Limited Sequence Research Articles

Methylome of circulating cell free DNA as a novel biomarker tool: from acute coronary syndrome to broader risk stratification of cardiovascular disease

Abstract Background Cell death during tissue injury leads to release of chromosomal DNA which is promptly degraded. However, short DNA fragments wrapped around nucleosomes survive longer in the bloodstream and can be isolated as circulating cell-free DNA (ccfDNA). Oncology field pioneered the use of cfDNA as clinical biomarker by identifying sequence modifications induced in cancer cells. Importantly, ccfDNA also retains the same epigenetic information as the tissue of origin. Despite major advances for disease diagnosis and therapy, coronary artery diseases (CAD) and associated conditions such acute myocardial infarction (AMI) or heart failure (HF) are responsible for leading number of deaths worldwide. Preventive and surveillance strategies with non-invasive testing are needed. Purpose Evaluation of ccfDNA methylation profiles as prognostic non-invasive biomarker for risk stratification of acute coronary syndromes and their potential application in preventive and surveillance strategies in other cardiovascular disorders. Methods CcfDNA isolated from citrate-plasma collected from our discovery cohort, comprising healthy controls, STEMI, NSTEMI and unstable angina (UA) patients, was analyzed applying Whole Genome Bisulfite Sequencing (WGBS) using a low-input BS-seq (PBAT) protocol. Computational analysis generated differentially methylated regions (DMRs) by tiling CpG areas and setting a threshold of minimal 25% methylation difference compared to the healthy group. To assess the validity of our findings, a selection of disease-specific DMRs were tested using targeted methylation sequencing protocol in a new set of patients. This procedure consisted in enzymatic conversion of ccfDNA methylated sites followed by probe-specific enrichment libraries and sequencing at a Novaseq 6000 platform. Results The study identified a total of 1941 DMRs and from those 486 were STEMI, 223 NSTEMI and 684 UA specific. From the identified DMRs, 74.13% from STEMI, 72.68% from NSTEMI and 61.62% from UA annotated with heart-related diseases using DisGeNET gene-disease associations. Interestingly, most of identified DMRs located within intronic regions (~60%) and also annotated as enhancers and lncRNAs. The independent validation with targeted methylation sequencing identified 566, 306 and 248 DMRs, respectively, and reduced the number of disease-specific DMRs to 171 in STEMI, 115 in NSTEMI and 82 in UA. Conclusion and Future Approaches Methylation profiles of ccfDNA showed potential as a biomarker to stratify the severity of acute coronary syndromes, a novel non-invasive detection methodology that could be extrapolated to risk stratification in other cardiovascular disease such as heart failure or coronary artery disease.

Adaptation of a Model Spike Aptamer for Isothermal Amplification-Based Sensing

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer’s dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

Steered molecular dynamics simulation as a post-process to optimize the iBRAB-designed Fab model.

Therapeutic monoclonal antibodies are an effective method of treating acute infectious diseases. However, knowing which of the produced antibodies in the vast number of human antibodies can cure the disease requires a long time and advanced technology. The previously introduced iBRAB method relies on studied antibodies to design a broad-spectrum antibody capable of neutralizing antigens of many different Influenza A viral strains. To evaluate the antigen-binding fragment as an applicable drug, the therapeutic antibody profiles providing guidelines collected from clinically staged therapeutic antibodies were used to access different measurements. Although the evaluated values were within an accepted range, the modification in the amino acid sequence is required for better properties. Thus, using the steered molecular dynamics(SMD) simulation to determine the binding capacity of amino acids in the functional region, the profile of interacted amino acids of Fab with the antigen was established for modified reference. As a result, the model was modified with amino acids elimination at positions 96-97 in the heavy chain and 26-27, 91, 96-97, and 102-103 in the light chain, which has better Therapeutic Antibody Profiler evaluations than the original designation. Thus again, SMD simulation is a promising computational approach for post-modification in rational drug design.

Long sequence Hopfield memory*

Abstract Sequence memory is an essential attribute of natural and artificial intelligence that enables agents to encode, store, and retrieve complex sequences of stimuli and actions. Computational models of sequence memory have been proposed where recurrent Hopfield-like neural networks are trained with temporally asymmetric Hebbian rules. However, these networks suffer from limited sequence capacity (maximal length of the stored sequence) due to interference between the memories. Inspired by recent work on Dense Associative Memories, we expand the sequence capacity of these models by introducing a nonlinear interaction term, enhancing separation between the patterns. We derive novel scaling laws for sequence capacity with respect to network size, significantly outperforming existing scaling laws for models based on traditional Hopfield networks, and verify these theoretical results with numerical simulation. Moreover, we introduce a generalized pseudoinverse rule to recall sequences of highly correlated patterns. Finally, we extend this model to store sequences with variable timing between states’ transitions and describe a biologically-plausible implementation, with connections to motor neuroscience.

An efficient C-glycoside production platform enabled by rationally tuning the chemoselectivity of glycosyltransferases

Despite the broad potential applications of C-glycosides, facile synthetic methods remain scarce. Transforming glycosyltransferases with promiscuous or natural O-specific chemoselectivity to C-glycosyltransferases is challenging. Here, we employ rational directed evolution of the glycosyltransferase MiCGT to generate MiCGT-QDP and MiCGT-ATD mutants which either enhance C-glycosylation or switch to O-glycosylation, respectively. Structural analysis and computational simulations reveal that substrate binding mode govern C-/O-glycosylation selectivity. Notably, directed evolution and mechanism analysis pinpoint the crucial residues dictating the binding mode, enabling the rational design of four enzymes with superior non-inherent chemoselectivity, despite limited sequence homology. Moreover, our best mutants undergo testing with 34 substrates, demonstrating superb chemoselectivities, regioselectivities, and activities. Remarkably, three C-glycosides and an O-glycoside are produced on a gram scale, demonstrating practical utility. This work establishes a highly selective platform for diverse glycosides, and offers a practical strategy for creating various types of glycosylation platforms to access pharmaceutically and medicinally interesting products.

Detection and characterization of Campylobacter in air samples from poultry houses using shot-gun metagenomics – a pilot study

BackgroundFoodborne pathogens such as Campylobacter jejuni are responsible for a large proportion of the gastrointestinal infections worldwide associated with poultry meat. Campylobacter spp. can be found in the chicken fecal microbiome and can contaminate poultry meat during the slaughter process. Commonly used sampling methods to detect Campylobacter spp. at poultry farms use fecal droppings or boot swabs in combination with conventional culture techniques or PCR. In this pilot study, we have used air filtering and filters spiked with mock communities in combination with shotgun metagenomics to detect Campylobacter and test the applicability of this approach for the detection and characterization of foodborne pathogens. To the best of our knowledge is this the first study that combines air filtering with shotgun metagenomic sequencing for detection and characterization of Campylobacter.ResultsAnalysis of air filters spiked with different levels of Campylobacter, into a background of mock or poultry house communities, indicated that we could detect as little as 200 colony forming units (CFU) Campylobacter per sample using our protocols. The results indicate that even with limited sequencing effort we could detect Campylobacter in the samples analysed in this study. We observed significant amounts of Campylobacter in real-life samples from poultry houses using both real-time PCR as well as shotgun metagenomics, suggesting that the flocks in both houses were infected with Campylobacter spp. Interestingly, in both houses we find diverse microbial communities present in the indoor air which reflect the fecal microbiome of poultry. Some of the identified genera such as Staphylococcus, Escherichia and Pseudomonas are known to contain opportunistic pathogenic species.ConclusionsThese results show that air sampling of poultry houses in combination with shotgun metagenomics can detect and identify Campylobacter spp. present at low levels. This is important since early detection of Campylobacter enables measures to be put in place to ensure the safety of broiler products, animal health and public health. This approach has the potential to detect any pathogen present in poultry house air.

Syntenic lncRNA locus exhibits DNA regulatory functions with sequence evolution

Syntenic long non-coding RNAs (lncRNAs) often show limited sequence conservation across species, prompting concern in the field. This study delves into functional signatures of syntenic lncRNAs between humans and zebrafish. Syntenic lncRNAs are highly expressed in zebrafish, with ∼90 % located near protein-coding genes, either in sense or antisense orientation. During early zebrafish development and in human embryonic stem cells (H1-hESC), syntenic lncRNA loci are enriched with cis-regulatory repressor signatures, influencing the expression of development-associated genes. In later zebrafish developmental stages and specific human cell lines, these syntenic lncRNA loci function as enhancers or transcription start sites (TSS) for protein-coding genes. Analysis of transposable elements (TEs) in syntenic lncRNA sequences revealed intriguing patterns: human lncRNAs are enriched in simple repeat elements, while their zebrafish counterparts show enrichment in LTR elements. This sequence evolution likely arises from post-rearrangement mutations that enhance DNA elements or cis-regulatory functions. It may also contribute to vertebrate innovation by creating novel transcription factor binding sites within the locus. This study highlights the conserved functionality of syntenic lncRNA loci through DNA elements, emphasizing their conserved roles across species despite sequence divergence.

Breeding season temporal and spatial trends in continental-scale migration of the monarch butterfly.

The monarch butterfly (Danaus plexippus) is a vagile species that undertakes an annual, multi-generational migration across North America. The abundance of this species at both eastern and western overwintering sites in Central Mexico and California indicates a population decline. Success of continental-scale conservation programs for a migratory species depends on providing, maintaining, and protecting habitats at appropriate temporal and spatial scales. Here, dynamics of monarch continental-scale migration and gene flow were obtained by combined stable isotope, morphological, and genetic analyses. These analyses were applied to temporal monarch samples collected from May to September during 2016-2021 at locations in Iowa, USA and spatial collections from Pennsylvania, Delaware, Iowa, Ohio, Nevada, Idaho, Hawaii, 3 Australian locations during July and August 2016, and Texas in April 2021. Evidence of seasonal multi-generational migration was obtained through δ2H analyses of spatial collections, which was corroborated by decreased wing hue (a morphological marker for non-migratory individuals). In Iowa, 10-15% of monarchs represented migrants from southern areas throughout the breeding season and 6% were migrants from the North in midsummer. Limited sequence variation detected across the mitochondrial genome impacted the capability to detect significant population genetic variation in our North American samples. However, 2 novel substitutions were identified and predicted to be fixed among Australia samples, contributing to intercontinental differentiation from counterparts in North America. Our assessment of temporal and spatial population dynamics across the North American monarch breeding range provides insight into continental-scale migration and previously undetected mitochondrial DNA variation among globally distributed populations.

YkuR functions as a protein deacetylase in Streptococcus mutans

Protein acetylation is a common and reversible posttranslational modification tightly governed by protein acetyltransferases and deacetylases crucial for various biological processes in both eukaryotes and prokaryotes. Although recent studies have characterized many acetyltransferases in diverse bacterial species, only a few protein deacetylases have been identified in prokaryotes, perhaps in part due to their limited sequence homology. In this study, we identified YkuR, encoded by smu_318, as a unique protein deacetylase in Streptococcus mutans. Through protein acetylome analysis, we demonstrated that the deletion of ykuR significantly upregulated protein acetylation levels, affecting key enzymes in translation processes and metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, and biofilm formation. In particular, YkuR modulated extracellular polysaccharide synthesis and biofilm formation through the direct deacetylation of glucosyltransferases (Gtfs) in the presence of NAD+. Intriguingly, YkuR can be acetylated in a nonenzymatic manner, which then negatively regulated its deacetylase activity, suggesting the presence of a self-regulatory mechanism. Moreover, in vivo studies further demonstrated that the deletion of ykuR attenuated the cariogenicity of S. mutans in the rat caries model, substantiating its involvement in the pathogenesis of dental caries. Therefore, our study revealed a unique regulatory mechanism mediated by YkuR through protein deacetylation that regulates the physiology and pathogenicity of S. mutans.

Immunomodulatory effects of PACAP sequence modifications in juvenile white shrimp Litopenaeus vannamei

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved multifunctional neuropeptide belonging to the secretin/glucagon/vasoactive intestinal peptide superfamily. In recent years, the role of PACAP in immunoregulation has been partially elucidated in mammals and teleost fishes, demonstrating that PACAP modulates both innate and acquired immunity. However, in invertebrates, little is known about its immunostimulant and antimicrobial properties. In previous research, studies performed in Litopenaeus (Penaeus) vannamei have revealed that Clarias gariepinus PACAP-38 improves immune functions like lectins, superoxide dismutase and nitric oxide synthase derived metabolites in postlarvae shrimp. In this study, the immunomodulatory effects of C. gariepinus PACAP-38 and four PACAP sequence modified peptides in juvenile L. vannamei were evaluated. The effect of several administrations of PACAP and its variants on shrimp immune effectors, as well as the effect of different doses was examined. Results revealed that PACAP can influence hemolymph immune parameters, and immune-related gene mRNA levels in the hemocytes, gill and hepatopancreas with different effects depending on the modification of the peptide sequence, the dose and the tissue assessed. Altogether, these findings provide valuable insights regarding the role of PACAP in crustacean innate immunity and highlight the need for further research into its potential application as an alternative to antibiotics in aquaculture.

Fatigue delamination damage analysis in composite materials through a rule of mixtures approach

The present study investigates delamination damage initiation and propagation within a homogenization theory of mixtures, using the concept of virtual layers and virtual interfaces. It eliminates spatial discretization of layers, introducing a resultant damage variable to capture structure’s bulk response under both monotonic and cyclic loads. Fatigue-induced deterioration is classified into sub-critical, critical, and over-critical stages based on interfacial stresses. Calibration is conducted employing the widely-available Wöhler curves for each loading mode independently. An advance-in-time strategy is included in the model to enhance the simulation speed. The reliability of the approach is assessed for crack initiation and propagation separately through standard test coupons, showing good correlation with experimental data in mode I, mode II, and mixed-mode loading conditions. Depending on the calibration procedure adopted, the model is applicable to a wide range of stress ratios. In addition, it could be integrated into any standard finite element framework using the desired number of elements through the thickness regardless of the physical amount of layers. This allows easy modification of stacking sequences or the number of layers within the constitutive law without mesh structure changes, facilitating simulation of large-scale composite laminates with minimal accuracy loss and reduced computational costs.

Breaking Down the Bottlebrush: Atomically Detailed Structural Dynamics of Mucins.

Mucins, the biomolecular components of mucus, are glycoproteins that form a thick physical barrier at all tissue-air interfaces, forming a first line of defense against pathogens. Structural features of mucins and their interactions with other biomolecules remain largely unexplored due to the challenges associated with their high-resolution characterization. Combining limited mass spectrometry glycomics and protein sequencing data, we present all-atom, explicitly solvated molecular dynamics simulations of a major respiratory mucin, MUC5B. We detail key forces and degrees of freedom imposed by the extensive O-glycosylation, which imbue the canonically observed bottlebrush-like structures to these otherwise intrinsically disordered protein backbones. We compare our simulation results to static structures observed in recent scanning tunneling microscopy experiments as well as other published experimental efforts. Our work represents the demonstration of a workflow applied to a mucin example, which we hope will be employed by other groups to investigate the dynamics and interactions of other mucins, which can inform on structural details currently inaccessible to experimental techniques.

A Synthetic Biology Approach to Transgene Expression in Insects.

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3' untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

Multifunctional Ethyl Violet@NH2-MIL-88B(Fe) Hybrids: CRISPR-Cas12a-Assisted PEC-FL-CL Triple-Mode Sensitive Detection of HPV-16.

The multimode assay based on multiple response mechanisms has received great attention to effectively improve the accuracy of a sensing platform. However, multifunctional sensing materials for simultaneously satisfying the multiple-mode detections are still in shortage due to the incompatibility of the signal transduction mechanisms in different modes. Here, taking human papillomavirus 16 (HPV-16) DNA (TDNA) as the model due to its important role in cervical cancer, a novel multifunctional material, ethyl violet (EV)@NH2-MIL-88B(Fe) (ENM) hybrids, have been successfully prepared, which could simultaneously satisfy CRISPR-Cas12a-assisted photoelectrochemical (PEC)-fluorescent (FL)-colorimetric (CL) triple-mode detection of TDNA. Based on the TDNA-induced trans-cleavage ability of CRISPR-Cas12a and efficient separation of magnetic beads, ENM was obtained from the single-stranded DNA-surrounded streptavidin-modified magnetic beads-ENM (SMB-ssDNA-ENM) and decomposed by pyrophosphate to get free EV, 2-aminoterephthalic acid (NH2-BDC), and Fe3+. Thus, TDNA was sensitively detected based on the EV-enhanced PEC signal of SnS2 nanosheets (PEC mode), fluorescent signal of NH2-BDC (FL mode), and characteristic absorption peak at about 720 nm of Fe3+-induced Prussian blue (PB) (CL mode). The designed PEC-FL-CL triple-mode biosensing platform had good performance for the detection of TDNA with a wide linear range (0.1 fM-100 nM) and ultralow detection limits (0.07 fM for PEC, 0.03 fM for FL and 0.09 fM for CL). Additionally, the developed PEC-FL-CL triple-mode biosensing platform has great potential for applications in early disease diagnosis and bioanalysis, as it can be easily extended to other DNA assays through modification of the crRNA sequence within the CRISPR-Cas12a system.

Local powers of least‐squares‐based test for panel fractional Ornstein–Uhlenbeck process

In recent years, significant advancements have been made in the field of identifying financial asset price bubbles, particularly through the development of time‐series unit‐root tests featuring fractionally integrated errors and panel unit‐root tests. This study introduces an innovative approach for assessing the sign of the persistence parameter () within a panel fractional Ornstein‐Uhlenbeck process, based on the least squares estimator of . This method incorporates three distinct test statistics based on the Hurst parameter (), which can take values in the range of , be equal to , or fall within the interval of . The null hypothesis corresponds to . Based on a panel of continuous records of observations, the null asymptotic distributions are obtained when the time span () is fixed and the number of cross sections () goes to infinity. The power function of the tests is obtained under the local alternative where is close to zero in the order of . This alternative covers the departure from the unit root hypothesis from the explosive side, enabling the calculation of lower power in bubble tests. The hypothesis testing problem and the local power function are also considered when a panel of discrete‐sampled observations is available under a sequential limit, that is, the sampling interval shrinks to zero followed by the goes to infinity.

Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.

Potential of training of anti-Staphylococcus aureus therapeutic phages against Staphylococcus epidermidis multidrug-resistant isolates is restricted by inter- and intra-sequence type specificity.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

The classification, origin, and evolutionary dynamics of severe fever with thrombocytopenia syndrome virus circulating in East Asia.

The classification of severe fever with thrombocytopenia syndrome virus (SFTSV) lacked consistency due to limited virus sequences used across previous studies, and the origin and transmission dynamics of the SFTSV remains not fully understood. In this study, we analyzed the diversity and phylodynamics of SFTSV using the most comprehensive and largest dataset publicly available for a better understanding of SFTSV classification and transmission. A total of 1267 L segments, 1289 M segments, and 1438 S segments collected from China, South Korea, and Japan were included in this study. Maximum likelihood trees were reconstructed to classify the lineages. Discrete phylogeographic analysis was conducted to infer the phylodynamics of SFTSV. We found that the L, M, and S segments were highly conserved, with mean pairwise nucleotide distances of 2.80, 3.36, and 3.35% and could be separated into 16, 13, and 15 lineages, respectively. The evolutionary rate for L, M, and the S segment was 0.61 × 10-4 (95% HPD: 0.48-0.73 × 10-4), 1.31 × 10-4 (95% HPD: 0.77-1.77 × 10-4) and 1.27 × 10-4 (95% HPD: 0.65-1.85 × 10-4) subs/site/year. The SFTSV most likely originated from South Korea around the year of 1617.6 (95% HPD: 1513.1-1724.3), 1700.4 (95% HPD: 1493.7-1814.0), and 1790.1 (95% HPD: 1605.4-1887.2) for L, M, and S segments, respectively. Hubei Province in China played a critical role in the geographical expansion of the SFTSV. The effective population size of SFTSV peaked around 2010 to 2013. We also identified several codons under positive selection in the RdRp, Gn-Gc, and NS genes. By leveraging the largest dataset of SFTSV, our analysis could provide new insights into the evolution and dispersal of SFTSV, which may be beneficial for the control and prevention of severe fever with thrombocytopenia syndrome.

Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.