Gujarat. Wallaceophis gen. et. gujaratenesis sp. nov. is a members of a clade of old world racers. The monotypic genus represents a distinct lineage among old world racers is recovered as a sister taxa to Lytorhynchus based on ~3047bp of combined nuclear (cmos) and mitochondrial molecular data (cytb, ND4, 12s, 16s). The snake is distinct morphologically in having a unique dorsal scale reduction formula not reported from any known colubrid snake genus. Uncorrected pairwise sequence divergence for nuclear gene cmos between Wallaceophis gen. et. gujaratenesis sp. nov. other members of the clade containing old world racers and whip snake is 21–36%.
Germplasm dynamics: the role of ecotypic diversity in shaping the patterns of genetic variation in Lolium perenne
Perennial ryegrass (Lolium perenne) is the most widely grown temperate grass species globally. Intensive plant breeding in ryegrass compared to many other crops species is a relatively recent exercise (last 100 years) and provides an interesting experimental system to trace the extent, impact and trajectory of undomesticated ecotypic variation represented in modern ryegrass cultivars. To explore germplasm dynamics in Lolium perenne, 2199 SNPs were genotyped in 716 ecotypes sampled from 90 European locations together with 249 cultivars representing 33 forage/amenity accessions. In addition three pseudo-cross mapping populations (450 individual recombinants) were genotyped to create a consensus genetic linkage map. Multivariate analyses revealed strong differentiation between cultivars with a small proportion of the ecotypic variation captured in improved cultivars. Ryegrass cultivars generated as part of a recurrent selection programme (RSP) are strongly associated with a small number of geographically localised Italian ecotypes which were among the founders of the RSP. Changes in haplotype frequency revealed signatures of selection in genes putatively involved in water-soluble carbohydrate (WSC) accumulation (a trait selected in the RSP). Retrospective analysis of germplasm in breeding programmes (germplasm dynamics) provides an experimental framework for the identification of candidate genes for novel traits such as WSC accumulation in ryegrass.
Structural Insight into Substrate Selectivity of Erwinia chrysanthemil Asparaginase
l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemil-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations—open and closed—corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.
FgSsn3 kinase, a component of the mediator complex, is important for sexual reproduction and pathogenesis in Fusarium graminearum
Fusarium graminearum is an important pathogen of wheat and barley. In addition to severe yield losses, infested grains are often contaminated with harmful mycotoxins. In this study, we characterized the functions of FgSSN3 kinase gene in different developmental and infection processes and gene regulation in F. graminearum. The FgSSN3 deletion mutant had a nutrient-dependent growth defects and abnormal conidium morphology. It was significantly reduced in DON production, TRI gene expression, and virulence. Deletion of FgSSN3 also resulted in up-regulation of HTF1 and PCS1 expression in juvenile cultures, and repression of TRI genes in DON-producing cultures. In addition, Fgssn3 was female sterile and defective in hypopodium formation and infectious growth. RNA-seq analysis showed that FgSsn3 is involved in the transcriptional regulation of a wide variety genes acting as either a repressor or activator. FgSsn3 physically interacted with C-type cyclin Cid1 and the cid1 mutant had similar phenotypes with Fgssn3, indicating that FgSsn3 and Cid1 form the CDK-cyclin pair as a component of the mediator complex in F. graminearum. Taken together, our results indicate that FgSSN3 is important for secondary metabolism, sexual reproduction, and plant infection, as a subunit of mediator complex contributing to transcriptional regulation of diverse genes.
Quercetin Directly Interacts with Vitamin D Receptor (VDR): Structural Implication of VDR Activation by Quercetin
The vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily. The VDR binds to active vitamin D3 metabolites, which stimulates downstream transduction signaling involved in various physiological activities such as calcium homeostasis, bone mineralization, and cell differentiation. Quercetin is a widely distributed flavonoid in nature that is known to enhance transactivation of VDR target genes. However, the detailed molecular mechanism underlying VDR activation by quercetin is not well understood. We first demonstrated the interaction between quercetin and the VDR at the molecular level by using fluorescence quenching and saturation transfer difference (STD) NMR experiments. The dissociation constant (Kd) of quercetin and the VDR was 21.15 ± 4.31 μM, and the mapping of quercetin subsites for VDR binding was performed using STD-NMR. The binding mode of quercetin was investigated by a docking study combined with molecular dynamics (MD) simulation. Quercetin might serve as a scaffold for the development of VDR modulators with selective biological activities.
Post glacial phylogeography and evolution of a wide ranging highly exploited keystone forest tree, eastern white pine (Pinus strobus) in North America: single refugium, multiple routes
Knowledge of the historical distribution and postglacial phylogeography and evolution of a species is important to better understand its current distribution and population structure and potential fate in the future, especially under climate change conditions, and conservation of its genetic resources. We have addressed this issue in a wide-ranging and heavily exploited keystone forest tree species of eastern North America, eastern white pine (Pinus strobus). We examined the range-wide population genetic structure, tested various hypothetical population history and evolutionary scenarios and inferred the location of glacial refugium and post-glacial recolonization routes. Our hypothesis was that eastern white pine survived in a single glacial refugium and expanded through multiple post-glacial recolonization routes. We studied the range-wide genetic diversity and population structure of 33 eastern white pine populations using 12 nuclear and 3 chloroplast microsatellite DNA markers. We used Approximate Bayesian Computation approach to test various evolutionary scenarios. We observed high levels of genetic diversity, and significant genetic differentiation (FST = 0.104) and population structure among eastern white pine populations across its range. A south to north trend of declining genetic diversity existed, consistent with repeated founder effects during post-glaciation migration northwards. We observed broad consensus from nuclear and chloroplast genetic markers supporting the presence of two main post-glacial recolonization routes that originated from a single southern refugium in the mid-Atlantic plain. One route gave rise to populations at the western margin of the species’ range in Minnesota and western Ontario. The second route gave rise to central-eastern populations, which branched into two subgroups: central and eastern. We observed minimal sharing of chloroplast haplotypes between recolonization routes but there was evidence of admixture between the western and west-central populations. Our study reveals a single southern refugium, two recolonization routes and three genetically distinguishable lineages in eastern white pine that we suggest to be treated as separate Evolutionarily Significant Units. Like many wide-ranging North American species, eastern white pine retains the genetic signatures of post-glacial recolonization and evolution, and its contemporary population genetic structure reflects not just the modern distribution and effects of heavy exploitation but also routes northward from its glacial refugium. The online version of this article (doi:10.1186/s12862-016-0624-1) contains supplementary material, which is available to authorized users.
Microsatellite based genetic diversity study in indigenous chicken ecotypes of Karnataka
The current study was the first of its kind taken upon indigenous ecotypes of the Karnataka in order to unravel the diversity details at 20 chicken microsatellite regions. 210 indigenous chicken belonging to six districts of Bangalore and Mysore division formed the target sample for the present study. The genomic deoxyribonucleic acid was isolated by phenol chloroform isoamyl alcohol method. A panel of 20 microsatellite regions, including 14 recommended by FAO and six identified from published scientific literature became the targeted chicken genomic region. 27-33 samples were successfully genotyped in each of the six ecotypes through simplex or multiplex polymerase chain reactions, polyacrylamide gel electrophoresis and silver staining for the selected microsatellite panel. The chickens of Ramanagara and Chamrajnagara were most distant with a Nei’s genetic distance value of 0.22. The chickens of Bangalore rural and Mysore were least distant with a value of 0.056. The Ramanagara and Chamrajnagara pair had Nei’s genetic identity value of 0.802, which is least among all pairs of ecotypes. There were five main nodes from which the six ecotypes evolved on the basis 20 microsatellite markers used in this study. This study indicates that the four ecotypes Ramnagara, Bangalore Rural, Chickaballapura and Mysore are genetically identical due to their common ancestral evolution while, Mandya and Chamrajnagara ecotypes formed a relatively different cluster due to a separate common ancestral chicken population and less number of generations since drifting from bifurcation node. Twenty microsatellite markers based genetic diversity study on six indigenous ecotypes indicated lower genetic distances as well as lower FST values compared to the distinguished breeds reported. There were two main clusters, which differentiated into six ecotypes. They may differentiate into more distinct varieties if bred in isolation for a longer number of generations.
Antimycobacterial Activity of a New Peptide Polydim I Isolated from Neotropical Social Wasp Polybia dimorpha
Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40–90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.
A Global Population Genetic Study of Pantala flavescens
Among terrestrial arthropods, the dragonfly species Pantala flavescens is remarkable due to their nearly global distribution and extensive migratory ranges; the largest of any known insect. Capable of migrating across oceans, the potential for high rates of gene flow among geographically distant populations is significant. It has been hypothesized that P. flavescens may be a global panmictic population but no sufficient genetic evidence has been collected thus far. Through a population genetic analysis of P. flavescens samples from North America, South America, and Asia, the current study aimed to examine the extent at which gene flow is occurring on a global scale and discusses the implications of the genetic patterns we uncovered on population structure and genetic diversity of the species. This was accomplished using PCR-amplified cytochrome oxidase one (CO1) mitochondrial DNA data to reconstruct phylogenetic trees, a haplotype network, and perform molecular variance analyses. Our results suggested high rates of gene flow are occurring among all included geographic regions; providing the first significant evidence that Pantala flavescens should be considered a global panmictic population.
Decoupled genomic elements and the evolution of partner quality in nitrogen‐fixing rhizobia
Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long‐term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long‐term nitrogen fertilization caused the evolution of less‐mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less‐mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long‐term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.
Genetic diversity and phylogenetic relationships in local cattle breeds of Senegal based on autosomal microsatellite markers
In Senegal, uncontrolled cross-breeding of cattle breeds and changes in production systems are assumed to lead to an increase of gene flow between populations. This might constitute a relevant threat to livestock improvement. Therewith, this study was carried out to assess the current genetic diversity and the phylogenetic relationships of the four native Senegalese cattle breeds (Gobra zebu, Maure zebu, Djakoré, and N’Dama). Genomic DNA was isolated from blood samples of 120 unrelated animals collected from three agro-ecological areas of Senegal according to their phenotypic traits. Genotyping was done using 11 specific highly polymorphic microsatellite makers recommended by Food and Agriculture Organization. The basic measures of genetic variation and phylogenetic trees were computed using bioinformatics’ software. A total of 115 alleles were identified with a number of alleles (Na) at one locus ranging from 6 to 16. All loci were polymorphic with a mean polymorphic information content of 0.76. The mean allelic richness (Rs) lay within the narrow range of 5.14 in N’Dama taurine to 6.10 in Gobra zebu. While, the expected heterozygosity (HE) per breed was high in general with an overall mean of 0.76±0.04. Generally, the heterozygote deficiency (FIS) of 0.073±0.026 was relatively due to inbreeding among these cattle breeds or the occurrence of population substructure. The high values of allelic and gene diversity showed that Senegalese native cattle breeds represented an important reservoir of genetic variation. The genetic distances and clustering trees concluded that the N’Dama cattle were most distinct among the investigated cattle populations. So, the principal component analyses showed qualitatively that there was an intensive genetic admixture between the Gobra zebu and Maure zebu breeds. The broad genetic diversity in Senegalese cattle breeds will allow for greater opportunities for improvement of productivity and adaptation relative to global changes. For the development of sustainable breeding and crossbreeding programs of Senegalese local breeds, effective management is needed towards genetic selection and transhumance to ensure their long-term survival.
Improving Protein Expression Prediction Using Extra Features and Ensemble Averaging
The article focus is the improvement of machine learning models capable of predicting protein expression levels based on their codon encoding. Support vector regression (SVR) and partial least squares (PLS) were used to create the models. SVR yields predictions that surpass those of PLS. It is shown that it is possible to improve the models predictive ability by using two more input features, codon identification number and codon count, besides the already used codon bias and minimum free energy. In addition, applying ensemble averaging to the SVR or PLS models also improves the results even further. The present work motivates the test of different ensembles and features with the aim of improving the prediction models whose correlation coefficients are still far from perfect. These results are relevant for the optimization of codon usage and enhancement of protein expression levels in synthetic biology problems.
From Observation to Information: Data Driven Understanding of on Farm Yield Variation
Agriculture research uses “recommendation domains” to develop and transfer crop management practices adapted to specific contexts. The scale of recommendation domains is large when compared to individual production sites and often encompasses less environmental variation than farmers manage. Farmers constantly observe crop response to management practices at a field scale. These observations are of little use for other farms if the site and the weather are not described. The value of information obtained from farmers’ experiences and controlled experiments is enhanced when the circumstances under which it was generated are characterized within the conceptual framework of a recommendation domain, this latter defined by Non-Controllable Factors (NCFs). Controllable Factors (CFs) refer to those which farmers manage. Using a combination of expert guidance and a multi-stage analytic process, we evaluated the interplay of CFs and NCFs on plantain productivity in farmers’ fields. Data were obtained from multiple sources, including farmers. Experts identified candidate variables likely to influence yields. The influence of the candidate variables on yields was tested through conditional forests analysis. Factor analysis then clustered harvests produced under similar NCFs, into Homologous Events (HEs). The relationship between NCFs, CFs and productivity in intercropped plantain were analyzed with mixed models. Inclusion of HEs increased the explanatory power of models. Low median yields in monocropping coupled with the occasional high yields within most HEs indicated that most of these farmers were not using practices that exploited the yield potential of those HEs. Varieties grown by farmers were associated with particular HEs. This indicates that farmers do adapt their management to the particular conditions of their HEs. Our observations confirm that the definition of HEs as recommendation domains at a small-scale is valid, and that the effectiveness of distinct management practices for specific micro-recommendation domains can be identified with the methodologies developed.
First report of Cryptosporidium canis in farmed Arctic foxes (Vulpes lagopus) in China
Cryptosporidium is an important genus of enteric zoonotic parasites, which can infect a wide range of animals including foxes. Little information is available concerning the prevalence and molecular characterisation of Cryptosporidium spp. in farmed Arctic foxes (Vulpes lagopus) in China. Thus, the objective of the present study was to investigate the prevalence of Cryptosporidium spp. in Arctic foxes in China using nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence of Cryptosporidium spp. in Arctic foxes was 15.9 % (48/302), with 12.9 % in male (18/139) and 18.4 % in female (30/163) foxes, respectively. The prevalence in different farms varied from 0 to 31.43 %. The prevalence of infection in different age groups varied from 14.1 % to 19.0 %. Foxes from Hebei Province (7.8 %, 11/141) had a significantly lower Cryptosporidium spp. prevalence than those from Heilongjiang Province (22.9 %, 16/70) and Jilin Province (23.1 %, 21/91) (P= 0.0015). Sequence analysis of the SSU rRNA gene indicated that all the 48 isolates represented C. canis. This is the first report of C. canis infection in farmed Arctic foxes in China, which also provides foundation data for preventing and controlling Cryptosporidium infection in foxes, other animals and humans.
Bacterial Cytological Profiling (BCP) as a Rapid and Accurate Antimicrobial Susceptibility Testing Method for Staphylococcus aureus
Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n = 71) within 1–2 h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n = 20) within 30 min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice. • Bacterial cytological profiling identifies antibiotic resistant S. aureus. • BCP predicts best treatment options for multidrug resistant MRSA. • Resistant strains are correctly identified within 1 h. • BCP does not require prior knowledge of resistance mechanism. Bacterial cytological profiling identifies antibiotic resistant S. aureus. BCP predicts best treatment options for multidrug resistant MRSA. Resistant strains are correctly identified within 1 h. BCP does not require prior knowledge of resistance mechanism. There is a great need for rapid antimicrobial susceptibility testing (AST) as it can dramatically improve clinical outcome for bacterial infections. Most currently proposed ASTs are dependent on knowledge of known resistance genes or based solely on growth/lysis. We have developed a new diagnostic method for rapidly determining antibiotic susceptibility of Staphylococcus aureus using quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. Our test has the potential to change the way antibiotic susceptibility testing is done in the future and is readily adaptable to different antibiotics and bacterial species regardless of the mechanisms of resistance.
Observer variability of absolute and relative thrombus density measurements in patients with acute ischemic stroke
Thrombus density may be a predictor for acute ischemic stroke treatment success. However, only limited data on observer variability for thrombus density measurements exist. This study assesses the variability and bias of four common thrombus density measurement methods by expert and non-expert observers. For 132 consecutive patients with acute ischemic stroke, three experts and two trained observers determined thrombus density by placing three standardized regions of interest (ROIs) in the thrombus and corresponding contralateral arterial segment. Subsequently, absolute and relative thrombus densities were determined using either one or three ROIs. Intraclass correlation coefficient (ICC) was determined, and Bland–Altman analysis was performed to evaluate interobserver and intermethod agreement. Accuracy of the trained observer was evaluated with a reference expert observer using the same statistical analysis. The highest interobserver agreement was obtained for absolute thrombus measurements using three ROIs (ICCs ranging from 0.54 to 0.91). In general, interobserver agreement was lower for relative measurements, and for using one instead of three ROIs. Interobserver agreement of trained non-experts and experts was similar. Accuracy of the trained observer measurements was comparable to the expert interobserver agreement and was better for absolute measurements and with three ROIs. The agreement between the one ROI and three ROI methods was good. Absolute thrombus density measurement has superior interobserver agreement compared to relative density measurement. Interobserver variation is smaller when multiple ROIs are used. Trained non-expert observers can accurately and reproducibly assess absolute thrombus densities using three ROIs. The online version of this article (doi:10.1007/s00234-015-1607-4) contains supplementary material, which is available to authorized users.
Untargeted plasma and tissue metabolomics in rats with chronic kidney disease given AST 120
Chronic kidney disease (CKD) results in the accumulation of metabolic waste products that are normally cleared by the kidney, known as uremia. Many of these waste products are from bacteria metabolites in the gut. Accumulation of uremic toxins in plasma and tissue, as well as the gut-plasma-tissue metabolic axis are important for understanding pathophysiological mechanisms of comorbidities in CKD. In this study, an untargeted metabolomics approach was used to determine uremic toxin accumulation in plasma, liver, heart and kidney tissue in rats with adenine-induced CKD. Rats with CKD were also given AST-120, a spherical carbon adsorbent, to assess metabolic changes in plasma and tissues with the removal of gut-derived uremic toxins. AST-120 decreased >55% of metabolites that were increased in plasma, liver and heart tissue of rats with CKD. CKD was primarily defined by 8 gut-derived uremic toxins, which were significantly increased in plasma and all tissues. These metabolites were derived from aromatic amino acids and soy protein including: indoxyl sulfate, p-cresyl sulfate, hippuric acid, phenyl sulfate, pyrocatechol sulfate, 4-ethylphenyl sulfate, p-cresol glucuronide and equol 7-glucuronide. Our results highlight the importance of diet and gut-derived metabolites in the accumulation of uremic toxins and define the gut-plasma-tissue metabolic axis in CKD.
The immunity related GTPase Irga6 dimerizes in a parallel head to head fashion
The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action. The online version of this article (doi:10.1186/s12915-016-0236-7) contains supplementary material, which is available to authorized users.
Modulation of mTOR Signalling Triggers the Formation of Stem Cell like Memory T Cells
Robust, long-lasting immune responses are elicited by memory T cells that possess properties of stem cells, enabling them to persist long-term and to permanently replenish the effector pools. Thus, stem cell-like memory T (TSCM) cells are of key therapeutic value and efforts are underway to characterize TSCM cells and to identify means for their targeted induction. Here, we show that inhibition of mechanistic/mammalian Target of Rapamycin (mTOR) complex 1 (mTORC1) by rapamycin or the Wnt-β-catenin signalling activator TWS119 in activated human naive T cells leads to the induction of TSCM cells. We show that these compounds switch T cell metabolism to fatty acid oxidation as favoured metabolic programme for TSCM cell generation. Of note, pharmacologically induced TSCM cells possess superior functional features as a long-term repopulation capacity after adoptive transfer. Furthermore, we provide insights into the transcriptome of TSCM cells. Our data identify a mechanism of pharmacological mTORC1 inhibitors, allowing us to confer stemness to human naive T cells which may be significantly relevant for the design of innovative T cell-based cancer immunotherapies. • Immunostimulatory effect of rapamycin on induction of TSCM cells • Previously unknown mTORC1 inhibiting drug effect of the Wnt activator TWS119 • Insights into the metabolic regulation of human CD4 + TSCM cells and into their transcriptome Immunostimulatory effect of rapamycin on induction of TSCM cells Previously unknown mTORC1 inhibiting drug effect of the Wnt activator TWS119 Insights into the metabolic regulation of human CD4 + TSCM cells and into their transcriptome Stem cell-like memory T (TSCM) cells represent the newly identified memory T cell subset, which harbours key therapeutic value by its self-renewal capacity and ability to generate memory and effector T cells. However, the signalling pathways controlling TSCM cell formation remain incompletely understood. Here, we present the induction of CD4 + TSCM cells from highly purified naïve cells by pharmacological inhibition of mTORC1 by either rapamycin or TWS119. We carried out comprehensive transcriptome and metabolic analyses of induced and naturally occurring TSCM cells. Targeted induction of TSCM cells by pharmacological means is highly relevant for the design of novel immunotherapeutic approaches.
Influence of the Biliary System on Biliary Bacteria Revealed by Bacterial Communities of the Human Biliary and Upper Digestive Tracts
Biliary bacteria have been implicated in gallstone pathogenesis, though a clear understanding of their composition and source is lacking. Moreover, the effects of the biliary environment, which is known to be generally hostile to most bacteria, on biliary bacteria are unclear. Here, we investigated the bacterial communities of the biliary tract, duodenum, stomach, and oral cavity from six gallstone patients by using 16S rRNA amplicon sequencing. We found that all observed biliary bacteria were detectable in the upper digestive tract. The biliary microbiota had a comparatively higher similarity with the duodenal microbiota, versus those of the other regions, but with a reduced diversity. Although the majority of identified bacteria were greatly diminished in bile samples, three Enterobacteriaceae genera (Escherichia, Klebsiella, and an unclassified genus) and Pyramidobacter were abundant in bile. Predictive functional analysis indicated enhanced abilities of environmental information processing and cell motility of biliary bacteria. Our study provides evidence for the potential source of biliary bacteria, and illustrates the influence of the biliary system on biliary bacterial communities.
Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma
Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/− CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs. • Cancer stem cell plasticity intersects with EMT to determine therapeutic resistance. • Co-treatment with TGFβ and RA enriches a plastic and drug resistant cancer stem cell sub-population for therapeutic testing. • Thapsigargin specifically targets the plastic and drug resistant cancer stem cell sub-population. Cancer stem cell plasticity intersects with EMT to determine therapeutic resistance. Co-treatment with TGFβ and RA enriches a plastic and drug resistant cancer stem cell sub-population for therapeutic testing. Thapsigargin specifically targets the plastic and drug resistant cancer stem cell sub-population. Cancer cells have the ability to switch between different identities that enhance their ability to proliferate and spread. In this report, we demonstrate that only some cancer cells possess this special ability to switch identity, and that these cells can resist current cancer therapies. We develop a method to enrich the cancer cells with this special ability, so that new drugs can be tested on them, and then use this method to identify a drug that can target these cells. These dangerous cells must be eradicated to enable long-term patient survival, and these are therefore significant findings for human health.
Usefulness of Flow Cytometric Analysis for Detecting Leptomeningeal Diseases in Non Hodgkin Lymphoma
The clinical usefulness of flow cytometry (FCM) for the diagnosis of leptomeningeal diseases (LMD) in non-Hodgkin lymphomas has been suggested in previous studies but needs to be further validated. With this regards, we evaluated the use of FCM for LMD in a series of Korean patients with non-Hodgkin lymphoma. FCM and cytomorphology were conducted using samples obtained from clinically suspected LMD patients, follow-up LMD patients, and those with high risk of developing tumorigenic diseases. We then compared results of FCM and cytomorphology. In total, 55 and 47 CSF samples were analyzed by FCM and cytomorphology, respectively. Of the samples analyzed, 25.5% (14/55) and 12.8% (6/47) were positive by FCM and cytomorphology, respectively. No samples were determined as negative by FCM but positive by cytomorphology. Seven patients were positive only by FCM and negative by cytomorphology, and six among them were clinically confirmed to have LMD either by follow-up cytomorphology or imaging study. We observed a high detection rate of tumor cells by FCM compared with cytomorphology. FCM study can be useful in early sensitive detection of LMD.
Social amoebae trap and kill bacteria by casting DNA nets
Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans. Neutrophils secrete net-like structures made of DNA and anti-microbial peptides, which can trap and kill extracellular pathogens. Here, the authors show that such nets are also produced by so-called Sentinel cells in the multicellular slug stage of the social amoeba Dictyostelium discoideum.
High Speed Imaging of Cavitation around Dental Ultrasonic Scaler Tips
Cavitation occurs around dental ultrasonic scalers, which are used clinically for removing dental biofilm and calculus. However it is not known if this contributes to the cleaning process. Characterisation of the cavitation around ultrasonic scalers will assist in assessing its contribution and in developing new clinical devices for removing biofilm with cavitation. The aim is to use high speed camera imaging to quantify cavitation patterns around an ultrasonic scaler. A Satelec ultrasonic scaler operating at 29 kHz with three different shaped tips has been studied at medium and high operating power using high speed imaging at 15,000, 90,000 and 250,000 frames per second. The tip displacement has been recorded using scanning laser vibrometry. Cavitation occurs at the free end of the tip and increases with power while the area and width of the cavitation cloud varies for different shaped tips. The cavitation starts at the antinodes, with little or no cavitation at the node. High speed image sequences combined with scanning laser vibrometry show individual microbubbles imploding and bubble clouds lifting and moving away from the ultrasonic scaler tip, with larger tip displacement causing more cavitation.
Structure Activity Relationship of Chlorotoxin Like Peptides
Animal venom (e.g., scorpion) is a rich source of various protein and peptide toxins with diverse physio-/pharmaco-logical activities, which generally exert their action via target-specific modulation of different ion channel functions. Scorpion venoms are among the most widely-known source of peptidyl neurotoxins used for callipering different ion channels, such as; Na+, K+, Ca+, Cl−, etc. A new peptide of the chlorotoxin family (i.e., Bs-Tx7) has been isolated, sequenced and synthesized from scorpion Buthus sindicus (family Buthidae) venom. This peptide demonstrates 66% with chlorotoxin (ClTx) and 82% with CFTR channel inhibitor (GaTx1) sequence identities reported from Leiurus quinquestriatus hebraeus venom. The toxin has a molecular mass of 3821 Da and possesses four intra-chain disulphide bonds. Amino acid sequence analysis of Bs-Tx7 revealed the presence of a scissile peptide bond (i.e., Gly-Ile) for human MMP2, whose activity is increased in the case of tumour malignancy. The effect of hMMP2 on Bs-Tx7, or vice versa, observed using the FRET peptide substrate with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher, designed and synthesized to obtain the lowest Km value for this substrate, showed approximately a 60% increase in the activity of hMMP2 upon incubation of Bs-Tx7 with the enzyme at a micromolar concentration (4 µM), indicating the importance of this toxin in diseases associated with decreased MMP2 activity.
MaGuS: a tool for quality assessment and scaffolding of genome assemblies with Whole Genome Profiling™ Data
Scaffolding is an essential step in the genome assembly process. Current methods based on large fragment paired-end reads or long reads allow an increase in contiguity but often lack consistency in repetitive regions, resulting in fragmented assemblies. Here, we describe a novel tool to link assemblies to a genome map to aid complex genome reconstruction by detecting assembly errors and allowing scaffold ordering and anchoring. We present MaGuS (map-guided scaffolding), a modular tool that uses a draft genome assembly, a Whole Genome Profiling™ (WGP) map, and high-throughput paired-end sequencing data to estimate the quality and to enhance the contiguity of an assembly. We generated several assemblies of the Arabidopsis genome using different scaffolding programs and applied MaGuS to select the best assembly using quality metrics. Then, we used MaGuS to perform map-guided scaffolding to increase contiguity by creating new scaffold links in low-covered and highly repetitive regions where other commonly used scaffolding methods lack consistency. MaGuS is a powerful reference-free evaluator of assembly quality and a WGP map-guided scaffolder that is freely available at https://github.com/institut-de-genomique/MaGuS. Its use can be extended to other high-throughput sequencing data (e.g., long-read data) and also to other map data (e.g., genetic maps) to improve the quality and the contiguity of large and complex genome assemblies. The online version of this article (doi:10.1186/s12859-016-0969-x) contains supplementary material, which is available to authorized users.
Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail
An attempt has been made to study the Myxovirus resistant (Mx1) gene polymorphism in Japanese quail. In the present, investigation four fragments viz. Fragment I of 185 bp (Exon 3 region), Fragment II of 148 bp (Exon 5 region), Fragment III of 161 bp (Exon 7 region), and Fragment IV of 176 bp (Exon 13 region) of Mx1 gene were amplified and screened for polymorphism by polymerase chain reaction-single-strand conformation polymorphism technique in 170 Japanese quail birds. Out of the four fragments, one fragment (Fragment II) was found to be polymorphic. Remaining three fragments (Fragment I, III, and IV) were found to be monomorphic which was confirmed by custom sequencing. Overall nucleotide sequence analysis of Mx1 gene of Japanese quail showed 100% homology with common quail and more than 80% homology with reported sequence of chicken breeds. The Mx1 gene is mostly conserved in Japanese quail. There is an urgent need of comprehensive analysis of other regions of Mx1 gene along with its possible association with the traits of economic importance in Japanese quail.
Venom of Parasitoid Pteromalus puparum Impairs Host Humoral Antimicrobial Activity by Decreasing Host Cecropin and Lysozyme Gene Expression
Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Here, we report that Pteromalus puparum venom impairs the antimicrobial activity of its host Pieris rapae. Inhibition zone results showed that bead injection induced the antimicrobial activity of the host hemolymph but that venom inhibited it. The cDNAs encoding cecropin and lysozyme were screened. Relative quantitative PCR results indicated that all of the microorganisms and bead injections up-regulated the transcript levels of the two genes but that venom down-regulated them. At 8 h post bead challenge, there was a peak in the transcript level of the cecropin gene, whereas the peak of lysozyme gene occurred at 24 h. The transcripts levels of the two genes were higher in the granulocytes and fat body than in other tissues. RNA interference decreased the transcript levels of the two genes and the antimicrobial activity of the pupal hemolymph. Venom injections similarly silenced the expression of the two genes during the first 8 h post-treatment in time- and dose-dependent manners, after which the silence effects abated. Additionally, recombinant cecropin and lysozyme had no significant effect on the emergence rate of pupae that were parasitized by P. puparum females. These findings suggest one mechanism of impairing host antimicrobial activity by parasitoid venom.
Premature termination codons in modern human genomes
The considerable range of genetic variation in human populations may partly reflect distinctive processes of adaptation to variable environmental conditions. However, the adaptive genomic signatures remain to be completely elucidated. This research explores candidate loci under selection at the population level by characterizing recently arisen premature termination codons (PTCs), some of which indicate a human knockout. From a total of 7595 participants from two population exome projects, 246 PTCs were found where natural selection has resulted in new alleles with a high frequency (from 1% to 96%) of derived alleles and various levels of population differentiation (FST = 0.00139–0.626). The PTC genes formed protein and regulatory networks limited to 15 biological processes or gene families, of which seven categories were previously unreported. PTC mutations have a strong tendency to be introduced into members of the same gene family, even during modern human evolution, although the exact nature of the selection is not fully known. The findings here suggest the ongoing evolutionary plasticity of modern humans at the genetic level and also partly provide insights into common human knockouts.
Novel small molecules targeting ciliary transport of Smoothened and oncogenic Hedgehog pathway activation
Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics.
Heat Shock Protein 27 is down regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH)
Ballooning degeneration (BD) of hepatocytes is a distinguishing histological feature associated with the progression of nonalcoholic fatty liver disease (NAFLD). Under the assumption that NAFLD severity is associated with metabolic-stress we explored the hypothesis that heat shock 27 kDa protein 1 (HSP27), a protein chaperone involved in stress resistance and cytoskeletal-remodeling, might be deregulated in ballooned hepatocytes. We observed that fasting plasma glucose (fpG) (p = 0.00002), total cholesterol (p = 0.02) and triglycerides (p = 0.01) levels, and female sex (p = 0.01) were significantly associated with the presence of BD. A logistic regression model showed that BD was independently associated with fpG (p = 0.002); OR per unit of glucose concentration 1.05, 95% confidence interval 1.02–1.09. Furthermore, BD was associated with a significant 2.24-fold decrease in the expression level of HSP27-mRNA in comparison with absence of ballooning, p = 0.002. Ballooned hepatocytes showed very low HSP27 immunoreactivity compared with hepatocyes without ballooning (p = 0.009); HSP27 immunoreactivity was inversely correlated with fpG levels (R: −0.49, p = 0.01). In conclusion, BD is associated with down-regulation of liver HSP27 gene and protein expression, suggesting that ballooned hepatocytes fail to ensure a robust physiological response to metabolic-induced stress.
T Cell Response in Patients with Implanted Biological and Mechanical Prosthetic Heart Valves
The study was aimed at assessing T cell subsets of peripheral blood from recipients of long-term functioning (more than 60 months) biological and mechanical heart valve prostheses. The absolute and relative number of CD4 and CD8 T cell subsets was analyzed: naïve (N, CD45RA+CD62L+), central memory (CM, CD45RA−CD62L+), effector memory (EM, CD45RA−CD62L−), and terminally differentiated CD45RA-positive effector memory (TEMRA, CD45RA+CD62L−) in 25 persons with biological and 7 with mechanical prosthesis compared with 48 apparently healthy volunteers. The relative and absolute number of central memory and naïve CD3+CD8+ in patients with biological prosthesis was decreased (p < 0.001). Meanwhile the number of CD45RA+CD62L−CD3+CD8+ and CD3+CD4+ was increased (p < 0.001). Patients with mechanical prosthesis had increased absolute and relative number of CD45RA+CD62L−CD3+CD8+ cells (p = 0.006). Also the relative number of CD3+CD4+ cells was reduced (p = 0.04). We assume that altered composition of T cell subsets points at development of xenograft rejection reaction against both mechanical and biological heart valve prostheses.
Whole Genome Sequencing for Public Health Surveillance of Shiga Toxin Producing Escherichia coli Other than Serogroup O157
Shiga toxin-producing Escherichia coli (STEC) are considered to be a significant threat to public health due to the severity of gastrointestinal symptoms associated with human infection. In England STEC O157 is the most commonly detected STEC serogroup, however, the implementation of PCR at local hospital laboratories has resulted in an increase in the detection of non-O157 STEC. The aim of this study was to evaluate the use of whole genome sequencing (WGS) for routine public health surveillance of non-O157 STEC by comparing this approach to phenotypic serotyping and PCR for subtyping the stx-encoding genes. Of the 102 isolates where phenotypic and genotypic serotyping could be compared, 98 gave fully concordant results. The most common non-O157 STEC serogroups detected were O146 (22) and O26 (18). All but one of the 38 isolates that could not be phenotypically serotyped (designated O unidentifiable or O rough) were serotyped using the WGS data. Of the 73 isolates where a flagella type was available by traditional phenotypic typing, all results matched the H-type derived from the WGS data. Of the 140 sequenced non-O157 isolates, 52 (37.1%) harboured stx1 only, 42 (30.0%) had stx2 only, 46 (32.9%) carried stx1 and stx2. Of these, stx subtyping PCR results were available for 131 isolates and 121 of these had concordant results with the stx subtype derived from the WGS data. Of the 10 discordant results, non-specific primer binding during PCR amplification, due to the similarity of the stx2 subtype gene sequences was the most likely cause. The results of this study showed WGS provided a reliable and robust one-step process for characterization of STEC. Deriving the full serotype from WGS data in real time has enabled us to report a higher level of strain discrimination while stx subtyping provides data on the pathogenic potential of each isolate, enabling us to predict clinical outcome of each case and to monitor the emergence of hyper-virulent strains.
Biological and molecular characterization of classical swine fever challenge virus from India
The aim of this study was biological and molecular characterization of classical swine fever (CSF) challenge virus from India. CSF challenge virus maintained at Division of Biological standardization was experimentally infected to two seronegative piglets. The biological characterization was done by clinical sign and symptoms along with postmortem findings. For molecular characterization 5’-nontranslated region, E2 and NS5B regions were amplified by reverse transcription polymerase chain reaction and sequenced. The sequences were compared with that of reference strains and the local field isolates to establish a phylogenetic relation. The virus produced symptoms of acute disease in the piglets with typical post-mortem lesions. Phylogenetic analysis of the three regions showed that the current Indian CSF Challenge virus is having maximum similarity with the BresciaX strain (USA) and Madhya Pradesh isolate (India) and is belonging to subgroup 1.2 under Group 1. Based on biological and molecular characterization of CSF challenge virus from India is described as a highly virulent virus belonging to subgroup 1.2 under Group 1 along with some field isolates from India and Brescia strain.
The Lateralization of Intrinsic Networks in the Aging Brain Implicates the Effects of Cognitive Training
Lateralization of function is an important organization of the human brain. The distribution of intrinsic networks in the resting brain is strongly related to cognitive function, gender and age. In this study, a longitudinal design with 1 year’s duration was used to evaluate the cognitive training effects on the lateralization of intrinsic networks among healthy older adults. The subjects were divided into two groups randomly: one with multi-domain cognitive training over 3 months and the other as a wait-list control group. Resting state fMRI data were acquired before training and 1 year after training. We analyzed the functional lateralization in 10 common resting state fMRI networks. We observed statically significant training effects on the lateralization of two important RSNs related to high-level cognition: right- and left- frontoparietal networks (FPNs). The lateralization of the left-FPN was retained especially well in the training group but decreased in the control group. The increased lateralization with aging was observed in the cerebellum network (CereN), in which the lateralization was significantly increased in the control group, although the same change tendency was observed in the training group. These findings indicate that the lateralization of the high-level cognitive intrinsic networks is sensitive to multi-domain cognitive training. This study provides neuroimaging evidence to support the hypothesis that cognitive training should have an advantage in preventing cognitive decline in healthy older adults.
Modulatory Action by the Serotonergic System: Behavior and Neurophysiology in Drosophila melanogaster
Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity.
Proteomic dataset of the organohalide respiring bacterium Dehalococcoides mccartyi strain CBDB1 grown on hexachlorobenzene as electron acceptor
The proteome of the anaerobic organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 was analyzed by nano liquid chromatography coupled to mass spectrometry (LC-MS/MS). Two different preparation methods, (i) in-solution and (ii) in-gel proteolytic digestion were assessed to elucidate the core and the functional proteome of bacterial cultures grown in synthetic anaerobic medium with hexachlorobenzene as sole electron acceptor. A detailed analysis of the data presented is available (Schiffmann et al., 2014) [1].
Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus
The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.
Genome wide transcriptome profiling of nitrogen fixation in Paenibacillus sp. WLY78
Diazotrophic (nitrogen-fixing) Gram-positive and endospore-formed Paenibacillus spp. have potential uses as a bacterial fertilizer in agriculture. The transcriptional analysis of nitrogen fixation in Paenibacillus is lacking, although regulation mechanisms of nitrogen fixation have been well studied in Gram-negative diazotrophs. Here we report a global transcriptional profiling analysis of nitrogen fixation in Paenibacillus sp. WLY78 cultured under N2-fixing condition (without O2 and NH4+) and non-N2-fixing condition (air and 100 mM NH4+). The nif (nitrogen fixation) gene operon composed of 9 genes (nifBHDKENXhesAnifV) in this bacterium was significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition, indicating that nif gene transcription is strictly controlled by NH4+ and O2. qRT-PCR confirmed that these nif genes were differently expressed. Non-nif genes specifically required in nitrogen fixation, such as mod, feoAB and cys encoding transporters of Mo, Fe and S atoms, were coordinately transcribed with nif genes in N2-fixing condition. The transcript abundance of suf operon specific for synthesis of Fe-S cluster was up-regulated in N2-fixing condition, suggesting that Sul system, which takes place of nifS and nifU, plays important role in the synthesis of nitrogenase. We discover potential specific electron transporters which might provide electron from Fe protein to MoFe protein of nitrogenase. The glnR whose predicted protein might mediate nif transcription regulation by NH4+ is significantly up-regulated in N2-fixing condition. The transcription levels of nitrogen metabolism and anaerobic respiration were also analyzed. The nif gene operon (nifBHDKENXhesAnifV) in Paenibacillus sp. WLY78 is significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition. Non-nif genes specifically required in nitrogen fixation were also significantly up-regulated in N2-fixing condition. Fur and Fnr which are involved in anaerobic regulation and GlnR which might mediate nif gene transcription regulation by NH4+ were significantly up-regulated in N2-fixing condition. This study provides valuable insights into nitrogen fixation process and regulation in Gram-positive firmicutes. The online version of this article (doi:10.1186/s12866-016-0642-6) contains supplementary material, which is available to authorized users.
Cardiac Light Sheet Fluorescent Microscopy for Multi Scale and Rapid Imaging of Architecture and Function
Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.