γδ T Cells Are Reduced and Rendered Unresponsive by Hyperglycemia and Chronic TNFα in Mouse Models of Obesity and Metabolic Disease

Epithelial cells provide an initial line of defense against damage and pathogens in barrier tissues such as the skin; however this balance is disrupted in obesity and metabolic disease. Skin γδ T cells recognize epithelial damage, and release cytokines and growth factors that facilitate wound repair. We report here that hyperglycemia results in impaired skin γδ T cell proliferation due to altered STAT5 signaling, ultimately resulting in half the number of γδ T cells populating the epidermis. Skin γδ T cells that overcome this hyperglycemic state are unresponsive to epithelial cell damage due to chronic inflammatory mediators, including TNFα. Cytokine and growth factor production at the site of tissue damage was partially restored by administering neutralizing TNFα antibodies in vivo. Thus, metabolic disease negatively impacts homeostasis and functionality of skin γδ T cells, rendering host defense mechanisms vulnerable to injury and infection

Structural characterization of helitrons and their stepwise capturing of gene fragments in the maize genome

<p>Abstract</p> <p>Background</p> <p>As a newly identified category of DNA transposon, <it>helitrons </it>have been found in a large number of eukaryotes genomes. <it>Helitrons </it>have contributed significantly to the intra-specific genome diversity in maize. Although many characteristics of <it>helitrons </it>in the maize genome have been well documented, the sequence of an intact autonomous <it>helitrons </it>has not been identified in maize. In addition, the process of gene fragment capturing during the transposition of <it>helitrons </it>has not been characterized.</p> <p>Results</p> <p>The whole genome sequences of maize inbred line B73 were analyzed, 1,649 <it>helitron</it>-like transposons including 1,515 helAs and 134 helBs were identified. <it>ZmhelA1</it>, <it>ZmhelB1 </it>and <it>ZmhelB2 </it>all encode an open reading frame (ORF) with intact replication initiator (Rep) motif and a DNA helicase (Hel) domain, which are similar to previously reported autonomous <it>helitrons </it>in other organisms. The putative autonomous <it>ZmhelB1 </it>and <it>ZmhelB2 </it>contain an extra replication factor-a protein1 (RPA1) transposase (RPA-TPase) including three single strand DNA-binding domains (DBD)-A/-B/-C in the ORF. Over ninety percent of maize <it>helitrons </it>identified have captured gene fragments. HelAs and helBs carry 4,645 and 249 gene fragments, which yield 2,507 and 187 different genes respectively. Many <it>helitrons </it>contain mutilple terminal sequences, but only one 3'-terminal sequence had an intact "CTAG" motif. There were no significant differences in the 5'-termini sequence between the veritas terminal sequence and the pseudo sequence. <it>Helitrons </it>not only can capture fragments, but were also shown to lose internal sequences during the course of transposing.</p> <p>Conclusions</p> <p>Three putative autonomous elements were identified, which encoded an intact Rep motif and a DNA helicase domain, suggesting that autonomous <it>helitrons </it>may exist in modern maize. The results indicate that gene fragments captured during the transposition of many <it>helitrons </it>happen in a stepwise way, with multiple gene fragments within one <it>helitron </it>resulting from several sequential transpositions. In addition, we have proposed a potential mechanism regarding how <it>helitrons </it>with multiple termini are generated.</p

TSC1/2 Signaling Complex Is Essential for Peripheral Naïve CD8+ T Cell Survival and Homeostasis in Mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral naïve CD8+ T cells but not CD4+ T cells were severely reduced. Tsc1 KO naïve CD8+ T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8+ T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO naïve CD8+T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO naïve CD8+T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral naïve CD8+ T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment

Characterization of the Metabolic Phenotype of Rapamycin-Treated CD8+ T Cells with Augmented Ability to Generate Long-Lasting Memory Cells

Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized.. than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells.Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy

A Critical Analysis of Atoh7 (Math5) mRNA Splicing in the Developing Mouse Retina

The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3′RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although ∼10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments

Mechanisms of T cell organotropism

F.M.M.-B. is supported by the British Heart Foundation, the Medical Research Council of the UK and the Gates Foundation

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta