Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters.

RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

A 2D-proteomic analysis identifies proteins differentially regulated by two different dengue virus serotypes.

The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

Greater ecophysiological stress tolerance in the core environment than in extreme environments of wild chickpea (Cicer reticulatum).

Global climate change and land use change underlie a need to develop new crop breeding strategies, and crop wild relatives (CWR) have become an important potential source of new genetic material to improve breeding efforts. Many recent approaches assume adaptive trait variation increases towards the relative environmental extremes of a species range, potentially missing valuable trait variation in more moderate or typical climates. Here, we leveraged distinct genotypes of wild chickpea (Cicer reticulatum) that differ in their relative climates from moderate to more extreme and perform targeted assessments of drought and heat tolerance. We found significance variation in ecophysiological function and stress tolerance between genotypes but contrary to expectations and current paradigms, it was individuals from more moderate climates that exhibited greater capacity for stress tolerance than individuals from warmer and drier climates. These results indicate that wild germplasm collection efforts to identify adaptive variation should include the full range of environmental conditions and habitats instead of only environmental extremes, and that doing so may significantly enhance the success of breeding programs broadly.

Development of a lateral flow assay for rapid and accurate detection of chicken anemia virus.

Significant challenges to poultry health are posed by chicken anemia virus (CAV), which induces immunosuppression and causes increased susceptibility to secondary infections. The effective management and containment of CAV within poultry stocks require precise and prompt diagnosis. However, a deficiency persists in the availability of low-cost, rapid, and portable CAV detection devices. In this study, an immunochromatographic lateral-flow test strip-based assay was developed for CAV detection using in-house generated monoclonal antibodies (MABs) against CAV viral protein 1 (VP1). The recombinant truncated VP1 protein (Δ60VP1), with amino acid residues 1 to 60 of the native protein deleted, was produced via a prokaryotic expression system and utilized for immunizing BALB/c mice. Subsequently, high-affinity MABs against Δ60VP1 were generated and screened using conventional hybridoma technology combined with serial dilution assays. Two MABs, MAB1, and MAB3, both binding to distinct epitopes of Δ60VP1, were selected for the development of a lateral-flow assay. Sensitivity analysis demonstrated that the Δ60VP1 antigen could be detected by our homemade lateral-flow assay at concentrations as low as 625 ng/mL, and this sensitivity was maintained for at least 6 mo. The assay exhibited high specificity, as evidenced by its lack of reactivity with surrogate recombinant proteins and the absence of cross-reactivity with other chicken viruses and viral antigens. Comparative analysis with quantitative PCR data demonstrated substantial agreement, with a Kappa coefficient of 0.66, utilizing a sample set comprising 305 clinical chicken serum samples. In conclusion, the first lateral-flow assay for CAV detection was developed in this study, utilizing 2 specific anti-VP1 MABs. It is characterized by simplicity, rapidity, sensitivity, and specificity.

Use of nominal group technique methods in the virtual setting: A reflective account and recommendations for practice.

Nominal group technique methods involve the use of structured activities within groups comprised of purposefully selected stakeholders (nominal groups), with the broad aim of achieving a level of consensus and prioritising information. In this paper, we will report how we facilitated nominal groups, using Microsoft Teams, to prioritise content for a theory-based behaviour change intervention to improve responses to clinically deteriorating patients. Our methods incorporated development and piloting of research materials, facilitation of online nominal groups with different stakeholders, and a structured approach to ranking behaviour change strategies. Practical suggestions are offered based on our experience of using this method in a virtual context.

Metabolomics and Microbial Metabolism: Toward a Systematic Understanding.

Over the past decades, our understanding of microbial metabolism has increased dramatically. Metabolomics, a family of techniques that are used to measure the quantities of small molecules in biological samples, has been central to these efforts. Advances in analytical chemistry have made it possible to measure the relative and absolute concentrations of more and more compounds with increasing levels of certainty. In this review, we highlight how metabolomics has contributed to understanding microbial metabolism and in what ways it can still be deployed to expand our systematic understanding of metabolism. To that end, we explain how metabolomics was used to (a) characterize network topologies of metabolism and its regulation networks, (b) elucidate the control of metabolic function, and (c) understand the molecular basis of higher-order phenomena. We also discuss areas of inquiry where technological advances should continue to increase the impact of metabolomics, as well as areas where our understanding is bottlenecked by other factors such as the availability of statistical and modeling frameworks that can extract biological meaning from metabolomics data.

Ecophysiological adaptations shape distributions of closely related trees along a climatic moisture gradient.

Tradeoffs between the energetic benefits and costs of traits can shape species and trait distributions along environmental gradients. Here we test predictions based on such tradeoffs using survival, growth, and 50 photosynthetic, hydraulic, and allocational traits of ten Eucalyptus species grown in four common gardens along an 8-fold gradient in precipitation/pan evaporation (P/Ep) in Victoria, Australia. Phylogenetically structured tests show that most trait-environment relationships accord qualitatively with theory. Most traits appear adaptive across species within gardens (indicating fixed genetic differences) and within species across gardens (indicating plasticity). However, species from moister climates have lower stomatal conductance than others grown under the same conditions. Responses in stomatal conductance and five related traits appear to reflect greater mesophyll photosynthetic sensitivity of mesic species to lower leaf water potential. Our data support adaptive cross-over, with realized height growth of most species exceeding that of others in climates they dominate. Our findings show that pervasive physiological, hydraulic, and allocational adaptations shape the distributions of dominant Eucalyptus species along a subcontinental climatic moisture gradient, driven by rapid divergence in species P/Ep and associated adaptations.

Strain Variation Can Significantly Modulate the miRNA Response to Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-transmitted virus that has emerged as a major public health concern due to its association with neurological disorders in humans, including microcephaly in fetuses. ZIKV infection has been shown to alter the miRNA profile in host cells, and these changes can contain elements that are proviral, while others can be antiviral in action. In this study, the expression of 22 miRNAs in human A549 cells infected with two different ZIKV isolates was investigated. All of the investigated miRNAs showed significant changes in expression at at least one time point examined. Markedly, 18 of the miRNAs examined showed statistically significant differences in expression between the two strains examined. Four miRNAs (miR-21, miR-34a, miR-128 and miR-155) were subsequently selected for further investigation. These four miRNAs were shown to modulate antiviral effects against ZIKV, as downregulation of their expression through anti-miRNA oligonucleotides resulted in increased virus production, whereas their overexpression through miRNA mimics reduced virus production. However, statistically significant changes were again seen when comparing the two strains investigated. Lastly, candidate targets of the miRNAs miR-34a and miR-128 were examined at the level of the mRNA and protein. HSP70 was identified as a target of miR-34a, but, again, the effects were strain type-specific. The two ZIKV strains used in this study differ by only nine amino acids, and the results highlight that consideration must be given to strain type variation when examining the roles of miRNAs in ZIKV, and probably other virus infections.

Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression.

OBJECTIVE: Studies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield. RESULTS: Our results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required.

The vitamin D receptor agonist EB1089 can exert its antiviral activity independently of the vitamin D receptor.

Vitamin D has been shown to have antiviral activity in a number of different systems. However, few studies have investigated whether the antiviral activity is exerted through the vitamin D receptor (VDR). In this study, we investigated whether the antiviral activity of a vitamin D receptor agonist (EB1089) towards dengue virus (DENV) was modulated by VDR. To undertake this, VDR was successively overexpressed, knocked down and retargeted through mutation of the nuclear localization signal. In no case was an effect seen on the level of the antiviral activity induced by EB1089, strongly indicating that the antiviral activity of EB1089 is not exerted through VDR. To further explore the antiviral activity of EB1089 in a more biologically relevant system, human neural progenitor cells were differentiated from induced pluripotent stem cells, and infected with Zika virus (ZIKV). EB1089 exerted a significant antiviral effect, reducing virus titers by some 2Log10. In support of the results seen with DENV, no expression of VDR at the protein level was observed. Collectively, these results show that the vitamin D receptor agonist EB1089 exerts its antiviral activity independently of VDR.

Functional classification of DDOST variants of uncertain clinical significance in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are rare genetic disorders with a spectrum of clinical manifestations caused by abnormal N-glycosylation of secreted and cell surface proteins. Over 130 genes are implicated and next generation sequencing further identifies potential disease drivers in affected individuals. However, functional testing of these variants is challenging, making it difficult to distinguish pathogenic from non-pathogenic events. Using proximity labelling, we identified OST48 as a protein that transiently interacts with lysyl oxidase (LOX), a secreted enzyme that cross-links the fibrous extracellular matrix. OST48 is a non-catalytic component of the oligosaccharyltransferase (OST) complex, which transfers glycans to substrate proteins. OST48 is encoded by DDOST, and 43 variants of DDOST are described in CDG patients, of which 34 are classified as variants of uncertain clinical significance (VUS). We developed an assay based on LOX N-glycosylation that confirmed two previously characterised DDOST variants as pathogenic. Notably, 39 of the 41 remaining variants did not have impaired activity, but we demonstrated that p.S243F and p.E286del were functionally impaired, consistent with a role in driving CDG in those patients. Thus, we describe a rapid assay for functional testing of clinically relevant CDG variants to complement genome sequencing and support clinical diagnosis of affected individuals.

Mosquito surveillance on U.S military installations as part of a Japanese encephalitis virus detection program: 2016 to 2021.

Japanese encephalitis virus (JEV) continues to circulate throughout Southeast Asia and the Western Pacific where approximately 3 billion people in 24 countries are at risk of infection. Surveillance targeting the mosquito vectors of JEV was conducted at four military installations on Okinawa, Japan, between 2016 and 2021. Out of a total of 10,426 mosquitoes from 20 different species, zero were positive for JEV. The most abundant mosquito species collected were Aedes albopictus (36.4%) followed by Culex sitiens (24.3%) and Armigeres subalbatus (19%). Statistically significant differences in mosquito species populations according to location were observed. Changes in land use over time appear to be correlated with the species and number of mosquitoes trapped in each location. JEV appears to be absent from mosquito populations on Okinawa, but further research on domestic pigs and ardeid birds is warranted.

Transcription-Replication Conflicts as a Source of Genome Instability.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

Linking stem rehydration kinetics to hydraulic traits using a novel method and mechanistic model.

BACKGROUND: Despite the recognized importance of hydraulic capacitance as a mechanism used by plants to maintain hydraulic functioning during high transpiration, characterizing the dynamics of capacitance remains a challenge. METHODS: We used a novel 'two-balance method' to investigate relationships between stem rehydration kinetics and other hydraulic traits in multiple tree species, and we developed a model to explore stem rehydration kinetics further. KEY RESULTS: We found that: (1) rehydration time constants and the amount of water uptake occurring during rehydration differed significantly across species; (2) time constants did not change with declining water potential (Ψ), while water uptake increased at lower Ψ in some species; (3) longer time constants were associated with lower wood density, higher capacitance and less negative stem pressures causing 50 % loss of hydraulic conductivity (P50); (4) greater water uptake occurred in stems with lower wood density and less negative P50 values; and (5) the model could estimate the total hydraulic resistance of the rehydration path, which cannot be measured directly. CONCLUSIONS: Overall, the two-balance method can be used to examine rehydration dynamics quickly and thoroughly in detached woody stems. This method has the potential to improve our understanding of how capacitance functions across tree species, which is an often-overlooked component of whole-plant hydraulics.

Resveratrol Enhances Cytotoxic Effects of Cisplatin by Inducing Cell Cycle Arrest and Apoptosis in Ovarian Adenocarcinoma SKOV-3 Cells through Activating the p38 MAPK and Suppressing AKT.

In the current study, we identified a mechanism of resveratrol (RES) underlying its anti-cancer properties against human ovarian adenocarcinoma SKOV-3 cells. We investigated its anti-proliferative and apoptosis-inducing effects in combination with cisplatin, using cell viability assay, flow cytometry, immunofluorescence study and Western blot analysis. We discovered that RES suppressed cancer cell proliferation and stimulated apoptosis, especially when combined with cisplatin. This compound also inhibited SKOV-3 cell survival, which may partly be due to its potential to inhibit protein kinase B (AKT) phosphorylation and induce the S-phase cell cycle arrest. RES in combination with cisplatin strongly induced cancer cell apoptosis through activating the caspase-dependent cascade, which was associated with its ability to stimulate nuclear phosphorylation of p38 mitogen-activated protein kinase (MAPK), well recognized to be involved in transducing environmental stress signals. RES-induced p38 phosphorylation was very specific, and the activation status of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was not mainly affected. Taken together, our study provides accumulated evidence that RES represses proliferation and promotes apoptosis in SKOV-3 ovarian cancer cells through activating the p38 MAPK pathway. It is interesting that this active compound may be used as an effective agent to sensitize ovarian cancer to apoptosis induced by standard chemotherapies.