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Next generation sequencing methods represent the latest era of molecular genetic diagnostics. After a general introduction on primary immunodeficiencies, the author summarizes the importance of molecular genetic studies, especially next generation sequencing in the diagnosis of primary immunodeficiencies. Another purpose of the manuscript is to give a brief summary on the methodological basis of next generation sequencing. The author analyzes the advantages and disadvantages of primary immunodeficiency gene-panel sequencing and whole-exome and wholegenome sequencing. Primary immunodeficiency genes and diseases recognized by next generation sequencing is also summarized. Finally, the author emphasizes the indispensability of gene level diagnostics in primary immunodeficiencies and presents the results achieved in this field in Hungary.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes(1)(-11), but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far(12)(,)(13). Although single splicing events have been described for <= 200 single cells with statistical confidence(14,)(15), full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3' sequencing enables the identification of cellular subtypes(16)(-21), but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites(6)(-9.)(22)(,)(23) We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.

Next-generation sequencing (NGS) generates large amounts of genomic data and reveals about 20 000 genetic coding variants per individual studied. Several mutation damage prediction scores are available to prioritize variants, but there is currently no application to help investigators to determine the relevance of the candidate genes and variants quickly and visually from population genetics data and deleteriousness scores. Here, we present PopViz, a user-friendly, rapid, interactive, mobile-compatible webserver providing a gene-centric visualization of the variants of any human gene, with (i) population-specific minor allele frequencies from the gnomAD population genetic database; (ii) mutation damage prediction scores from CADD, EIGEN and LINSIGHT and (iii) amino-acid positions and protein domains. This application will be particularly useful in investigations of NGS data for new disease-causing genes and variants, by reinforcing or rejecting the plausibility of the candidate genes, and by selecting and prioritizing, the candidate variants for experimental testing.

Background: There has been a dramatic shift in use of bariatric procedures, but little is known about their long-term comparative effectiveness. Objective: To compare weight loss and safety among bariatric procedures. Design: Retrospective observational cohort study, January 2005 to September 2015. (ClinicalTrials.gov: NCT02741674) Setting: 41 health systems in the National Patient-Centered Clinical Research Network. Participants: 65 093 patients aged 20 to 79 years with body mass index (BMI) of 35 kg/m(2) or greater who had bariatric procedures. Intervention: 32 208 Roux-en-Y gastric bypass (RYGB), 29 693 sleeve gastrectomy (SG), and 3192 adjustable gastric banding (AGB) procedures. Measurements: Estimated percent total weight loss (TWL) at 1, 3, and 5 years; 30-day rates of major adverse events. Results: Total numbers of eligible patients with weight measures at 1, 3, and 5 years were 44 978 (84%), 20 783 (68%), and 7159 (69%), respectively. Thirty-day rates of major adverse events were 5.0% for RYGB, 2.6% for SG, and 2.9% for AGB. One- year mean TWLs were 31.2% (95% CI, 31.1% to 31.3%) for RYGB, 25.2% (CI, 25.1% to 25.4%) for SG, and 13.7% (CI, 13.3% to 14.0%) for AGB. At 1 year, RYGB patients lost 5.9 (CI, 5.8 to 6.1) percentage points more weight than SG patients and 17.7 (CI, 17.3 to 18.1) percentage points more than AGB patients, and SG patients lost 12.0 (CI, 11.6 to 12.5) percentage points more than AGB patients. Five-year mean TWLs were 25.5% (CI, 25.1% to 25.9%) for RYGB, 18.8% (CI, 18.0% to 19.6%) for SG, and 11.7% (CI, 10.2% to 13.1%) for AGB. Patients with diabetes, those with BMI less than 50 kg/m(2), those aged 65 years or older, African American patients, and Hispanic patients lost less weight than patients without those characteristics. Limitation: Potential unobserved confounding due to nonrandomized design; electronic health record databases had missing outcome data. Conclusion: Adults lost more weight with RYGB than with SG or AGB at 1, 3, and 5 years; however, RYGB had the highest 30-day rate of major adverse events. Small subgroup differences in weight loss outcomes were observed.

Population densities of a species measured in different locations are often correlated over time, a phenomenon referred to as synchrony. Synchrony results from dispersal of individuals among locations and spatially correlated environmental variation, among other causes. Synchrony is often measured by a correlation coefficient. However, synchrony can vary with timescale. We demonstrate theoretically and experimentally that the timescale-specificity of environmental correlation affects the overall magnitude and timescale-specificity of synchrony, and that these effects are modified by population dispersal. Our laboratory experiments linked populations of flour beetles by changes in habitat size and dispersal. Linear filter theory, applied to a metapopulation model for the experimental system, predicted the observed timescale-specific effects. The timescales at which environmental covariation occurs can affect the population dynamics of species in fragmented habitats.

PURPOSE. The purpose of the research was to elucidate the role of folic acid (B9) deficiency in the development of nutritional optic neuritis and to characterize the neurophysiological consequences of optic nerve degeneration in the cortical visual system. METHODS. A combined behavioral and electrophysiological approach was applied to study luminance contrast sensitivity in two macaque monkeys affected by nutritional optic neuritis and in two healthy monkeys for comparison. For one monkey, a follow-up approach was applied to compare visual performance before onset of optic neuropathy, during the disease, and after treatment. RESULTS. Optic nerve degeneration developed as a consequence of insufficient dietary intake of folic acid in two exemplars of macaque monkeys. The degeneration resulted in markedly reduced luminance contrast sensitivity as assessed behaviorally. In one monkey, we also measured visual activity in response to varying contrast at the level of single neurons in the cortical visual system and found a striking reduction in contrast sensitivity, as well as a marked increase in the latency of neuronal responses. Prolonged daily folate supplementation resulted in a significant recovery of function. CONCLUSIONS. Folic acid deficiency per se can lead to the development of optic nerve degeneration in otherwise healthy adult animals. The optic nerve degeneration strongly affects contrast sensitivity and leads to a distinct reduction in the strength and velocity of the incoming signal to cortical visual areas of the macaque brain, without directly affecting excitability and functional properties of cortical neurons.

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase. bacteriophage lambda helicase loader (lambda P) complex. We show that five copies of lambda P bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.

We report the molecular, cellular, and clinical features of 5 patients from 3 kindreds with biallelic mutations in the autosomal LIG1 gene encoding DNA ligase 1. The patients exhibited hypogammaglobulinemia, lymphopenia, increased proportions of circulating gamma dT cells, and erythrocyte macrocytosis. Clinical severity ranged from a mild antibody deficiency to a combined immunodeficiency requiring hematopoietic stem cell transplantation. Using engineered LIG1-deficient cell lines, we demonstrated chemical and radiation defects associated with the mutant alleles, which variably impaired the DNA repair pathway. We further showed that these LIG1 mutant alleles are amorphic or hypomorphic, and exhibited variably decreased enzymatic activities, which lead to premature release of unligated adenylated DNA. The variability of the LIG1 genotypes in the patients was consistent with that of their immunological and clinical phenotypes. These data suggest that different forms of autosomal recessive, partial DNA ligase 1 deficiency underlie an immunodeficiency of variable severity.