The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand (-)strand of ...viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand (+)strand DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.
The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
Reproductive tract pathology caused by
infection is an important global cause of human infertility. To better understand the mechanisms associated with
-induced genital tract pathogenesis in humans, ...we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to
Disruption of TLR3 function in these cells significantly diminished the
-induced synthesis of several inflammation biomarkers, including interferon beta (IFN-β), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rβ, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the
-induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that
infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within
inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.
A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, ...and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.
β-cell microRNA-21 (miR-21) is increased by islet inflammatory stress but it decreases glucose-stimulated insulin secretion (GSIS). Thus, we sought to define the effects of miR-21 on β-cell function ...using in vitro and in vivo systems.
We developed a tetracycline-on system of pre-miR-21 induction in clonal β-cells and human islets, along with transgenic zebrafish and mouse models of β-cell-specific pre-miR-21 overexpression.
β-cell miR-21 induction markedly reduced GSIS and led to reductions in transcription factors associated with β-cell identity and increased markers of dedifferentiation, which led us to hypothesize that miR-21 induces β-cell dysfunction by loss of cell identity. In silico analysis identified transforming growth factor-beta 2 (Tgfb2) and Smad family member 2 (Smad2) mRNAs as predicted miR-21 targets associated with the maintenance of β-cell identity. Tgfb2 and Smad2 were confirmed as direct miR-21 targets through RT-PCR, immunoblot, pulldown, and luciferase assays. In vivo zebrafish and mouse models exhibited glucose intolerance, decreased peak GSIS, decreased expression of β-cell identity markers, increased insulin and glucagon co-staining cells, and reduced Tgfb2 and Smad2 expression.
These findings implicate miR-21-mediated reduction of mRNAs specifying β-cell identity as a contributor to β-cell dysfunction by the loss of cellular differentiation.
•In vitro and in vivo β-cell miR-21 induction reduced glucose-stimulated insulin secretion and led to hyperglycemia.•β-cell miR-21-induction reduced transcription factors linked to β-cell identity and increased markers of dedifferentiation.•miR-21 mediated effects on β-cell identity and function were mediated in part by direct targeting of Tgfb2 and Smad2 mRNAs.
J. Neurochem. (2011) 116, 1112-1121. ABSTRACT: Dendritic trafficking and translation of brain-derived neurotrophic factor (BDNF) transcripts play a key role in mediating synaptic plasticity. ...Recently, we demonstrated that siRNA-mediated knockdown of translin, an RNA-binding protein, impairs KCl-induced dendritic trafficking of BDNF mRNA in cultured hippocampal neurons. We have now assessed whether translin deletion impairs dendritic trafficking of BDNF mRNA in hippocampal neurons in vivo. We have found that translin and its partner protein, trax, undergo dendritic translocation in response to treatment with pilocarpine, a pro-convulsant muscarinic agonist that increases dendritic trafficking of BDNF mRNA in hippocampal neurons. In translin knockout mice, the basal level of dendritic BDNF mRNA is decreased in CA1 pyramidal neurons. However, translin deletion does not block pilocarpine's ability to increase dendritic trafficking of BDNF mRNA indicating that the requirement for translin in this process varies with the stimulus employed to drive it. Consistent with this inference, we found that dendritic trafficking of BDNF mRNA induced by bath application of recombinant BDNF in cultured hippocampal neurons, is not blocked by siRNA-mediated knockdown of translin. Taken together, these in vivo and in vitro findings indicate that dendritic trafficking of BDNF mRNA can be mediated by both translin-dependent and -independent mechanisms.
Abstract Objective Preclinical and human family studies clearly link monoamine oxidase A (MAOA) to aggression and antisocial personality (ASP). The 30–base pair variable number tandem repeat in the ...MAOA promoter regulates MAOA levels, but its effects on ASP in humans are unclear. Methods We evaluated the association of the variable number tandem repeat of the MAOA promoter with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition , ASP disorder (ASPD) traits in a community sample of 435 participants from the Hopkins Epidemiology of Personality Disorders Study. Results We did not find an association between the activity of the MAOA allele and ASPD traits; however, among whites, when subjects with a history of childhood physical abuse were excluded, the remaining subjects with low-activity alleles had ASPD trait counts that were 41% greater than those with high-activity alleles ( P < .05). Conclusion The high-activity MAOA allele is protective against ASP among whites with no history of physical abuse, lending support to a link between MAOA expression and antisocial behavior.
Thanks to many government incentives, China has provided, at relatively low prices, a number of solar PV products to the world and enhanced domestic solar PV power. While China's growing solar PV ...industry has brought about both domestic and international environmental benefits, the provision of heavy subsidies has motivated the discussion of social and economic benefits and costs of this technology. Using the LCA approach, this paper performs an in-depth analysis on the environmental benefits and costs of this industry for the period of 2011–2016. Differing from existing literature, this paper broadens system boundaries to cover 11 stages of the solar PV industry life cycle, taking module sources and market directions of PV system into consideration, and quantifies the costs of environmental emissions of the industry by shadow pricing. The key finding of this study is that during 2011–2016 the total environment benefit was smaller than the total cost of China's solar PV industry. Meanwhile, coinciding with the rise of the domestic solar PV market, the resulting net environmental benefits have witnessed dramatic yearly growth. Policy implications and conclusions from findings are provided at the end of this paper.
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•We quantify the effectiveness of transformer-based models for clinical trial search. This is the first to have compared these methods under the same retrieval framework for a fair ...comparison.•We evaluate a broad selection of re-ranking models to uncover the role of pre-training corpus in evidence search for precision medicine.•Our evaluation points to a neural re-ranking system which achieves state-of-the-art results.•Our search system is fully automatic; its effectiveness does not rely on manual query reformulation strategies, nor problem-specific heuristic approaches.•With limited training data, leveraging the transfer learning inherent to transformer-based models makes the proposed model competitive to heuristic, manually optimised systems.
Bidirectional Encoder Representations from Transformers (BERT) have achieved state-of-the-art effectiveness in some of the biomedical information processing applications. We investigate the effectiveness of these techniques for clinical trial search systems. In precision medicine, matching patients to relevant experimental evidence or prospective treatments is a complex task which requires both clinical and biological knowledge. To assist in this complex decision making, we investigate the effectiveness of different ranking models based on the BERT models under the same retrieval platform to ensure fair comparisons. An evaluation on the TREC Precision Medicine benchmarks indicates that our approach using the BERT model pre-trained on scientific abstracts and clinical notes achieves state-of-the-art results, on par with highly specialised, manually optimised heuristic models. We also report the best results to date on the TREC Precision Medicine 2017 ad hoc retrieval task for clinical trial search.
Data suggest that β cell exosomes, extracellular vesicles released by exocytosis of multivesicular endosomes (MVEs) , may act as paracrine effectors in islet health. However, mechanisms linking β ...cell stress to changes in exosomes and whether activation of these pathways can impact diabetes remain less explored. In other cells, the enzyme neutral spingomyelinase 2 (nSMase2) induces ceramide dependent MVE vesicle formation and release of ceramide-enriched exosomes. We hypothesized that β cell inflammatory stress engages ceramide-dependent exosomal formation pathways, and that ceramide-enriched exosomes could then impact surrounding β cells. To test this we treated INS-1 β cells with 24 hrs of 5 ng/mL of IL1β. nSMase 2 and ceramide expression were quantified with immunoblot and/or flow cytometry and exosomes were isolated using a tetraspanin bead-based pulldown. IL1β increased β cell nSMase2 expression and ceramide expression in concert. Direct nSMase2 activation with 24 hrs of 5-ng/mL Caffeic acid phenethyl ester (CAPE) also increased β cell ceramide staining. IL1β and CAPE treatments also yielded increased ceramide expression on β cell exosomes. Human islets exhibited similar increases in cellular nSMase2 and ceramide staining after 24-hr cytokine mix treatment (5ng/mL IL1β, 10ng/mL TNFα, and 100ng/mL IFNγ) . To test the potential for transfer of β cell exosomes to surrounding cells, we generated INS-1 cells with GFP tagged CD9 (exosome transmembrane protein) , which release GFP labeled exosomes. Flow cytometry staining in wild-type INS-1 cells after 24-hr transwell coculture with CD9-GFP cells showed that cocultured wild-type cells exhibited increased green fluorescence, suggesting transfer of CD9-GFP exosomes. Our findings suggest that nSMase2 activity may regulate β cell exosome subpopulations under conditions of inflammatory stress. Future work will interrogate impacts on exosome cargo and physiologic impacts of transfer on recipient cells.
Disclosure
J.Xu: None. E.K.Sims: Other Relationship; Medscape, Patent.
Funding
5 R01DK121929
The transcriptional activity of Pdx1 is modulated by a diverse array of coregulatory factors that govern chromatin accessibility, histone modifications, and nucleosome distribution. We previously ...identified the Chd4 subunit of the nucleosome remodeling and deacetylase complex as a Pdx1-interacting factor. To identify how loss of Chd4 impacts glucose homeostasis and gene expression programs in β-cells in vivo, we generated an inducible β-cell-specific Chd4 knockout mouse model. Removal of Chd4 from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature-to-mature insulin granules in Chd4-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA sequencing and assay for transposase-accessible chromatin with sequencing showed that lineage-labeled Chd4-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell-enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function.
Pdx1-Chd4 interactions were previously shown to be compromised in β-cells from human donors with type 2 diabetes. β-Cell-specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key β-cell functional genes and chromatin accessibility are compromised in Chd4-deficient β-cells. Chromatin remodeling activities enacted by Chd4 are essential for β-cell function under normal physiological conditions.
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