Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers

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Highlights? RAG-dependent monoallelic loop formation is linked to monoallelic RAG cleavage ? RAG enrichment, cleavage, and higher-order loop formation occur at the 3′ end of Tcra ? Looping out is a determinant of directed RAG targeting ? ATM-mediated control of looping out is linked to the maintenance of genome stability     

Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes

Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2–1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10⁴–10⁵ genomes ml⁻¹ for the samples from the photic zone and 10²–10³ genomes ml⁻¹ for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.     

Age and menopause affect the expression of specific cytokines/chemokines in plasma and cervical lavage samples from female sex workers in Nairobi,Kenya

Background

Aging of the immune system, known as immunosenescence, is associated with profound changes in both innate and adaptive immune responses, resulting in increased susceptibility to infection and a decreased ability to respond to vaccination. The purpose of this study was to investigate the effect of age and menopause on the expression of 22 different cytokines/chemokines in both plasma and cervical lavage samples from female sex-worker cohort from Nairobi, Kenya (age range 20–65).

Results

Cytokine/chemokine levels were measured using a Miliplex multiplex assay (Millipore). We found that age positively correlated with MCP-1 (p?=?0.0002) and IP-10 (p?=?0.03) systemic cytokine expression, and that women over 50 expressed the highest levels of these cytokines, but also had elevated expression of MIG (ANOVA p?=?0.0096) and MIP-3β(ANOVA p?=?0.0434). We also found that IL-8 (p?=?0.047) and sCD40L (p?=?0.01) systemic expression negatively correlated with age. Further, MIG (p?=?0.0081) and MCP-1 (p?=?0.0157) were present at higher levels in post-menopausal women suggesting a potential estrogen dependant systemic regulation of these cytokines. In cervical lavage samples, age did not directly correlate with the expression of any of the tested cytokines/chemokines, however sIL-2Rα (ANOVA p?=?0.0170) and IL-15 (ANOVA p?=?0.0251)were significantly higher in women over 50. Menopause was shown to have a more profound effect on cytokine expression in the cervical mucosa with MIG (p?=?0.0256), MIP-3α (p?=?0.0245), IL-1β (p?=?0.0261), IL-6 (p?=?0.0462), IL-8 (p?=?0.007), IP-10 (p?=?0.0357) and MCP-1 (p?=?0.0427) all significantly under-expressed in post-menopausal women.

Conclusions

This study demonstrates that aging and menopause-associated hormonal changes are associated with significant changes in systemic and mucosal cytokine/chemokine expression, which may have implications for the age-related decline in the ability to fight against infections.

     

Immunological and nutritional factors in elderly people in low-level care and their association with mortality

Background

This study examines associations between markers of nutritional status and lymphocyte subsets and seeks to determine if lymphocyte profile is predictive of survival in elderly Australians residing in aged care facilities. Aged yet still ambulatory subjects (n?=?88, 73% female) living in low-level care and requiring minimal assistance were studied for 143 weeks. At baseline when participants were aged (mean?±?SD) 86.0?±?5.9 years, dietary intake was determined by 3-day weighed food record, body composition was assessed by dual energy X-ray absorptiometry (DXA) and a venous blood sample was taken.

Results

At baseline assessment, study participants were consuming nutrient-poor diets and most had symptoms of chronic disease. Although overweight, 40% exhibited sarcopenia. Markers of nutritional status did not relate closely to immune cell numbers (absolute or relative), which on average were within the normal range. Men had lower numbers of CD3⁺CD4⁺ cells (CD4⁺ T cells), a higher proportion of CD3^? CD16^± CD56^± (natural killer (NK) cells) and a higher ratio of NK: CD4⁺ T cells than women (all P?<?0.05). The main age-related changes evident were decreased T cells, particularly low CD4⁺ T cell counts, and increased numbers of CD19⁺ (B-cell) and NK cells. During the 143 week duration of follow-up, about one quarter of the study participants died, with death more likely in men than women (P?<?0.01). Poor survival was predicted by the presence of decreased numbers of CD4⁺ T cells (hazard ratio (HR) 0.919, P?<?0.01) and expanded numbers of NK cells (HR 1.085, P?<?0.05) in the blood, and therefore the presence of a high NK: CD4⁺ T cell ratio (HR 30.521, P?<?0.01).

Conclusions

The NK: CD4⁺ T cell ratio may potentially have clinical utility for predicting longevity in elderly populations. Further studies are needed in other elderly populations to confirm this finding.

     

Germline Mutations in the Polyposis-Associated Genes BMPR1A,SMAD4, PTEN,MUTYH and GREM1 Are Not Common in Individuals with Serrated Polyposis Syndrome

Background

Recent reports have observed that individuals with serrated polyps, some of whom meet the clinical diagnostic criteria for Serrated Polyposis Syndrome (SPS), are among those who carry germline mutations in genes associated with polyposis syndromes including; (1) genes known to underlie hamartomatous polyposes (SMAD4, BMPR1A, and PTEN), (2) MUTYH-associated polyposis and (3) GREM1 in Hereditary Mixed Polyposis Syndrome (HMPS). The aim of this study was to characterise individuals fulfilling the current WHO criteria for SPS for germline mutations in these polyposis-associated genes.

Methods

A total of 65 individuals with SPS (fulfilling WHO criteria 1 or 3), were recruited to the Genetics of Serrated Neoplasia study between 2000 and 2012, through multiple Genetics or Family Cancer Clinics within Australia, or from the New Zealand Familial Gastrointestinal Cancer Service. Individuals with SPS were tested for coding mutations and large deletions in the PTEN, SMAD4, and BMPR1A genes, for the MUTYH variants in exons 7 (Y179C) and 13 (G396D), and for the duplication upstream of GREM1.

Results

We found no variants that were likely to be deleterious germline mutations in the SPS cases in the PTEN, SMAD4, and BMPR1A genes. A novel variant in intron 2 (c.164+223T>C) of PTEN was identified in one individual and was predicted by in silico analysis to have no functional consequences. One further individual with SPS was found to be mono-allelic for the MUTYH G396D mutation. No individuals carried the recently reported duplication within GREM1.

Conclusions

Genes involved in the gastrointestinal hamartomatous polyposis, Hereditary Mixed Polyposis Syndrome and MUTYH-associated polyposis syndromes are not commonly altered in individuals with SPS.     

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.     

Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.