Vascular smooth muscle TRPC3 channels facilitate the inverse hemodynamic response during status epilepticus

Human status epilepticus (SE) is associated with a pathological reduction in cerebral blood flow termed the inverse hemodynamic response (IHR). Canonical transient receptor potential 3 (TRPC3) channels are integral to the propagation of seizures in SE, and vascular smooth muscle cell (VSMC) TRPC3 channels participate in vasoconstriction. Therefore, we hypothesize that cerebrovascular TRPC3 channels may contribute to seizure-induced IHR. To examine this possibility, we developed a smooth muscle-specific TRPC3 knockout (TRPC3smcKO) mouse. To quantify changes in neurovascular coupling, we combined laser speckle contrast imaging with simultaneous electroencephalogram recordings. Control mice exhibited multiple IHRs, and a limited increase in cerebral blood flow during SE with a high degree of moment-to-moment variability in which blood flow was not correlated with neuronal activity. In contrast, TRPC3smcKO mice showed a greater increase in blood flow that was less variable and was positively correlated with neuronal activity. Genetic ablation of smooth muscle TRPC3 channels shortened the duration of SE by eliminating a secondary phase of intense seizures, which was evident in littermate controls. Our results are consistent with the idea that TRPC3 channels expressed by cerebral VSMCs contribute to the IHR during SE, which is a critical factor in the progression of SE.Fil: Cozart, Michael A.. University of Arkansas for Medical Sciences; Estados UnidosFil: Phelan, Kevin D.. University of Arkansas for Medical Sciences; Estados UnidosFil: Wu, Hong. University of Arkansas for Medical Sciences; Estados UnidosFil: Mu, Shengyu. University of Arkansas for Medical Sciences; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rusch, Nancy J.. University of Arkansas for Medical Sciences; Estados UnidosFil: Zheng, Fang. University of Arkansas for Medical Sciences; Estados Unido

Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study.

BACKGROUND: The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and containment strategies. METHODS: In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years. Transmission dynamics, infectiousness, and drug susceptibility were analysed in a subset of patients from the Western Cape using whole-genome sequencing (WGS; n=149), a cough aerosol sampling system (CASS; n=26), and phenotypic testing for 18 drugs (n=179). FINDINGS: Between Oct 1, 2008, and Oct 31, 2012, we enrolled and followed up 273 patients for a median of 20·3 months (IQR 9·6-27·8). 203 (74%) had programmatically incurable tuberculosis and unfavourable outcomes (treatment failure, relapse, default, or death despite treatment with a regimen based on capreomycin, aminosalicylic acid, or both). 172 (63%) patients were discharged home, of whom 104 (60%) had an unfavourable outcome. 54 (31%) home-discharged patients had failed treatment, with a median time to death after discharge of 9·9 months (IQR 4·2-17·4). 35 (20%) home-discharged cases were smear-positive at discharge. Using CASS, six (23%) of 26 home-discharged cases with data available expectorated infectious culture-positive cough aerosols in the respirable range (<5 μm), and most reported inter-person contact with suboptimal protective mask usage. WGS identified 17 (19%) of the 90 patients (with available sequence data) that were discharged home before the diagnosis of 20 downstream cases of extensively drug-resistant tuberculosis with almost identical sequencing profiles suggestive of community-based transmission (five or fewer single nucleotide polymorphisms different and with identical resistance-encoding mutations for 14 drugs). 11 (55%) of these downstream cases had HIV co-infection and ten (50%) had died by the end of the study. 22 (56%) of 39 isolates in patients discharged home after treatment failure were resistant to eight or more drugs. However, five (16%) of 31 isolates were susceptible to rifabutin and more than 90% were likely to be sensitive to linezolid, bedaquiline, and delamanid. INTERPRETATION: More than half of the patients with programmatically incurable tuberculosis were discharged into the community where they remained for an average of 16 months, were at risk of expectorating infectious cough aerosols, and posed a threat of transmission of extensively drug-resistant tuberculosis. Urgent action, including appropriate containment strategies, is needed to address this situation. Access to delamanid, bedaquiline, linezolid, and rifabutin, when appropriate, must be accelerated along with comprehensive drug susceptibility testing. FUNDING: UK Medical Research Council, South African Medical Research Council, South African National Research Foundation, European & Developing Countries Clinical Trials Partnership, Oppenheimer Foundation, Newton Fund, Biotechnology and Biological Sciences Research Council, King Abdullah University of Science & Technology

XIAP Protection of Photoreceptors in Animal Models of Retinitis Pigmentosa

BACKGROUND: Retinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels. CONCLUSIONS/SIGNIFICANCE: Our gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments.

International audienceSHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice

Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. METHODS: We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. RESULTS: We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. CONCLUSIONS: We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).This research was supported by the Intramural Research Program of the NIH, Center for Cancer Research, National Cancer Institute and by a National Cancer Institute Strategic Partnering to Evaluate Cancer Signatures (SPECS II) grant (5U01CA157581-05). R.S. was supported by the Dr Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe). D.J.H. was a Kay Kendall Leukaemia Fund Intermediate research fellow. M.K. was supported by the National Institutes of Health Oxford-Cambridge Scholars Program and the Washington University in St. Louis Medical Scientist Training Progra

Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer.

BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases and 23,236 controls, we measured genetic associations of 1,351 SNPs in Treg cell pathway genes with odds of ovarian cancer and tested pathway and gene-level associations, overall and by histotype, for the 25 genes, using the admixture likelihood (AML) method. The most significant single SNP associations were tested for correlation with expression levels in 44 ovarian cancer patients. RESULTS: The most significant global associations for all genes in the pathway were seen in endometrioid ( p = 0.082) and clear cell ( p = 0.083), with the most significant gene level association seen with TGFBR2 ( p = 0.001) and clear cell EOC. Gene associations with histotypes at p < 0.05 included: IL12 ( p = 0.005 and p = 0.008, serous and high-grade serous, respectively), IL8RA ( p = 0.035, endometrioid and mucinous), LGALS1 ( p = 0.03, mucinous), STAT5B ( p = 0.022, clear cell), TGFBR1 ( p = 0.021 endometrioid) and TGFBR2 ( p = 0.017 and p = 0.025, endometrioid and mucinous, respectively). CONCLUSIONS: Common inherited gene variation in Treg cell pathways shows some evidence of germline genetic contribution to odds of EOC that varies by histologic subtype and may be associated with mRNA expression of immune-complex receptor in EOC patients

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The Role of Canonical Transient Receptor Potential Channels in Seizure and Excitotoxicity

Canonical transient receptor potential (TRPC) channels are a family of polymodal cation channels with some degree of Ca2+ permeability. Although initially thought to be channels mediating store-operated Ca2+ influx, TRPC channels can be activated by stimulation of Gq-coupled G-protein coupled receptors, or by an increase in intracellular free Ca2+ concentration. Thus, activation of TRPC channels could be a common downstream event of many signaling pathways that contribute to seizure and excitotoxicity, such as N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ influx, or metabotropic glutamate receptor activation. Recent studies with genetic ablation of various TRPC family members have demonstrated that TRPC channels, in particular heteromeric TRPC1/4 channels and homomeric TRPC5 channels, play a critical role in both pilocarpine-induced acute seizures and neuronal cell death. However, exact underlying mechanisms remain to be fully elucidated, and selective TRPC modulators and antibodies with better specificity are urgently needed for future research