Access Microbiology

BackgroundHepatitis B virus infection remains a major public health challenge, particularly among people living with human immunodeficiency virus, due to shared transmission routes and the potential for accelerated liver disease progression. Molecular characterization of circulating HBV strains is essential for understanding viral epidemiology, mutation patterns, and implications for diagnostics and vaccination. MethodsThis study investigated the prevalence of hepatitis B infection and molecular characteristics of the hepatitis B virus surface gene among HIV-infected individuals receiving antiretroviral therapy in Nairobi County, Kenya. Plasma samples were screened for hepatitis B surface antigen using enzyme-linked immunosorbent assay. Hepatitis B viral DNA was extracted from HBsAg-positive samples and the surface gene region amplified by polymerase chain reaction. Amplified products were subjected to Sanger sequencing. Sequence assembly, genotype determination, and mutation analysis. ResultsThe prevalence of HIV/HBV co-infection among HIV-positive individuals was determined to be 8.97%. Genotype analysis revealed the circulation of HBV genotype A (sub-genotypes A1 and A4) and genotype D (sub-genotypes D4 and D10) among the studied population. Amino acid sequence analysis of the major hydrophilic region of the surface gene identified several mutations, with R122K and Y134F being the most frequently observed substitutions. ConclusionHepatitis B infection remains prevalent among HIV-infected individuals receiving antiretroviral therapy in Nairobi County. The circulation of multiple hepatitis B virus genotypes and the presence of mutations within the surface gene highlight the importance of continuous molecular surveillance to monitor viral evolution and its potential implications for hepatitis B virus diagnosis, vaccination strategies, and clinical management in HIV-infected populations

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Background The COVID-19 pandemic has caused significant global mortality. Despite declining infection rates, new variants of SARS-CoV-2 continue to emerge, necessitating new prevention strategies. Objective This study aimed to evaluate the effect of four over-the-counter (OTC) antiseptic mouthwash/gargling solutions in the U.S., compared with a distilled water control, on SARS-CoV-2 viral load across multiple oral and oropharyngeal sample types. Methods This pilot single-center randomized controlled clinical trial enrolled adults in the San Francisco Bay Area, California, who tested positive for COVID-19. Participants were randomized to distilled water, chlorine dioxide, hydrogen peroxide, cetylpyridinium chloride, and essential oils. Participants were instructed to rinse and gargle four times daily for four weeks using standardized instructions to ensure protocol adherence. Samples were collected on Days 1, 7, and 28 and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The primary outcome was the change in SARS-CoV-2 viral load from baseline to Day 28, assessed using cycle threshold (Ct) values. Secondary outcomes included self-reported clinical symptoms and hospitalization. Results Forty-nine participants completed the study. No mouthwash demonstrated a statistically significant reduction in SARS-CoV-2 viral load over time. Cetylpyridinium chloride showed a transient increase in Ct values on Day 7 that was not sustained on Day 28. At baseline, throat swab samples had the lowest Ct values across all sample types. Due to limited subgroup sample sizes for secondary outcome measures, no statistical or moderator analyses were conducted. Conclusion Further large-scale randomized trials are needed before recommending antiseptic mouthwashes for SARS-CoV-2 prevention or management.

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

Staphylococcus aureus (S. aureus) is the most common pathogen associated with skin infections worldwide. Significant efforts have been made to identify and develop innovative therapeutic strategies against S. aureus as alternatives to conventional antibiotics. Physical plasma has a broad range of potential uses, with non-destructive disinfection being one of its earliest applications. Although the literature emphasizes the antibacterial properties of cold atmospheric plasma (CAP), the effect of plasma on S. aureus on damaged skin susceptible to S. aureus invasion through the itch-scratch cycle has not been studied to date. Thus, we examined the effectiveness of CAP treatment on S. aureus bacteria in atopic dermatitis lesions using floating electrode dielectric barrier discharge devices, as well as helium and argon plasma jets. Heat distribution on the skin target, ultraviolet C radiation, and ozone generation of plasma jets for the operator of plasma sources were evaluated. Microbial tests confirmed the presence of S. aureus on the lesions of the groups before treatment. The groups exposed to plasma treatment showed a notable reduction in bacterial population compared to the model group (p<0.05). Furthermore, our investigation indicated that plasma treatment reduced pruritus behavior. The findings suggest that cold atmospheric plasma treatment may potentially target skin infections caused by S. aureus in addition to conventional therapies.

Pseudomonas aeruginosa is a clinically significant bacterial pathogen that poses a serious threat to aquaculture. However, there are limited information on Massilia isolates against pathogenic P. aeruginosa in aquaculture. In the present study, a facultative predator, M. varians isolate P2-4, was isolated from aquaculture sediment using Chinese mitten crab Eriocheir sinensis-pathogenic P. aeruginosa as the prey bacterium, and its genomic feature, bacteriolysis-related genes, safety, bacteriolytic spectrum, and in vitro and in vivo antibacterial effects against pathogenic P. aeruginosa in E. sinensis were further characterized. Isolate P2-4 consisted of one chromosome and one plasmid (with a total of 75 tRNAs, 7 5S rRNAs, 7 16S rRNAs, 7 23S rRNAs, 34 sRNAs, 5,238 coding genes, 20 genomic islands, 1 prophage, 23 insertion sequences, and 102 repeat sequences), and harbored 19 bacteriolysis-related genes (pilA, pilB, pilC, pilD, pilF, pilG, pilH, pilM, pilO, pilP, pilQ, pilS, pilR, pilT, mltA, mltB, mltC, mltD, and dacB) associated with cellular motility and cell wall lysis. In addition, the isolate carried no virulence genes, was unable to produce haemolysin, hydrogen sulfide, nitrite and ammonia, and avirulent in E. sinensis with a 7-day acute intraperitoneal LD50 value of above 5.0 x 108 CFU/mL. Furthermore, the isolate possessed a wide bacteriolytic spectrum against pathogenic Shewanella algae, Aeromonas caviae, A. hydrophila, and Photobacterium damselae besides P. aeruginosa, exhibited bacteriolysis rates of 99.35% to 99.99% towards the pathogenic P. aeruginosa at 1.0x103 to 1.0x10{square} CFU/mL, and displayed relative percentage survivals of 42.31% to 73.08% against P. aeruginosa infection in E. sinensis at doses of 6.0 x 103 to 6.0 x 105 CFU/g diet. To our knowledge, this study for the first time demonstrates a M. varians strain as a potential biocontrol agent against pathogenic P. aeruginosa in aquaculture.

O_LIPlants and fungi are major sources of natural products beneficial to society, making the study of distinct species essential for discovering new drugs and bioactive compounds. The medicinal mushroom "Lingzhi" or "Reishi" (Ganoderma lingzhi) is widely used in traditional medicine and extensively studied for its bioactive triterpenoids, yet it is commonly identified as Ganoderma lucidum, the type species of the genus, which lacks a type specimen. C_LIO_LIWe sequenced a G. lucidum specimen preserved in the Kew fungarium, which agreed with the original description and was collected from wood of Corylus avellana in southern England. Using this reference specimen, we compiled genomic and ITS barcoding datasets to explore the genetic and geographic variation within this species. C_LIO_LIWe showed that G. lingzhi and G. lucidum diverged more than 12 million years ago and that all seven "G. lucidum" genomes deposited in public databases belong to other species. More than 1000 barcoding sequences showed that the widely used homology-based ITS barcoding is not working in this group, which can be mitigated by a phylogenetic placement approach. The 149 sequences assigned to G. lucidum with high confidence showed a Eurasian distribution and introductions to North and South America and Africa. C_LIO_LIOur study underscores the importance of accurate species identification and provides guidance for a group of pharmaceutical and socially significant species. To further support future studies and the wider public in differentiating between G. lingzhi and G. lucidum, we propose using "False Lingzhi" as the English name for G. lucidum. C_LI Societal Impact StatementTraditional Chinese Medicine has expanded far beyond Asia, with growing markets in North America and Europe for supplements and functional foods. Lingzhi or Reishi (Ganoderma lingzhi), a well-known medicinal mushroom, is valued for its anti-inflammatory and anticancer properties. However, it is often misidentified with species that may not provide the same health benefits. This confusion poses risks to consumer safety, product regulation, and research. Here, we establish a reference using morphological and molecular tools for the most commonly misidentified species (Ganoderma lucidum) and propose the name "False Lingzhi" to distinguish it, supporting accurate identification, safer product development, and reliable research.

BackgroundThe global rise of multidrug-resistant (MDR) bacteria represents a critical public health threat, and Romania ranks amongst the most affected countries in Europe. As conventional therapy increasingly fails, bacteriophage therapy has re-emerged as a promising alternative to antibiotics. Urban rivers, contaminated with resistant bacterial strains, represent an underexplored and accessible reservoir for the isolation of lytic phages with therapeutic potential. MethodsTwo bacteriophages, 17M_Ec17_D and 22C_Ec22_D, were isolated from the Dambovita River, Bucharest, Romania, using MDR E. coli as host bacteria. Phage characterization included plaque morphology, transmission electron microscopy, and host range assessment by spot assay against 30 MDR E. coli isolates. Whole genome sequencing was performed on Illumina MiSeq and Oxford Nanopore Technologies MinION platforms, followed by bioinformatic analysis including taxonomic classification, lifestyle prediction, and functional annotation. ResultsBoth phages formed clear plaques and were classified as Kayfunavirus (17M_Ec17_D, Podoviridae-like) and Kagunavirus (22C_Ec22_D, Siphoviridae-like) with nucleotide similarities of 89.2% and 71.4% to their closest relatives, respectively, suggesting both are candidates for novel species. Host range analysis revealed lytic activity against 13% and 10% of tested MDR isolates, with complementary infection profiles. Genomic analysis confirmed a strictly lytic lifestyle for both phages, supported by the presence of holin and spanin genes and the absence of lysogenic modules, antibiotic resistance genes, and virulence factors. ConclusionsTo the best of our knowledge, this is the first study conducted in Romania to isolate and genomically characterize lytic bacteriophages targeting MDR E. coli. The characterized phages represent safe therapeutic candidates whose complementary host ranges suggest potential application as part of phage cocktail to broaden antimicrobial coverage against MDR infections.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

BackgroundInfectious bovine keratoconjunctivitis is the most important cattle ocular disease worldwide. The infection is primarily caused by Moraxella bovis and is a highly contagious disease that significantly affects cattle welfare. Currently, antibiotic medication is the primary treatment for infectious bovine keratoconjunctivitis. However, with rising concerns over antibiotic resistance, we propose developing a more targeted therapeutic strategy using bacteriophages (phages). Materials and MethodsWe have isolated the first known Moraxella bovis phages, characterised them according to their genome sequence, local virulence index and with transmission electron microscopy. The host ranges were assessed using 41 clinical M. bovis strains isolated from infected cows. ResultsFour phages were isolated and characterised. Comparative analysis identified a high degree of genomic similarity between the phages MB15, MB16, MB26 and MB43. MB43 was the most distinct, with the smallest host range phenotype. ConclusionsThe isolated phages show therapeutic potential for further development against Moraxella infections.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

BackgroundEnvironmental conditions shape the evolutionary trajectories of RNA viruses, yet little is known about how complex physical stressors such as microgravity influence host-virus interactions and viral evolution. Here, we investigated the short-term evolutionary consequences of simulated microgravity on the Caenorhabditis elegans - Orsay virus (OrV) system. MethodsOrV was subjected to six serial passages in hosts acclimated to low-shear modeled microgravity, with parallel evolution under standard-gravity. Evolutionary outcomes were evaluated using virulence, transmission, and replication traits, all measured under standard-gravity conditions. ResultsViral load fluctuated across passages in both environments, with lower mean accumulation in microgravity-evolved lineages. After evolution, we detected no significant changes in virulence. Transmission increased in standard-gravity lineages but not in microgravity-evolved ones, while viral replication decreased in all lineages, with a stronger decline in those evolved under microgravity. However, the magnitude of phenotypic changes was generally modest. DiscussionThese results indicate that evolution under microgravity can alter viral phenotypic trajectories over short timescales. However, because all traits were assayed under standard-gravity conditions, we cannot directly assess local adaptation to microgravity, and the observed differences may reflect environment-specific trade-offs rather than reduced fitness per se. Furthermore, the limited number of passages and the modest magnitude of phenotypic change suggest that evolutionary responses may still be in an early stage. ConclusionOverall, our findings provide initial evidence that simulated microgravity can influence the evolutionary dynamics of an RNA virus, while highlighting the need for reciprocal fitness assays and longer-term experiments to fully characterize adaptation to altered gravitational environments.

Pairwise analysis of genomic and spatial data offers opportunities to identify and estimate the associations between covariates and the transmission of pathogens between individuals. However, such pairwise analyses are computationally intensive, and may not be feasible to conduct given the high dyad count in even moderately sized datasets. Here we compare two approaches to increase the efficiency of pairwise analysis for large datasets. We quantify and compare the performance of divide-and-conquer Bayesian model fitting and pairwise case-control approaches for estimating associations between individual- and pair-level covariates and shared membership in a transmission cluster. We utilize a large dataset (n=4,154) of spatially-referenced, genomically-sequenced Mycobacterium tuberculosis isolates collected from a single city for this analysis. We find that the case-control approach produces unbiased estimates of effect sizes with expected credible interval coverage and is more robust than the divide-and-conquer method when effect sizes are large. Thus, we recommend using the case-control approach with at least three controls per case to downscale datasets for pairwise analysis when analysis of the entire dataset is not possible. This approach mitigates the computational challenges of pairwise Bayesian modeling on datasets that require significant computational resources while maintaining desired inferential properties. Author SummaryPairwise analyses of large datasets to study pathogen transmission are computationally demanding because they typically require simultaneous analysis of each possible pair of individuals in a dataset; as datasets become larger these analyses often are not feasible to conduct even with access to high-performance computing resources. In this work, we compare a case-control approach and divide-and-conquer approaches for more efficient pairwise analysis of large datasets. Using a large dataset of Mycobacterium tuberculosis isolates including genetic and spatial data, we investigate the performance of each method for estimating the associations between host covariates and genetic clustering of isolates. We find that the case-control approach is generally preferred over methods which first divide the data into subsets and then combine results. While additional extensions of these analyses are needed to test the generality of these findings to other data settings, this work provides a practical way forward for the pairwise analysis of large datasets to study pathogen transmission.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 g/ml for CAZ, 8 g/ml for LVX, 16 g/ml for MEM, and 8 g/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 g/ml, 8 g/ml, and 16 g/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCEThe Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

Nearest neighbor parameters are widely used in software for estimating the conformational stability of an RNA sequence folding into a specific structure. Folding stability for RNA with canonical nucleotides A, C, G, and U has been widely studied, but the same is not true for most modified nucleotides. In this work, we present a comprehensive set of nearest neighbor parameters for estimating the folding stability of RNA including pseudouridine in helical or loop contexts. These parameters are derived from 210 optical melting experiments involving helices with pseudouridine-A and pseudouridine-G pairs and with pseudouridine in loop motifs. The experiments include sequences with pseudouridine and U in the same strand, including U-A and U-G pairs, allowing us to consider the folding stability of sequences with both U and pseudouridine. On average, pseudouridine stabilizes RNA folding compared to U in an analogous motif, although this effect is sequence-context dependent. These parameters improve the modeling of folding stability for RNA secondary structures containing pseudouridine. We demonstrate that these parameters successfully model the secondary structure change for Saccharomyces cerevisiae U2 snRNA when two additional inducible pseudouridines are present. These parameters are freely available and incorporated into the RNAstructure software package. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725682v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@e1167aorg.highwire.dtl.DTLVardef@18ac7f0org.highwire.dtl.DTLVardef@4c909eorg.highwire.dtl.DTLVardef@aa8bca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is well known for life-threatening acute infections among the human population. The bacterium can withstand most antibiotics by using their high levels of inherent and acquired resistance mechanisms such as Biofilm-EPS, Persistence, and Quorum sensing (QS). Owing to the importance of adaptive antibiotic multi-drug resistance of P. aeruginosa, the current investigation is aimed to explore the phytochemicals derived from mangrove plants as potential agents to control biofilm and drug resistance mechanisms through a multi-mechanistic computational approach. For identifying potential compounds and target, In-silico drug repurposing technique is implemented by docking/virtual screening of 49 phytochemical compounds against 18 proteins involved in the Persister Cell formation, QS, and EPS synthesis in P. aeruginosa which resulted the proteins RelA and SpoT (persistence), PqsA, and PqSR (QS), and PelA and PelB (EPS synthesis) and compounds Taraxerone and Taraxerol to be potential. The results of docking were well corroborated with MD simulations. These targets and compounds explored through in-silico approach, are found to target potential antimicrobial pathways involving EPS synthesis, persistence genes, and QS, aiming to enhance antibiotic efficacy. Further, this study could be reference for in-vivo and in-vitro investigations to evaluate the further effectiveness of the compounds and potentiality of the proteins for MDR therapeutics of P. aeruginosa.