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Stable Intronic Sequence RNA (sisRNA) is a relatively new class of non-coding RNA. Found in many organisms, these sisRNA produced from their host genes are generally involved in regulatory roles, controlling gene expression at multiple levels through active involvement in regulatory feedback loops. Large scale identification of sisRNA via genome-wide RNA sequencing has been difficult, largely in part due to its low abundance. Done on its own, RNA sequencing often yields a large mass of information that is ironically uninformative; the potential sisRNA reads being masked by other highly abundant RNA species like ribosomal RNA and messenger RNA. In this review, we present a practical workflow for the enrichment of circular sisRNA through the use of transcriptionally quiescent systems, rRNA-depletion, and RNase R treatment prior to deep sequencing. This workflow allows circular sisRNA to be reliably detected. We also present various methods to experimentally validate the circularity and stability of the circular sisRNA identified, as well as a few methods for further functional characterisation.

Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.

Autophagic decline is considered a hallmark of ageing. The activity of this intracytoplasmic degradation pathway decreases with age in many tissues and autophagy induction ameliorates ageing in many organisms, including mice. Autophagy is a critical protective pathway in neurons and ageing is the primary risk factor for common neurodegenerative diseases. Here, we describe that autophagosome biogenesis declines with age in mouse brains and that this correlates with increased expression of the SORBS3 gene (encoding vinexin) in older mouse and human brain tissue. We characterise vinexin as a negative regulator of autophagy. SORBS3 knockdown increases F-actin structures, which compete with YAP/TAZ for binding to their negative regulators, angiomotins, in the cytosol. This promotes YAP/TAZ translocation into the nucleus, thereby increasing YAP/TAZ transcriptional activity and autophagy. Our data therefore suggest brain autophagy decreases with age in mammals and that this is likely, in part, mediated by increasing levels of vinexin.

Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies a potentially pre-adaptive residue in a promiscuous N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzyme from Amycolatopsis sp. T-1–60. This enzyme belongs to a branch of the OSBS family which includes many catalytically promiscuous NSAR/OSBS enzymes. R266 is conserved in all members of the NSAR/OSBS subfamily. However, the homologous position is usually hydrophobic in other OSBS subfamilies, whose enzymes lack NSAR activity. The second-shell amino acid R266 is close to the catalytic acid/base K263, but it does not contact the substrate, suggesting that R266 could affect the catalytic mechanism. Mutating R266 to glutamine in Amycolatopsis NSAR/OSBS profoundly reduces NSAR activity, but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation-π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.

The Human Proteoform Atlas (HPfA) is a web-based repository of experimentally verified human proteoforms on-line at http://human-proteoform-atlas.org and is a direct descendant of the Consortium of Top-Down Proteomics’ (CTDP) Proteoform Atlas. Proteoforms are the specific forms of protein molecules expressed by our cells and include the unique combination of post-translational modifications (PTMs), alternative splicing and other sources of variation deriving from a specific gene. The HPfA uses a FAIR system to assign persistent identifiers to proteoforms which allows for redundancy calling and tracking from prior and future studies in the growing community of proteoform biology and measurement. The HPfA is organized around open ontologies and enables flexible classification of proteoforms. To achieve this, a public registry of experimentally verified proteoforms was also created. Submission of new proteoforms can be processed through email vianrtdphelp@northwestern.edu, and future iterations of these proteoform atlases will help to organize and assign function to proteoforms, their PTMs and their complexes in the years ahead.

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

Echinobase (www.echinobase.org) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledgebase, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.

Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

Subjective belief elicitation about uncertain events has a long lineage in the economics and statistics literatures. Recent developments in the experimental elicitation and statistical estimation of subjective belief distributions allow inferences about whether these beliefs are biased relative to expert opinion, and the confidence with which they are held. Beliefs about COVID-19 prevalence and mortality interact with risk management efforts, so it is important to understand relationships between these beliefs and publicly disseminated statistics, particularly those based on evolving epidemiological models. The pandemic provides a unique setting over which to bracket the range of possible COVID-19 prevalence and mortality outcomes given the proliferation of estimates from epidemiological models. We rely on the epidemiological model produced by the Institute for Health Metrics and Evaluation together with the set of epidemiological models summarised by FiveThirtyEight to bound prevalence and mortality outcomes for one-month, and December 1, 2020 time horizons. We develop a new method to partition these bounds into intervals, and ask subjects to place bets on these intervals, thereby revealing their beliefs. The intervals are constructed such that if beliefs are consistent with epidemiological models, subjects are best off betting the same amount on every interval. We use an incentivised experiment to elicit beliefs about COVID-19 prevalence and mortality from 598 students at Georgia State University, using six temporally-spaced waves between May and November 2020. We find that beliefs differ markedly from epidemiological models, which has implications for public health communication about the risks posed by the virus.

Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position., Ty3 retrotransposon integrates with an exquisite specificity upstream of RNA Polymerase III-transcribed genes, such as transfer RNAs. Here the authors resolve a cryo-EM structure of an active Ty3 intasome in complex with a TFIIIB-bound tRNA promoter, shedding light into the molecular determinants of harmless retrotransposition.

Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo., E. coli MukBEF is an SMC complex that plays key roles in chromosome organization-segregation. Here the authors show that the interaction between MukBEF and the Acyl Carrier Protein (AcpP) is essential for MukBEF activity in vitro (ATPase) and in vivo.

Recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transpired into pandemic coronavirus disease 2019 (COVID-19). SARS-CoV-2 has been rapidly transmitted across the globe within a short period of time, with more than 106 million cases and 2.3 million deaths. The continuous rise in worldwide cases of COVID-19, transmission dynamics of SARS-CoV-2 including re-infections and enormous case-fatality rates emphasizes the urgent need of potential preventive and therapeutic measures. The development of effective therapeutic and preventive measures relies on understanding the molecular and cellular mechanism of replication exhibited by SARS-CoV-2. The structure of SARS-CoV-2 is ranging from 90/120 nm that comprises surface viral proteins including spike, envelope, membrane which are attached in host lipid bilayer containing the helical nucleocapsid comprising viral RNA. Spike (S) glycoprotein initiates the attachment of SARS-CoV-2 with a widely expressed cellular receptor angiotensin-converting enzyme 2 (ACE2), and subsequent S glycoprotein priming via serine protease TMPRSS2. Prominently, comprehensive analysis of structural insights into the crucial SARS-CoV-2 proteins may lead us to design effective therapeutics molecules. In the present article, we will emphasize the mechanistic insights of SARS-CoV-2 replication including insights into the structural perspective of crucial SARS-CoV-2 proteins.

Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2-producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared with MUC2 silenced or gene edited cells. Similarly, colonoids from

The test positivity (TP) rate has emerged as an important metric for gauging the illness burden due to COVID-19. Given the importance of COVID-19 TP rates for understanding COVID-related morbidity, researchers and clinicians have become increasingly interested in comparing TP rates across countries. The statistical methods for performing such comparisons fall into two general categories: frequentist tests and Bayesian methods. Using data from Our World in Data (ourworldindata.org), we performed comparisons for two prototypical yet disparate pairs of countries: Bolivia versus the United States (large vs. small-to-moderate TP rates), and South Korea vs. Uruguay (two very small TP rates of similar magnitude). Three different statistical procedures were used: two frequentist tests (an asymptotic z-test and the 'N-1' chi-square test), and a Bayesian method for comparing two proportions (TP rates are proportions). Results indicated that for the case of large vs. small-to-moderate TP rates (Bolivia versus the United States), the frequentist and Bayesian approaches both indicated that the two rates were substantially different. When the TP rates were very small and of similar magnitude (values of 0.009 and 0.007 for South Korea and Uruguay, respectively), the frequentist tests indicated a highly significant contrast, despite the apparent trivial amount by which the two rates differ. The Bayesian method, in comparison, suggested that the TP rates were practically equivalent-a finding that seems more consistent with the observed data. When TP rates are highly similar in magnitude, frequentist tests can lead to erroneous interpretations. A Bayesian approach, on the other hand, can help ensure more accurate inferences and thereby avoid potential decision errors that could lead to costly public health and policy-related consequences.

Because the spread of pandemics depends heavily on human choices and behaviors, dealing with COVID-19 requires insights from cognitive science which integrates psychology, neuroscience, computer modeling, philosophy, anthropology, and linguistics. Cognitive models can explain why scientists adopt hypotheses about the causes and treatments of disease based on explanatory coherence. Irrational deviations from good reasoning are explained by motivated inference in which conclusions are influenced by personal goals that contribute to emotional coherence. Decisions about COVID-19 can also be distorted by well-known psychological and neural mechanisms. Cognitive science provides advice about how to improve human behavior in pandemics by changing beliefs and by improving behaviors that result from intention-action gaps.

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1–3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes. Using time-controlled CRISPR screens, the authors identify AKIRIN2 as a factor involved in the nuclear import of the proteasome.

cGMP interactors play a role in several pathologies and may be targets for cGMP analog-based drugs, but the success of targeting depends on the biochemical stereospecificity between the cGMP-analog and the interactor. The stereospecificity between general cGMP analogs-or such that are selectivity-modified to obtain, for example, inhibitory actions on a specific target, like the cGMP-dependent protein kinase-have previously been investigated. However, the importance of stereospecificity for cGMP-analog binding to interactors is not known. We, therefore, applied affinity chromatography on mouse cortex proteins utilizing analogs with cyclic phosphate (8-AET-cGMP, 2-AH-cGMP, 2'-AHC-cGMP) and selectivity-modified analogs with sulfur-containing cyclic phosphorothioates (Rp/Sp-8-AET-cGMPS, Rp/Sp-2'-AHC-cGMPS) immobilized to agaroses. The results illustrate the cGMP analogs' stereospecific binding for PKG, PKA regulatory subunits and PKA catalytic subunits, PDEs, and EPAC2 and the involvement of these in various KEGG pathways. For the seven agaroses, PKG, PKA regulatory subunits, and PKA catalytic subunits were more prone to be enriched by 2-AH-, 8-AET-, Rp-8-AET-, and Sp-8-AET-cGMP, whereas PDEs and EPAC2 were more likely to be enriched by 2-AH-, Rp-2'-AHC-, and Rp-8-AET-cGMP. Our findings help elucidate the stereospecific-binding sites essential for the interaction between individual cGMP analogs and cGMP-binding proteins, as well as the cGMP analogs' target specificity, which are two crucial parameters in drug design.

The inclusion of peptide retention time prediction promises to remove peptide identification ambiguity in complex LC-MS identification workflows. However, due to the way peptides are encoded in current prediction models, accurate retention times cannot be predicted for modified peptides. This is especially problematic for fledgling open modification searches, which will benefit from accurate retention time prediction for modified peptides to reduce identification ambiguity. We here therefore present DeepLC, a novel deep learning peptide retention time predictor utilizing a new peptide encoding based on atomic composition that allows the retention time of (previously unseen) modified peptides to be predicted accurately. We show that DeepLC performs similarly to current state-of-the-art approaches for unmodified peptides, and, more importantly, accurately predicts retention times for modifications not seen during training. Moreover, we show that DeepLC’s ability to predict retention times for any modification enables potentially incorrect identifications to be flagged in an open modification search of CD8-positive T-cell proteome data. DeepLC is available under the permissive Apache 2.0 open source license and comes with a user-friendly graphical user interface, as well as a Python package on PyPI, Bioconda, and BioContainers for effortless workflow integration.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 A four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Forster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

In a temperate climate, the mycotoxins deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROC) and zearalenone (ZEN) are often found in maize silage. Although rumen microbiota are able to degrade some mycotoxins (e.g. DON), others are known to stay mainly intact (e.g. ROC). In addition, mycotoxin degradation can be hampered by a low ruminal pH or decrease in rumen microbial activity. Hence, these mycotoxins can reach the small intestine and exert a cytotoxic effect on intestinal epithelial cells. In this study, a real-time kinetic analysis of the cytotoxicity of these six mycotoxins and some of their metabolites (α- and β-zearalenol, α- and β-ZOL) was performed in a calf small intestinal epithelial cell line (CIEB). Confluency as well as the cell death parameters apoptosis and necrosis were determined to evaluate the mycotoxin-induced cytotoxicity. A combination of Annexin-V green and Cytotox red staining was used to determine early and late apoptosis as well as necrosis. Six different concentrations were tested ranging from 0.78 to 12.5 μM. Compared to cells not exposed to mycotoxins, DON and NIV exert a fast toxic effect with DON being more toxic than NIV within the first hours of incubation, whereas the inverse was observed at 16 h of incubation. On the other hand, MPA and ZEN induced increased Annexin V green positive cells within several hours of incubation with higher toxicity over time. Increased Annexin V green and Cytotox red positive cells were seen for ROC only at the highest concentration tested. For ENN B, increased Annexin V green positive cells were observed only after 12 h and α- and β-ZOL did not show cytotoxic effects. Hence, mycotoxin exposure causes either severe (DON and NIV) or more limited (ZEN, ROC, MPA, and ENN B) risk of bovine intestinal epithelial damage.

Genome sequence analysis of Atlantic salmon bafinivirus (ASBV) revealed a small open reading frame (ORF) predicted to encode a Type I membrane protein with an N-terminal cleaved signal sequence (110 aa), likely an envelope (E) protein. Bioinformatic analyses showed that the predicted protein is strikingly similar to the coronavirus E protein in structure. This is the first report to identify a putative E protein ORF in the genome of members of the Oncotshavirus genus (subfamily Piscavirinae, family Tobaniviridae, order Nidovirales) and, if expressed would be the third family (after Coronaviridae and Arteriviridae) within the order to have the E protein as a major structural protein.

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway–Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.

The CRISPR/Cas9 system has been harnessed to cleave a targeted DNA fragment via its Cas nuclease activity under the direction of guide RNA for rendering gene insertions, deletions, and point mutations in basic research and clinical applications. There are a number of vehicles, including lipofectamine, viruses, and nanoparticles, that can deliver the CRISPR/Cas9 system, but all these methods face numerous challenges during their application in life science contexts. Here, we focus on the delivery of CRISPR/Cas9 via nanoparticles because this method has shown great advantages in terms of safety, simplicity and flexibility.

A protein complex is a collection of two or more associated proteins that interact with each other in a stable long-term interaction. Protein complexes have essential roles in regulatory processes, cellular functions and signaling cascades. This paper examines how well-known collections of protein complexes are supported in protein–protein interaction (PPI) networks, i.e. whether they form connected subnetworks in a particular PPI network. For that purpose, we apply a variable neighbourhood search (VNS) metaheuristic algorithm for adding the minimum number of interactions in order to support protein complexes. Experimental results obtained on several PPI networks (BioGRID, WI-PHI and String) and four protein complex standards (MIPS, TAP06, SGD and CYC2008) show that considered networks do not include enough PPIs to support all complexes from complex standards. Deeper analysis indicates that there exists common PPIs which are probably missing in the considered networks. These findings can be useful for further biological interpretation and developing of PPI prediction models.

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatch amplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For prime editing, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the target region with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapid detection of insertion or deletion events in under 2.5 h from genomic DNA extraction. We show that our method of SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets in BHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing and next-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editing efficiencies. Our workflow details three different methods for the detection of rare base and prime editing events, enabling a tiered approach from low to high resolution that makes use of gel electrophoresis, Sanger sequencing, and NGS.

Whole genome sequence analysis of Epstein-Barr virus genomes from tumours and healthy individuals identified three amino acid changes in EBNA1 that are strongly associated with gastric carcinoma and nasopharyngeal carcinoma. Here we show that, while these mutations do not impact EBNA1 plasmid maintenance function, one of them (Thr85Ala) decreases transcriptional activation and results in a gain of function interaction with PLOD1 and PLOD3. PLOD family proteins are strongly linked to multiple cancers, and PLOD1 is recognized as a prognostic marker of gastric carcinoma. We identified the PLOD1 binding site in EBNA1as the N-terminal transactivation domain and show that lysine 83 is critical for this interaction. The results provide a novel link between EBV infection and the cancer-associated PLOD proteins.

In vivo genome editing meets numerous challenges including efficiency and safety. Here we describe an efficient in vivo genome editing method of delivering CRISPR-Cas9 into vascular endothelial cells with adeno-associated viruses (AAVs). In this system, expression of SpCas9 is driven by a specific endothelial promoter of intercellular adhesion molecule 2 (pICAM2) to restrict this foreign enzyme in vascular endothelial cells, which can be efficiently infected by AAV1. We exemplify this approach by editing VEGFR2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy, and expect that this simplified protocol can be expanded to other researches on editing endothelial genome in vivo.

Mammalian hibernation is a period that involves substantial metabolic change in order to promote survival in harsh conditions, with animals typically relying on non-carbohydrate fuel stores during long bouts of torpor. However, the use and maintenance of carbohydrate fuel stores remains important during periods of arousal from torpor as well as when exiting hibernation. Gluconeogenesis plays a key role in maintaining glucose stores; however, little is known about this process within the muscles of hibernating mammals. Here, we used 13-lined ground squirrels (Ictidomys tridecemlineatus) as our model for mammalian hibernation, and showed that skeletal muscle fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), the rate-limiting enzyme for the gluconeogenic pathway, was suppressed during torpor as compared to the euthermic control. A physical assessment of partially purified FBPase via exposure to increasing concentrations of the denaturant urea indicated that FBPase from the two conditions were structurally distinct. Western blot analysis suggests that the kinetic and physical differences between euthermic and torpid FBPase may be derived from differential acetylation, whereby increased acetylation of the torpid enzyme makes FBPase more rigid and less active. This study increases our understanding of skeletal muscle carbohydrate metabolism during mammalian hibernation and sets forth a potentially novel mechanism for the regulation of FBPase during environmental stress.

The development of CRISPR/Cas genome editing tools has revolutionized the life sciences by providing transformative applications in many biological fields, including the field of neurological disorders. Compared with previous CRISPR-Cas systems targeting DNA, a new field of RNA editing using CRISPR-Cas13 systems is gaining immense popularity. CRISPR-Cas13 is a robust, precise, versatile and safe RNA guided RNA-targeting system, which uniquely targets single-strand RNA. Recently, RNA-targeted gene editing tools have been refined by the introduction of an AAV (adeno-associated virus)-based CRISPR-Cas13 system for in vivo therapeutic cell fate conversion, which has been used to treat animal models of Parkinson's disease. This flavor of gene editing showed promising effects on glia-to-neuron conversion in both intact and damaged mature retinas in a mouse model. Herein, we summarize the CRISPR-Cas13 system and its potential for applications in neurological diseases, focusing on the method of applying the AAV-mediated CRISPR-Cas13 system to the conversion of glia-to-neuron.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based base editors have been developed for precisely installing point mutations in genomes with high efficiency. Two editing systems of cytosine base editors (CBEs) and adenine base editors (ABEs) have been developed for conversion of C.G-to-T.A and A.T-to-G.C, respectively, showing the prominence in genomic DNA correction and mutation. Here, we summarize recent optimized approaches in improving base editors, including the evolution of Cas proteins, the choice of deamination enzymes, modification on linker length, base-editor expression, and addition of functional domains. Specifically, in this paper we highlight a strategy of split-intein mediated base-editor reconstitution for its adeno-associated virus (AAV) delivery. The purpose of this article is to offer readers with a better understanding of AAV-mediated base editors, and facilitate them to use this tool in in vivo experiments and potential clinical applications.

Base editing technology is an efficient tool for genome editing, particularly in the correction of base mutations. Diverse base editing systems were developed according to the dCas9 or nCas9 linked with different deaminase or reverse transcriptase in the editors, including ABEs, CBEs, PEs and dual-functional of base editor (such as CGBE1, A&C-BEmax, ACBE, etc.). Currently, Base editing technology has been widely applied to various fields such as microorganisms, plants, animals and medicine for basic research and therapeutics. Here, we reviewed the advancement of base editing technology. We also discussed the application of base editors in different areas in the future.

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

The homospermidine synthases from P. aeruginosa and B. viridis, as well as their single-residue variants, are compared based on crystal structures and activity assays. A high structural similarity is demonstrated, suggesting the equivalent involvement of relevant residues in the reaction mechanism and catalytic dependence on cation–π interaction., Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (‘ionic slide’) and near the active site (‘inner amino site’) to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

Conversion between phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate on endosomal membranes is critical for the maturation of early endosomes to late endosomes/lysosomes and is regulated by the PIKfyve-Vac14-Fig4 complex. Despite the importance of this complex for endosomal homeostasis and vesicular trafficking, there is little known about how its activity is regulated or how it interacts with other cellular proteins. Here, we screened for the cellular interactome of Vac14 and Fig4 using proximity-dependent biotin labeling (BioID). After independently screening the interactomes of Vac14 and Fig4, we identified 89 high-confidence protein hits shared by both proteins. Network analysis of these hits revealed pathways with known involvement of the PIKfyve-Vac14-Fig4 complex, including vesicular organization and PI3K/Akt signaling, as well as novel pathways including cell cycle and mitochondrial regulation. We also identified subunits of coatomer complex I (COPI), a Golgi-associated complex with an emerging role in endosomal dynamics. Using proximity ligation assays, we validated the interaction between Vac14 and COPI subunit COPB1 and between Vac14 and Arf1, a GTPase required for COPI assembly. In summary, this study used BioID to comprehensively map the Vac14-Fig4 interactome, revealing potential roles for these proteins in diverse cellular processes and pathways, including preliminary evidence of an interaction between Vac14 and COPI. Data are available via ProteomeXchange with the identifier PXD027917.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

Occurrence of mycotoxins in agricultural products represents a risk for human and animal health. Therefore, there is a requirement of strategies to mitigate their harmful impacts. This study investigated the effects of phytic acid (IP6) on the immunological response of pro-(interleukin (IL)-1β, IL-6, IL-8, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α) and anti-inflammatory (IL-10) cytokines and β-defensins 1 (pBD-1) and 2 (pBD-2) in porcine jejunal explants exposed to deoxynivalenol (DON) and fumonisin B1 (FB1). The explants were exposed to the following treatments: control, DON (10 μM), DON plus IP6 2.5 mM or 5 mM, FB1 (70 μM), FB1 IP6 plus 2.5 or 5 mM. The expression levels of the cytokines were measured by RT-qPCR. The exposure to FB1 and DON induced intestinal lesions. The presence of 2.5 and 5 mM IP6 inhibited the morphological changes induced by the mycotoxins. The explants exposed to DON showed an increase in the expression of IL-1β and IL-8 and a decrease in the levels of IL-6, IFN-γ, IL-10 and pBD-2. IP6 (5 mM) decreased the expression of IL-8 and increased the expression in pBD-1 and 2 compared to DON alone. FB1 induced a significant decrease in the levels of most of the pro-inflammatory cytokines, IL-10 and pBD-1, and an increase in IL-1β expression. The addition of IP6 5 mM induced significant increase in TNF-α expression compared to FB1. Taken together, the results suggest IP6 modulates immunological changes induced by DON and FB1 on intestinal mucosa resulting in beneficial effects that contribute to intestinal homeostasis and health.

Внеклеточный матрикс биопленок обеспечивает фиксацию биопленки на биологической поверхности и защиту ее бактерий от внешних неблагоприятных факторов. Основным структурным компонентом био­пленок является внеклеточное полисахаридное вещество. Ключевыми молекулярными структурами, поддерживающими трехмерную структуру биопленки, считают экзополисахариды и амилоидоподобные волокна. До недавнего времени предполагалось, что большинство механизмов диспергирования биопленок ассоциировано с функционированием матриксных деградирующих ферментов, таких как гликозидгидролазы, полисахаридные лиазы и протеазы. Однако продемонстрировано, что в процессе разрушения матриксных экзополисахаридов и амилоидоподобных волокон играют самостоятельную роль малые молекулы. Среди соединений, нарушающих матрикс биопленки, различают антиматриксные молекулы, соединения, взаимодействующие с микродоменами бактериальной мембраны, и бактериальные поверхностно-активные вещества (биосурфагенты). Продемонстрировано, что соединения данных групп могут ингибировать образование биопленок и способствовать их диспергированию. Из группы антиматриксных молекул полиаминное соединение норспермидин взаимодействует с экзополисахаридами, а производные бензохинона AA-861 и сесквитерпеновый лактон партенолид — с TasA-амилоидоподобными волокнами. Норспермидин предотвращает образование и диспергирует биопленки различных бактерий, включая Acinetobacter baumannii, Bacillus subtili, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis. Соединение AA-861 проявляет активность против биопленок, которые образованы бактериями Streptococcus mutans, Bacillus cereus, Escherichia coli, а партенолид диспергирует биопленки, сформированные Escherichia coli и Bacillus cereus. Сарагосовые кислоты, взаимодействуя с микродоменами бактериальной мембраны, нарушают функционирование рафт-ассоциированных протеинов бактерий. Малые антиматриксные молекулы и нацеленные на микродомены мембраны бактерий, инициирующие диспергирование биопленки, безусловно, станут основанием для разработки эффективных антибиопленочных терапевтических лекарственных средств.

Capsid protein (CP) of Chikungunya virus (CHIKV) is a multifunctional protein with a conserved hydrophobic pocket that plays a crucial role in the capsid assembly and virus budding process. This study demonstrates antiviral activity of thymoquinone (TQ), a natural compound targeting the hydrophobic pocket of CP. The binding of TQ to the hydrophobic pocket of CHIKV CP was analysed by structure-based molecular docking, isothermal titration calorimetry and fluorescence spectroscopy. The binding constant KD obtained for TQ was 27 μM. Additionally, cell-based antiviral studies showed that TQ diminished CHIKV replication with an EC50 value 4.478 μM. Reduction in viral RNA copy number and viral replication as assessed by the qRT-PCR and immunofluorescence assay, confirmed the antiviral potential of TQ. Our study reveals that TQ is an effective antiviral targeting the hydrophobic pocket of CHIKV CP and may serve as the basis for development of a broad-spectrum therapy against alphaviral diseases.

Here, we discuss a collection of cutting-edge techniques and applications in use today by some of the leading experts in the field of correlative approaches in single-molecule biophysics. A key difference in emphasis, compared with traditional single-molecule biophysics approaches detailed previously, is on the emphasis of the development and use of complex methods which explicitly combine multiple approaches to increase biological insights at the single-molecule level. These so-called correlative single-molecule biophysics methods rely on multiple, orthogonal tools and analysis, as opposed to any one single driving technique. Importantly, they span both in vivo and in vitro biological systems as well as the interfaces between theory and experiment in often highly integrated ways, very different to earlier traditional non-integrative approaches. The first applications of correlative single-molecule methods involved adaption of a range of different experimental technologies to the same biological sample whose measurements were synchronised. However, now we find a greater flora of integrated methods emerging that include approaches applied to different samples at different times and yet still permit useful molecular-scale correlations to be performed. The resultant findings often enable far greater precision of length and time scales of measurements, and a more nuanced understanding of the interplay between different processes in the same cell. Many new correlative single-molecule biophysics techniques also include more complex, physiologically relevant approaches as well as an increasing number that combine of approaches advanced computational methods and mathematical analysis with experimental tools. Here, we review the motivation behind the development of correlative single-molecule microscopy methods, its history and recent progress in the field.

There is a pressing need for vaccines against mosquito-borne alphaviruses such as Venezuelan and eastern equine encephalitis viruses (VEEV, EEEV). We demonstrate an approach to vaccine development based on physicochemical properties (PCP) of amino acids to design a PCP-consensus sequence of the epitope rich B domain of the VEEV major antigenic E2 protein. The consensus “spike” domain was incorporated into a live-attenuated VEEV vaccine candidate (ZPC/IRESv1). Mice inoculated with either ZPC/IRESv1 or the same virus containing the consensus E2 protein fragment (VEEVconE2) were protected against lethal challenge with VEEV strains ZPC-738 and 3908, and Mucambo virus (MUCV, related to VEEV), and had comparable neutralizing antibody titers against each virus. Both vaccines induced partial protection against Madariaga virus (MADV), a close relative of EEEV, lowering mortality from 60% to 20%. Thus PCP-consensus sequences can be integrated into a replicating virus that could, with further optimization, provide a broad-spectrum vaccine against encephalitic alphaviruses.

Based on the previously reported 13-residue antibacterial peptide analog, brevinin-1EMa (FLGWLFKVASKVL, peptide B), we attempted to design a novel class of antiviral peptides. For this goal, we synthesized three peptides with different stapling positions (B–2S, B–8S, and B–5S). The most active antiviral peptide with the specific stapling position (B–5S) was further modified in combination with either cysteine (B–5S3C, B–5S7C, and B–5S10C) or hydrophilic amino acid substitution (Bsub and Bsub-5S). Overall, B, B–5S, and Bsub-5S peptides showed superior antiviral activities against enveloped viruses such as retrovirus, lentivirus, hepatitis C virus, and herpes simplex virus with EC50 values of 1–5 μM. Murine norovirus, a non-enveloped virus, was not susceptible to the virucidal actions of these peptides, suggesting the virus membrane disruption as their main antiviral mechanisms of action. We believe that these three novel peptides could serve as promising candidates for further development of membrane-targeting antiviral drugs in the future.

Single-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap’s inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged β-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding., Replicative hexameric helicases are fundamental components of replisomes. Here the authors resolve a cryo-EM structure of the E1 helicase from papillomavirus bound to a DNA replication fork, providing insights into the mechanism of DNA unwinding by these hexameric enzymes.

The fall of 2020 brought several new variants of SARS-CoV-2 circulating across the globe, and the steadily increasing COVID-19 cases are responsible for the emergence of these variants. All the SARS-CoV-2 variants reported to date have multiple mutations in the spike (S) protein, specifically in the receptor-binding domain (RBD). Here, we employed an integrated computational approach involving structure and sequence based predictions to study the effect of naturally occurring variations in the S-RBD on its stability and ACE2 binding affinity. The hotspot stabilizing residue mutations N501I, N501Y, Q493L, Q493H and K417R, strengthen the RBD-ACE2 complex by modulating the interaction statistics at the interface. Thus, we report here some critical mutations that could increase the binding affinity of the SARS-CoV-2 RBD with ACE2, increasing the viral infectivity and pathogenicity. Understanding the effect of these mutations will help in developing potential vaccines and therapeutics., Graphical abstract Image 1

Primary infection with Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease and severe disease in patients with X-linked lymphoproliferative disease; no therapies are approved to prevent EBV infection in these patients. Hyperimmune globulin has been used to prevent some virus infections in immunocompromised persons. Here, we identified plasma donors with high titers of EBV gp350 and EBV B cell neutralizing antibodies. Pooled IgG isolated from these donors was compared to intravenous immunoglobulin (IVIG) for its ability to reduce viral load in the blood in humanized mice challenged with EBV. Mice that received EBV hyperimmune globulin had significantly reduced EBV DNA copy numbers compared to animals that received saline control; however, while animals that received EBV hyperimmune globulin had lower EBV DNA copies than those that received IVIG, the difference was not significant. Thus, while EBV hyperimmune globulin reduced viral load compared to IVIG, the effect was modest.

Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.

Burkholderia sp. strain MS389, an endophytic bacterium, was isolated from a healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. Preliminary studies demonstrated that strain MS389 possesses antimicrobial activities against multiple plant pathogens. Burkholderia sp. strain MS389 was found to have three circular chromosomes of 3,563,380 bp, 3,002,449 bp, and 1,180,421 bp in size, respectively. The 7,746,250-bp genome, with 66.73% G+C content, harbors 6,756 protein coding genes in the predicted 6,985 genes. In total, 18 rRNAs, 68 tRNAs, and four ncRNAs were identified and 139 pseudogenes were annotated as well. The findings of this study will provide valuable data to explore the antimicrobial mechanisms of the endophytic bacterial strain.