Biology subjects

Tran, T. T.

Publications and source records attributed to Tran, T. T..

At least 19 recordsLinked to original sources

bioRxiv2026

The Cost to Scroll: Short-form Video Use and Episodic Memory

Short-form video platforms expose users to rapid streams of novel content; these features are posited to place heavy demands on cognitive processes. In a preregistered online study, we used device-reported screen time to examine whether short-form video use is associated with sustained attention and episodic memory in participants aged 18-24 yrs (N = 502). During a sustained attention task, participants made indoor/outdoor decisions on scene stimuli, with 90% frequent trials and 10% infrequent trials; the subsequent surprise recognition test assessed memory for these items. Among participants with device-reported TikTok or Instagram use (N = 398), short-form video use was not associated with attention performance. By contrast, greater short-form video use was associated with poorer episodic memory; this relationship was driven by poorer recognition of infrequent-category items. These findings connect heavier short-form video consumption with poorer episodic memory in young adults, showing how daily digital habits co-vary with memory function.

neuroscience↗

bioRxiv2026

Ketogenic diet is protective during endotoxin-induced lung injury through the elevation of BHB

Acute respiratory distress syndrome (ARDS) is marked by severe pulmonary edema and concomitant hypoxia, affecting hundreds of thousands of people a year, especially those in critical care conditions or suffering from septic shock. Previous studies have implicated that the ketogenic diet, a high-fat and low-carbohydrate diet, modulates inflammatory responses. However, the impact of the ketogenic diet on septic ARDS outcomes is unknown. Here, we demonstrated that mice on a ketogenic diet showed strikingly reduced lung injury and inflammation compared to those on a control diet during a murine model of endotoxin-induced lung injury, induced by intratracheal lipopolysaccharide (LPS) injection. Immune mass cytometry studies on lung tissue indicated that the ketogenic diet reduces immune cell infiltration. Treating mice with beta-hydroxybutyrate (BHB), the primary metabolite of ketogenesis, after the onset of ARDS reduced pulmonary edema and lung inflammation, as well as NF-kB activity, suggesting strong therapeutic potential. By multiplex analysis in bronchial alveolar lavage fluid, we observed that the ketogenic diet or BHB administration attenuates the chemotaxis and activation of immune cells. Altogether, our findings reveal that the ketogenic diet provides lung protection during endotoxin-induced lung injury through BHB.

physiology↗

bioRxiv2026

Dissociable structural and molecular pathways of age-related change in sustained attention

Sustained attention, the capacity to maintain goal-directed attention over extended periods, declines with age but with substantial individual variability across cognitively unimpaired (CU) older adults. We sought to determine the contributions of microstructural deterioration of the superior longitudinal fasciculus (SLF), the principal white-matter tract coupling prefrontal and parietal nodes of the dorsal attention network, as well as markers of preclinical Alzheimers disease (AD) to individual differences in sustained attention in ageing. In 162 CU older adults (mean (SD) age = 73.0 (4.9), %F = 64.8) drawn from two Stanford ageing cohorts, we measured sustained attention using the gradual-onset Continuous Performance Task (gradCPT). Performance was indexed by a composite score (Att-Z) derived from discriminability (d') and response-time variability (RTV). We used diffusion MRI tractography to quantify SLF fractional anisotropy (FA) and mean diffusivity (MD) alongside two control tracts, the corticospinal tract (CST) and cingulum (CGC). NULISA multiplex plasma immunoassays were used to measured AD-related pathology (pTau-181, pTau-217), neuroaxonal injury (NfL), and astrocytic reactivity (GFAP; plasma subsample N = 146). Parallel mediation and commonality analyses tested whether structural and plasma biomarker pathways contribute independently to individual differences in sustained attention function. Older age was associated with lower Att-Z ({beta} = -0.315, p < 0.001). In simultaneous three-tract regression models, only SLF microstructure uniquely predicted Att-Z (FA: {beta} = +0.275, p < 0.001; MD: {beta} = -0.320, p < 0.001). SLF microstructure mediated the age-attention relationship (FA indirect {beta}unstandardised = -0.01, pboot <0.01, MD indirect {beta}unstandardised = -0.009 to -0.01, pboot <0.05). Plasma pTau-181 ({beta} = -0.212, pFDR < 0.03), pTau-217 ({beta} = -0.163, pFDR < 0.05), and NfL ({beta} = -0.156, pFDR < 0.05) were negatively associated with Att-Z and each mediated effects of age on performance (pTau-181 indirect {beta}unstandardised = -0.008, pboot < 0.01; pTau-217 indirect {beta}unstandardised= -0.006, pboot< 0.05; NfL indirect {beta}unstandardised = -0.011, pboot< 0.05), whereas GFAP showed no association with sustained attention in any model. SLF microstructure was negatively associated with age (FA {beta} = -0.207, p < 0.01; MD {beta} = +0.270, p < 0.001) but was not significantly associated with plasma biomarkers (all p > 0.10). In parallel mediation models, SLF microstructure and plasma pTau exhibited significant independent indirect effects on Att-Z (SLF FA indirect {beta}unstandardised= -0.010, pboot< 0.01; SLF MD indirect {beta}unstandardised = -0.009 to -0.010, pboot< 0.05; pTau-181 indirect {beta}unstandardised = -0.008 to -0.009, pboot< 0.01; pTau-217 indirect {beta}unstandardised= -0.006 to -0.007, pboot< 0.05). Effects of NfL were attenuated when combined in a model with pTau (indirect {beta}unstandardised = -0.005 to -0.008, pboot> 0.15), suggesting shared variance among these markers. These findings reveal multiple pathways that contribute to individual differences in sustained attention in ageing that are detectable before clinical impairment, including SLF white matter integrity -- which is linked to the structural disconnection of the dorsal attention network -- and preclinical AD pathology. The independence of these pathways identifies two separable biological targets for preserving attentional capacity in CU older adults.

neuroscience↗

bioRxiv2026

Whole-body 3D kinematics of freely behaving Drosophila

Understanding how nervous systems generate coordinated movement requires precise measurement of body kinematics during natural behavior. The fruit fly, Drosophila, is a model organism with sophisticated behavior and well-studied neural circuits, but tracking fly movements in 3D remains challenging because of their teeny bodies, rapid movements, and frequent self-occlusions. Here we present a pipeline for markerless, full-body 3D pose estimation of fly terrestrial behavior, combining seven synchronized high-speed cameras to capture whole-body kinematics at 800 frames per second. We trained a hybrid 2D/3D deep learning model to track 50 keypoints, then refined them to produce anatomically feasible kinematic trajectories through a retargeting process that solved an inverse kinematics problem constrained by a biomechanical body model. Analysis of 3D kinematics revealed that flies perform grounded running across their full speed range, without transitioning between discrete gaits. Using multi-animal tracking, we found that courting males coordinate both wings during song and modulate body pitch to track the female's vertical position. Our open-source pipeline and large 3D kinematic dataset of fly behavior provide a foundation for neuromechanical modeling and mechanistic studies of motor control in a genetically tractable model organism.

neuroscience↗

bioRxiv2026

DETECTION OF BIOSYNTHETIC GENE CLUSTERS FROM METAGENOME OF NATURAL HONEY COLLECTED IN VIETNAM

The antimicrobial properties of natural honey are partly attributed to bioactive secondary metabolites produced by its associated microbial communities, yet the biosynthetic capacity of these communities remains poorly characterized. Here, we applied a metagenomic approach to investigate the biosynthetic gene cluster (BGC) diversity of bacteria associated with Apis cerana honey from the Northwest mountainous region of Vietnam - a biogeographically distinct and underexplored ecosystem. A total of 366 BGCs spanning 38 compound classes were identified, with terpenes, nonribosomal peptide synthetases (NRPS), and ribosomally synthesized and post-translationally modified peptides (RiPPs) being the most prevalent. Strikingly, 304 BGCs (>83%) lacked close matches in the MIBiG reference database, indicating a high degree of biosynthetic novelty relative to previously characterized natural product repertoires. Among the identified clusters, an azole-containing RiPP BGC recovered from a metagenome-assembled genome (MAG) assigned to Atlantibacter hermannii was predicted to exhibit strong antibacterial activity, with a probability score of 74.5%, representing a prioritized target for heterologous expression and bioactivity validation. These findings establish the Apis cerana honey microbiome as a tractable and largely untapped reservoir for applied microbial research, with direct implications for the discovery of novel antimicrobial agents from underexplored environmental niches. ImportanceAntimicrobial resistance represents one of the most pressing challenges in modern medicine, driving urgent demand for novel bioactive compounds from underexplored microbial sources. Honey-associated bacterial communities are recognized contributors to the antimicrobial properties of natural honey, yet their biosynthetic capacity remains poorly characterized at the metagenomic level. This study demonstrates that the microbiome of Apis cerana honey from a biogeographically distinct region of Vietnam harbors extensive and largely novel biosynthetic gene cluster diversity, with over 83% (304/366) of identified BGCs lacking database references. These findings position honey microbiomes as a tractable and underutilized reservoir for applied microbial research, with direct relevance to the discovery of new antimicrobial agents. The identification of a candidate antibacterial RiPP BGC in Atlantibacter hermannii provides a concrete target for future cultivation-based and heterologous expression studies, bridging metagenomic discovery with applied biotechnological pipelines.

microbiology↗

bioRxiv2026

Proteome-wide multi-omics profiling of osteosarcoma transcription factor networks

Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

biochemistry↗

bioRxiv2025

Clinical and Genomic Characterization of Recalcitrant Enterococcal Bacteremia: A Multicenter Prospective Cohort Study (VENOUS)

BackgroundPatients with recalcitrant enterococcal bloodstream infections are at greater risk of adverse outcomes. We identified patients in the 2016-2022 Vancomycin-Resistant Enterococcal Bacteremia Outcomes Study (VENOUS) cohort experiencing recalcitrant bloodstream infections for further clinical and genomic characterization. MethodsBacteremia episodes were considered "persistent" if there was a lack of clearance on day four while receiving [≥] 48 hours of active therapy and recurrent if there was clearance during hospitalization with a subsequent positive culture (collectively, "recalcitrant" bacteremia). A matched comparison group of non-recalcitrant bacteremia patients was chosen in a 2:1 control:case ratio. Isolates were subjected to short- and long-read whole-genome sequencing. Hybrid assemblies were created using a custom pipeline. Findings. A total of 46 recalcitrant infections from 41 patients were identified. Patients with persistent bacteremia were more often admitted to the ICU upon admission relative to controls. E. faecalis strains causing persistent infections had a significantly higher proportion of genes associated with carbohydrate utilization relative to controls. Representation of functional groups associated with mutated genes was disparate between E. faecium and E. faecalis index and persistent isolates, suggesting species-specific adaptation. DiscussionEnterococcal isolates causing recalcitrant bacteremia were genomically diverse, indicating that strain-specific signatures are not drivers of persistence. However, comparisons of index vs. persistent isolates revealed that E. faecium may be genetically pre-adapted to cause persistent infection, and site-specific structural variation during infection suggests the role of differential gene expression in adaptation and persistence. This data lays groundwork for future studies to define signatures of enterococcal adaptation during bacteremia.

genomics↗

bioRxiv2024

Supervised non-negative matrix factorization on cell-free DNA fragmentomic features enhances early cancer detection

BackgroundCell-free circulating DNA (cfDNA) fragments exhibit non-random patterns in their length (FLEN), end-motif (EM), and distance to nucleosome position (ND). While these cfDNA features have shown promise as inputs for machine learning and deep learning models in early cancer detection, most studies utilize them as raw inputs, overlooking the potential benefits of pre-processing to extract cancer-specific features. This study aims to enhance cancer detection accuracy by developing a novel approach to feature extraction from cfDNA fragmentomics. MethodsWe implemented a supervised non-negative matrix factorization (SNMF) algorithm to generate embedding vectors capturing cancer-specific signals within cfDNA fragmentomic features. These embeddings served as input for a machine learning model to classify cancer patients from healthy individuals. ResultsWe validated our framework using two datasets: an in-house cohort of 431 cancer patients and 442 healthy individuals (dataset 1), and a published cohort comprising 90 hepatocellular carcinoma (HCC) patients and 103 individuals with cirrhosis or hepatitis B (dataset 2). In dataset 1, we achieved an AUC of 94% in pan-cancer detection. In dataset 2, our framework achieved an AUC of 100% for HCC vs healthy classification, 99% for HCC vs non-HCC patients classification, and 96% for identifying HCC patients among a mixed group of non-HCC patients and healthy donors. ConclusionThis study demonstrates the efficiency of SNMF-transformed features in improving both pan-cancer detection and specific HCC detection. Our approach offers a significant advancement in leveraging cfDNA fragmentomics for early cancer detection, potentially enhancing diagnostic accuracy in clinical settings.

bioinformatics↗

bioRxiv2024

Top-down attention and Alzheimer's pathology impact cortical selectivity during learning,influencing episodic memory in older adults

Human aging affects the ability to remember new experiences, in part, because of altered neural function during memory formation. One potential contributor to age-related memory decline is diminished neural selectivity -- i.e., a decline in the differential response of cortical regions to preferred vs. non-preferred stimuli during event perception -- yet the factors driving variability in neural selectivity with age remain unclear. We examined the impact of top-down attention and preclinical Alzheimers disease (AD) pathology on neural selectivity during memory encoding in 156 cognitively unimpaired older participants who underwent fMRI while performing a word-face and word-scene associative memory task. Neural selectivity in face- and place-selective cortical regions was greater during events that were later remembered compared to forgotten. Critically, neural selectivity during learning positively scaled with memory-related variability in top-down attention, whereas selectivity negatively related to early AD pathology, evidenced by elevated plasma pTau181. Path analysis revealed that neural selectivity at encoding mediated the effects of age, top-down attention, and pTau181 on associative memory. Collectively, these data reveal multiple pathways that contribute to memory differences among older adults -- AD-independent reductions in top-down attention and AD-related pathology alter the precision of cortical representations of events during experience, with consequences for remembering.

neuroscience↗

bioRxiv2024

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin

The siderophore-cephalosporin cefiderocol(FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA or piuD from 498 unique isolates collected before the introduction of FDC from 4 clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n=15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

microbiology↗

bioRxiv2023

A (Sub)field Guide to Quality Control in Hippocampal Subfield Segmentation on High-resolution T2-weighted MRI

Inquiries into properties of brain structure and function have progressed due to developments in magnetic resonance imaging (MRI). To sustain progress in investigating and quantifying neuroanatomical details in vivo, the reliability and validity of brain measurements are paramount. Quality control (QC) is a set of procedures for mitigating errors and ensuring the validity and reliability of brain measurements. Despite its importance, there is little guidance on best QC practices and reporting procedures. The study of hippocampal subfields in vivo is a critical case for QC because of their small size, inter-dependent boundary definitions, and common artifacts in the MRI data used for subfield measurements. We addressed this gap by surveying the broader scientific community studying hippocampal subfields on their views and approaches to QC. We received responses from 37 investigators spanning 10 countries, covering different career stages, and studying both healthy and pathological development and aging. In this sample, 81% of researchers considered QC to be very important or important, and 19% viewed it as fairly important. Despite this, only 46% of researchers reported on their QC processes in prior publications. In many instances, lack of reporting appeared due to ambiguous guidance on relevant details and guidance for reporting, rather than absence of QC. Here, we provide recommendations for correcting errors to maximize reliability and minimize bias. We also summarize threats to segmentation accuracy, review common QC methods, and make recommendations for best practices and reporting in publications. Implementing the recommended QC practices will collectively improve inferences to the larger population, as well as have implications for clinical practice and public health.

neuroscience↗

bioRxiv2023

Protein Design by Directed Evolution Guided by Large Language Models

Directed evolution, a strategy for protein engineering, optimizes protein properties (i.e., fitness) by a rigorous and resource-intensive process of screening or selecting among a vast range of mutations. By conducting an in silico screening of sequence properties, machine learning-guided directed evolution (MLDE) can expedite the optimization process and alleviate the experimental workload. In this work, we propose a general MLDE framework in which we apply recent advancements of Deep Learning in protein representation learning and protein property prediction to accelerate the searching and optimization processes. In particular, we introduce an optimization pipeline that utilizes Large Language Models (LLMs) to pinpoint the mutation hotspots in the sequence and then suggest replacements to improve the overall fitness. Our experiments have shown the superior efficiency and efficacy of our proposed framework in the conditional protein generation, in comparision with other state-of-the-art baseline algorithms. We expect this work will shed a new light on not only protein engineering but also on solving combinatorial problems using data-driven methods. Our implementation is publicly available at https://github.com/HySonLab/Directed_Evolution.

bioinformatics↗

bioRxiv2023

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VREfm) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium (Efm) with an activated LiaFSR system. Unlike E. faecalis, Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

microbiology↗

bioRxiv2023

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimers disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the cortices that make up the parahippocampal gyrus (entorhinal and parahippocampal cortices) and the adjacent Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 {micro}m thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized (20X resolution) slices with 5 mm spacing. Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed more gradually. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed human neuroimaging research on the MTL cortex.

neuroscience↗

bioRxiv2023

Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A

ObjectivesTranscript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored. MethodsWe applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis. ResultsWe identified an intronic homozygous MFN2 c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-length MFN2 transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A). DiscussionThis case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.

genetics↗

bioRxiv2022

Multiplexed measurement of binding- and neutralizing antibodies to SARS-CoV-2 variants in 12.000 post-vaccine sera

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

immunology↗

bioRxiv2021

Evolution of Enterococcus faecium to A Combination of Daptomycin and Fosfomycin Reveals Distinct and Diverse Adaptive Strategies

Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) are an important public health threat. VREfm have become increasingly resistant to the front-line antibiotic, daptomycin (DAP). As such, the use of DAP combination therapies (like fosfomycin [FOS]), has received increased attention. Antibiotic combinations could extend the efficacy of current available antibiotics and potentially delay the onset of further resistance. We investigated the potential for E. faecium HOU503, a clinical VREfm isolate that is DAP and FOS susceptible, to develop resistance to a DAP-FOS combination. Of particular interest was whether the genetic drivers for DAP-FOS resistance might be epistatic and, thus, potentially decrease the efficacy of a combinatorial approach in either inhibiting VREfm or in delaying the onset of resistance. We show that resistance to DAP-FOS could be achieved by independent mutations to proteins responsible for cell wall synthesis for FOS and in altering membrane dynamics for DAP. However, we did not observe genetic drivers that exhibited substantial cross-drug epistasis that could undermine DAP-FOS combination. Of interest was that FOS resistance in HOU503 was largely mediated by changes in phosphoenolpyruvate (PEP) flux as a result of mutations in pyruvate kinase (pyk). Increasing PEP flux could be a readily accessible mechanism for FOS resistance in many pathogens. Importantly, we show that HOU503 were able to develop DAP resistance through a variety of biochemical mechanisms and were able to employ different adaptive strategies. Finally, we showed that the addition of FOS can prolong the efficacy of DAP, significantly extending the timeline to resistance in vitro. ImportanceWhile the discovery of antibiotics was one of the greatest health care advances in history, its success is being challenged by the emergence of multidrug-resistant pathogens, including vancomycin-resistant enterococci (VRE). Daptomycin (DAP), a lipopeptide antibiotic that targets cell membrane, is currently prescribed as a frontline drug to treat VRE infections. However, emergence of daptomycin-resistant VRE is concerning. Consequently, DAP-Fosfomycin (FOS) combination (DF) has been proposed as a potential method to maintain DAP efficacy. Here, we provide evidence that DF successfully delayed the emergence of resistance in vitro. Genetic data indicates that resistance was acquired independently, with little evidence of significant cross-drug epistasis that could undermine a combinatorial approach. We also uncovered a novel FOS resistance mechanism, through changes in phosphoenolpyruvate (PEP) flux, that may potentially be shared with other bacterial species. Additionally, we also have evidence showing that E. faecium was able to employ different resistance mechanisms.

evolutionary biology↗

bioRxiv2021

Public T-cell epitopes shared among SARS-CoV-2 variants are presented on prevalent HLA class I alleles

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians.

immunology↗