A novel antisense lncRNA, ARBAG harboring an RNA destabilizing GWAS variant for C-peptide dictates the transcript isoforms of GABRA6 in cerebellum.

Human disease-associated genetic variations often map to long non-coding RNA (lncRNA) genes; however, elucidation of their functional impact is challenging. We previously identified a new genetic variant rs4454083 (A/G) residing in exon of an uncharacterized lncRNA ARBAG that strongly associates with plasma levels of C-peptide, a hormone that regulates insulin bioavailability. On the opposite strand, rs4454083 also corresponds to an intron of a cerebellum-specific GABA receptor subunit gene GABRA6 that mediates strengthening of inhibitory synapses by insulin. Here, we show that alleles of rs4454083 modulate transcript levels of the antisense gene, ARBAG, which then controls the expression of the sense gene, GABRA6. Predisposing to low C-peptide, GG (a minor allele genotype across ethnicities) stabilizes ARBAG lncRNA causing higher transcript levels in cerebellum. ARBAG lncRNA abundance leads to cleavage of GABRA6 mRNA at the complementary region, resulting in a dysfunctional GABRA6 protein that would not be recruited for synapse strengthening. Together, our findings in human cerebellar cell-line and induced Pluripotent Stem Cells (iPSCs) demonstrate biological role of a novel lncRNA in determining the ratio of mRNA isoforms of a protein-coding gene and the ability of an embedded variant in modulating lncRNA stability leading to inter-individual differences in protein expression.

Gestational glucose intolerance (GGI) and gestational diabetes mellitus (GDM) among antenatal women attending urban community health centers of Lucknow: A cross-sectional study.

Background: Gestational diabetes mellitus (GDM) is an emerging public health concern in India, which has detrimental effects on both the mother and the baby. The data on prevalence of GDM was unavailable at secondary urban health facilities, from where a majority of pregnant women seek antenatal services, and the following study identifies this burden. Methods: A cross-sectional study was conducted from May 2019 to June 2020 among pregnant women attending the antenatal outpatient department (OPD) at secondary level health facilities in urban Lucknow. A semi-structured interview schedule was administered to the study subjects for collecting the relevant information and 75 g of oral glucose tolerance test was performed irrespective of the meal. The cut-off points taken for the diagnosis of GDM and gestational glucose intolerance (GGI) was as per the guidelines of the Ministry of Health and Family Welfare for diagnosis of GGI/GDM. Results: The overall prevalence of GDM and GGI in the study was 11.6% and 16.8%, respectively. Three-fourth of the women (22/29) were diagnosed with GDM in the second trimester of pregnancy. The prevalence of GDM (16.7%) was significantly higher in pregnant women aged more than 25 years and in those who were overweight. Mean birth weight (3.2 ± 8.1 kg) of the babies was significantly higher in the women with GDM. Among the fetal complications was respiratory distress observed among 28 pregnant women and 31% of them had GDM and this was statistically significant. Conclusion: The prevalence of GGI and GDM was found 16.8% and 11.6%, respectively. Gestational age, pre-pregnancy weight, pre-pregnancy BMI, weight gain during the pregnancy, family history of diabetes. PCOS, macrosomia and GDM in prior pregnancies was found to significant with GDM in the study.

Multi-species single-cell transcriptomic analysis of ocular compartment regulons.

The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.

Diversification of reprogramming trajectories revealed by parallel single-cell transcriptome and chromatin accessibility sequencing.

Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery.

Parallel bimodal single-cell sequencing of transcriptome and chromatin accessibility.

Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)-centered upstream regulatory networks for various cell states.

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

Correction: Delivery and evaluation of participatory education for animal keepers led by veterinarians and para-veterinarians around the Kanha Tiger Reserve, Madhya Pradesh, India.

[This corrects the article DOI: 10.1371/journal.pone.0200999.].

Delivery and evaluation of participatory education for animal keepers led by veterinarians and para-veterinarians around the Kanha Tiger Reserve, Madhya Pradesh, India.

AIM: We aim to investigate local perceptions of animal health challenges; current animal health knowledge; and methods to provide effective, relevant education to animal keepers in the Kanha Tiger Reserve area. MATERIALS AND METHODS: A farmer education programme was undertaken in the Kanha Tiger Reserve area. Local animal health priorities were investigated through participatory village meetings (n = 38), individual animal keeper questionnaires (n = 100) and a written survey of local paravets (n = 16). Educational interventions were: veterinary surgeon led education meeting (VE); paravet led education meeting (PVE); distribution of printed materials (PM). 230 village meetings were carried out across 181 villages, contacting 3791 animal keepers. 20 villages received printed materials. Information was gathered on perceptions of local animal health challenges and current remedies. Efficacy of knowledge transfer was assessed four to five months later using a purposeful sample of 38 villages. RESULTS: Group meetings identified ticks (35/38), foot and mouth disease (FMD) (31/38) and diarrhoea (30/38) as the greatest animal health challenges. Individual interviews identified haemorrhagic septicaemia (HS) (87/100), blackquarter (BQ) (66/100) and plastic ingestion (31/100). Paravets identified FMD (7/16), BQ (6/16) and HS (6/16), and also indicated that animal husbandry and socio-economic factors were important. Current treatments were primarily home remedies and herbalism, but also included contacting a paravet, use of pharmaceuticals and faith healing. Animal treatment knowledge prior to intervention was not significantly different between groups (P = 0.868). Following intervention animal health knowledge was assessed: PVE performed better than controls (P = 0.001) and PM (P = 0.003); VE performed better than controls (P = 0.009). There was no significant difference between VE and PVE (P = 0.666) nor PM and controls (P = 0.060). CONCLUSIONS AND RECOMMENDATIONS: Open access participatory village meetings are an effective way to provide animal health education. In this region distribution of posters and leaflets did not appear to be an effective way to contact animal keepers. Meetings led by paravets can be as effective as those led by veterinarians and paravets can rapidly and sustainably contact large numbers of animal keepers. Investigation of the local animal health situation is essential to ensure education is relevant and accessible to intended recipients. Interventions must be carefully planned to maximise engagement of all sections of the community, particularly women.

Regulation of ERVs in pluripotent stem cells and reprogramming.

Recent advances in our understanding of endogenous retroviruses (ERVs) regulation and its functional aspects have provided us with vast power to unravel its role in the host's genome. Co-evolutionary model of ERVs and Kruppel associated box-Zinc Finger Proteins (KRAB-ZFPs) provides a deeper knowledge of how the genome is shaped during the course of evolution. However, the role of ERVs in normal cellular function still remains an enigma. Here we review studies in recent years with a focus on the role of ERVs in maintaining stemness and cell fate reprogramming, along with the recent discoveries of novel regulatory factors which have been shown to mediate ERV expression in both canonical and non-canonical pathways.

Molecular insights into RNA-binding properties of Escherichia coli-expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

Stability of Two Antifungal Agents, Fluconazole and Miconazole, Compounded in HUMCO RECURA Topical Cream to Determine Beyond-Use Date.

A novel compounding vehicle (RECURA) has previously been proven to penetrate the nail bed when compounded with the antifungal agent miconazole or fluconazole, providing for an effective treatment for onychomycosis. In this study, miconazole and fluconazole were compounded separately in RECURA compounding cream, and they were tested at different time points (0, 7, 14, 28, 45, 60, 90, and 180 days) to determine the beyond-use date of those formulations. The beyond-use date testing of both formulations (10% miconazole in RECURA and 10% fluconazole in RECURA) proved them to be physically, chemically, and microbiologically stable under International Conference of Harmonisation controlled room temperature (25°C ± 2°C/60% RH ±5%) for at least 180 days from the date of compounding. Stability-indicating analytical method validation was completed for the simultaneous determination of miconazole and fluconazole in RECURA base using high-performance liquid chromatography coupled with photodiode array detector prior to the study.

Presence and transport of the antimicrobials triclocarban and triclosan in a wastewater-dominated stream and freshwater environment.

The presence of the antimicrobials triclocarban (TCC) and triclosan (TCS)in Fountain Creek, a wastewater-dominated stream, and the Arkansas River, Colorado, USA was measured in the surface water, suspended sediments, and bed sediments during spring runoff (high flow) and summer base flow (low flow) conditions. Fountain Creak is a tributary of the Arkansas River. Passive polar organic chemical integrative samplers (POCIS) were used along with active sampling (water grab samples) to measure and TCS concentrations in these surface waters. The concentration of TCC and TCS, based on POCIS measurements, ranged from 4.5 to 47.3 ng/L and 3.9 to 28.3 ng/L, respectively, at the five sample sites monitored in this study under both flow conditions. The range of concentrations of TCC and TCS in suspended sediments was 0.7-57.3 ng/g and 0.7-13.3 ng/g, respectively, and was closely tied to the quantity of organic carbon in the suspended sediment, which ranged from 1.6 to 14.5%. The quantity of organic carbon in suspended sediment during the summer base flow was influenced by runoff from the burn area of a large forest fire that occurred between the two sampling periods. The primary transport mechanism of TCC and TCS in these surface waters was in the dissolved phase, with 64-99% of TCC and 68-99% of TCS transported in the dissolved phase. The total amount of TCS and TCC in bed-sediments was relatively low, with the maximum amount at any one site being 0.38 ± 0.15 ng/g TCS and 4.09 ± 5.26 ng/g TCC. Fountain Creek contributed up to 76% and 69% of the TCC and TCS, respectively, that is transported directly below its confluence with Arkansas River. Fountain Creek drained approximately 3.0 g/day TCS (in spring), 2.9 g/day TCS (in summer) and 1.9 g/day TCC (in spring), 2.0 g/day TCC (in-summer) into the Arkansas River, which suggests consistent input of TCC and TCS into Fountain Creek, such as in discharge of treated wastewater that is independent of changing creek flow conditions.