Identification of a Marine Cyanophage in a Protist Single‐cell Metagenome Assembly

Analysis of microbial biodiversity is hampered by a lack of reference genomes from most bacteria, viruses, and algae. This necessitates either the cultivation of a restricted number of species for standard sequencing projects or the analysis of highly complex environmental DNA metagenome data. Single‐cell genomics (SCG) offers a solution to this problem by constraining the studied DNA sample to an individual cell and its associated symbionts, prey, and pathogens. We used SCG to study marine heterotrophic amoebae related to Paulinella ovalis (A. Wulff) P.W. Johnson, P.E. Hargraves & J.M. Sieburth (Rhizaria). The genus Paulinella is best known for its photosynthetic members such as P. chromatophora Lauterborn that is the only case of plastid primary endosymbiosis known outside of algae and plants. Here, we studied the phagotrophic sister taxa of P. chromatophora that are related to P. ovalis and found one SCG assembly to contain α‐cyanobacterial DNA. These cyanobacterial contigs are presumably derived from prey. We also uncovered an associated cyanophage lineage (provisionally named phage PoL_MC2). Phylogenomic analysis of the fragmented genome assembly suggested a minimum genome size of 200 Kbp for phage PoL_MC2 that encodes 179 proteins and is most closely related to Synechococcus phage S‐SM2. For this phage, gene network analysis demonstrates a highly modular genome structure typical of other cyanophages. Our work demonstrates that SCG is a powerful approach for discovering algal and protist biodiversity and for elucidating biotic interactions in natural samples.     

High Mobility Group Protein N5 (HMGN5) and Lamina-associated Polypeptide 2α (LAP2α) Interact and Reciprocally Affect Their Genome-wide Chromatin Organization

The interactions of nuclear lamins with the chromatin fiber play an important role in regulating nuclear architecture and chromatin function; however, the full spectrum of these interactions is not known. We report that the N-terminal domain of the nucleosome-binding protein HMGN5 interacts with the C-terminal domain of the lamin-binding protein LAP2α and that these proteins reciprocally alter their interaction with chromatin. Chromatin immunoprecipitation analysis of cells lacking either HMGN5 or LAP2α reveals that loss of either protein affects the genome-wide distribution of the remaining partner. Our study identifies a new functional link between chromatin-binding and lamin-binding proteins.     

Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild‐type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA‐dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum‐sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS‐regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non‐encapsulated wild‐type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

High Adult Sex Ratios and Risky Sexual Behaviors: A Systematic Review

Background

Thirty-four countries worldwide have abnormally high sex ratios (>102 men per 100 women), resulting in over 100 million missing women. Widespread sex selective abortion, neglect of young girls leading to premature mortality, and gendered migration have contributed to these persistent and increasing distortions. Abnormally high adult sex ratios in communities may drive sexually transmitted disease (STD) spread where women are missing and men cannot find stable partners. We systematically reviewed evidence on the association between high community sex ratios and individual sexual behaviors.

Methods and Findings

Seven databases (PubMed, Web of Science, Embase, Scopus, The Cochrane Database of Systematic Reviews, Sociological Abstracts, and PopLINE) were searched without restrictions on time or location. We followed PRISMA guidelines and evaluated quality according to STROBE criteria. 1093 citations were identified and six studies describing 57,054 individuals were included for review. All six studies showed an association between high community sex ratios and individual sexual risk behaviors. In high sex ratio communities, women were more likely to have multiple sex partners and men were more likely to delay first sexual intercourse and purchase sex. Only two studies included STD outcomes.

Conclusions

High community sex ratios were associated with increased individual sexual risk behavior among both men and women. However, none of the studies examined unprotected sex or appropriately adjusted for gendered migration. Further studies are needed to understand the effect of community sex ratios on sexual health and to inform comprehensive STD control interventions.     

Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.     

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.     

Increased Vaspin Levels Are Associated with Beneficial Metabolic Outcome Pre- and Post-Bariatric Surgery

Purpose

Vaspin (visceral-adipose-tissue-derived-serine-protease-inhibitor) is a recently identified adipokine with putative insulin-sensitizing properties. Plasma vaspin responses to surgery-induced weight loss are sparse and contradictory.

Design and Participants

We evaluated changes in vaspin levels and relationship to post-operative outcomes in men (n = 22) and women (n = 55) undergoing biliopancreatic-diversion/duodenal-switch bariatric surgery. Body composition and plasma parameters were measured at baseline, acutely (1 and 5 days) and medium-term (6 and 12 months) post-surgery.

Results

Fasting preoperative vaspin concentrations were comparable in men vs women. The distribution was biphasic (both men and women) with a nadir of 2.5 ng/ml. Subjects were divided into high (≥2.5 ng/mL, HI-group) and low (<2.5 ng/mL, LO-group) vaspin level. Both groups had comparable sex distribution, age and BMI, but the HI-vaspin group had lower insulin, HOMA, and triglyceride and higher HDL-cholesterol, acylation stimulating protein (ASP) and IL-6 levels (all p<0.05). Post-operatively, both groups decreased BMI comparably over 12 months; the HI-vaspin group maintained high vaspin levels, while the LO-vaspin group gradually increased their levels with weight loss over 12 months. The HI-vaspin group maintained a better glucose, insulin, HOMA, fructosamine, HDL-cholesterol, and triglyceride profile throughout. The HI-vaspin group also had higher gamma-glutamyltransferase and ASP profiles. Finally, baseline vaspin level inversely correlated significantly with baseline and 12-month insulin, HOMA, triglyceride and positively correlated with HDL and ASP. Twelve-month vaspin also correlated similarly, including an inverse correlation with BMI.

Conclusion

Globally, this study supports the concept of vaspin as a beneficial adipokine in obesity, which may potentially lead to possible therapeutic targets.     

Detection of recombination events,haplotype reconstruction and imputation of sires using half-sib SNP genotypes

Background

Identifying recombination events and the chromosomal segments that constitute a gamete is useful for a number of applications in genomic analyses. In livestock, genotypic data are commonly available for half-sib families. We propose a straightforward but computationally efficient method to use single nucleotide polymorphism marker genotypes on half-sibs to reconstruct the recombination and segregation events that occurred during meiosis in a sire to form the haplotypes observed in its offspring. These meiosis events determine a block structure in paternal haplotypes of the progeny and this can be used to phase the genotypes of individuals in single half-sib families, to impute haplotypes of the sire if they are not genotyped or to impute the paternal strand of the offspring’s sequence based on sequence data of the sire.

Methods

The hsphase algorithm exploits information from opposing homozygotes among half-sibs to identify recombination events, and the chromosomal regions from the paternal and maternal strands of the sire (blocks) that were inherited by its progeny. This information is then used to impute the sire’s genotype, which, in turn, is used to phase the half-sib family. Accuracy (defined as R²) and performance of this approach were evaluated by using simulated and real datasets. Phasing results for the half-sibs were benchmarked to other commonly used phasing programs – AlphaPhase, BEAGLE and PedPhase 3.

Results

Using a simulated dataset with 20 markers per cM, and for a half-sib family size of 4 and 40, the accuracy of block detection, was 0.58 and 0.96, respectively. The accuracy of inferring sire genotypes was 0.75 and 1.00 and the accuracy of phasing was around 0.97, respectively. hsphase was more robust to genotyping errors than PedPhase 3, AlphaPhase and BEAGLE. Computationally, hsphase was much faster than AlphaPhase and BEAGLE.

Conclusions

In half-sib families of size 8 and above, hsphase can accurately detect block structure of paternal haplotypes, impute genotypes of ungenotyped sires and reconstruct haplotypes in progeny. The method is much faster and more accurate than other widely used population-based phasing programs. A program implementing the method is freely available as an R package (hsphase).