Biology subjects

Lewis, K. A.

Publications and source records attributed to Lewis, K. A..

14 recordsLinked to original sources

bioRxiv2026

A refined sticker-spacer grammar marks disordered regions with interpretable pathogenic missense variation

Intrinsically disordered regions (IDRs) of proteins show low sequence conservation, and sequence position alone is a weak predictor of disease risk. Phase-separating IDRs (PS-IDRs) are an exception, showing nearly three times higher pathogenic mutation rates than typical IDRs. Here we determine which sequences within that class show the greatest enrichment of pathogenic missense variants and what local features they share. Using pathogenic mutation rates as an independent biological readout, we isolated a core PS-IDR subclass that maximizes pathogenic enrichment. These sequences are defined less by bulk amino acid content than by a refined sticker-spacer grammar in which aromatic residues are embedded in short glycine-rich motifs, while homotypic charge tracts are avoided. Motifs such as YGG/YGGG, FGG/FGGG, and related patterns are elevated 7- to >200-fold relative to conventional IDRs. Comparison of pathogenic and benign substitutions then shows why genetic variation in IDRs has been confounding: the grammar itself is almost never mutated. Instead, the principal pathogenic targets are glycines directly flanked by non-glycine residues. These findings link a specific local sequence architecture to mutational vulnerability and provide a practical framework for interpreting variants in disordered proteins.

biophysics↗

bioRxiv2026

Co-Expressed MicroRNAs Identify Potential Mechanisms Underlying Risk for Multimorbid Depression and Type 2 Diabetes in Midlife Women

BackgroundWomen are disproportionately affected by multimorbid depression and type 2 diabetes (T2D), with prevalence peaking during midlife (40-64 years), a biologically dynamic timeframe due to changes associated with reproductive aging. Yet, phenotypic and mechanistic factors contributing to midlife womens disproportionate risk for co-occurrence remain poorly defined. We previously identified co-expressed microRNAs (miRs) in midlife women with prediabetes that increased odds of assignment to a high psychometabolic risk phenotype. Here, we extend these findings by characterizing putative mRNA targets of these co-expressed miRs and pathways overrepresented among mRNAs, providing insights into potential mechanisms underlying psychometabolic risk in midlife women. MethodsThis study included baseline data from midlife women (ages 40-64 years) with prediabetes who participated in the Diabetes Prevention Program (DPP) (n = 603). In silico analyses were performed using miRTarBase to identify mRNAs regulated by 3 or more of the miRs that most prominently loaded a principal component previously identified to increase odds of assignment to a high psychometabolic risk phenotype defined in this sample. Pathway enrichment analysis was conducted to assess for overrepresentation of KEGG pathways among predicted mRNA targets. To enhance interpretability, pathways were thematically clustered based on their evidenced role in human physiology. ResultsWe identified a total of 13 mRNAs targeted by co-expressed miRs associated with increased odds of assignment to a high psychometabolic risk phenotype in midlife women with prediabetes. Pathway enrichment analysis revealed a total of 71 KEGG pathways with overrepresentation of identified mRNA targets. Four overarching biological themes emerged, reflecting involvement of metabolic, inflammatory, endocrine, and stress/biological weathering-related processes. ConclusionsExperimentally validated mRNA targets related biological pathways were identified, providing multisystem insights into potential mechanisms underlying risk for multimorbid depression and T2D in midlife women. Findings offer mechanistic targets for experimental validation and future precision health research focused on this high-risk population. Overall, this work positions the utility of miRs as context-sensitive biomarkers in the characterization of risk for complex, multimorbid conditions in women during biologically dynamic timeframes.

genomics↗

bioRxiv2025

Perinatal Shifts in Fecal-Derived Metabolites and Associations with Postpartum Depression

IntroductionResearch on maternal depression is largely limited to static, blood-derived biomarkers in the postpartum period and mechanistic targets derived from populations outside the physiological contexts of pregnancy and postpartum, resulting in critical gaps in understanding context-specific mechanisms underlying this debilitating condition. ObjectivesTo examine temporal shifts in the maternal gut metabolome and associations between pregnancy-specific shifts and postpartum depression (PPD). MethodsWe applied untargeted metabolomics (UPLC-MS/MS) to fecal samples collected from participants (N= 25) enrolled in the Maternal and Infant NutriTion (MINT) study. Random forest analysis was used to identify key pathways and metabolites contributing to temporal shifts. Fold change analysis and paired t-tests were used to quantify the magnitude and significance of metabolite changes. Associations between pregnancy-specific changes and PPD (Edinburgh Postnatal Depression Scale at 6 weeks) were identified using Pearsons correlation. ResultsLipid, amino acid, and xenobiotic metabolism emerged as core pathways driving temporal changes in the maternal gut metabolome. The most pronounced shifts occurred from 35 weeks gestation to postpartum, with 55 metabolites significantly altered compared to 24 from 24-to 35 weeks gestation and 26 from 24 weeks gestation to postpartum. Of the 29 metabolites associated with PPD; 68.9% were metabolic intermediates, primarily involved in lipid and amino acid metabolism (58.6%). ConclusionsThis study provides some of the first evidence of temporal shifts in the maternal gut metabolome and associations with PPD, highlighting the importance of lipid and amino acid metabolism and laying the groundwork for future multi-omics research.

systems biology↗

bioRxiv2025

Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

Human in vitro lung models represent advanced tools for studying respiratory infections, particularly those caused by emerging respiratory pathogens. Despite scientific advances, vaccine and therapeutics pre-clinical development has yet to fully adopt human-relevant testing platforms due in part to a lack of validation. In this study, we characterised how static and dynamic flow culture conditions influence microphysiological systems (MPS) generated using primary bronchial and alveolar epithelial cells. We assessed epithelial structure, functional differentiation, and infection dynamics. This study represents the first direct comparison of how dynamic flow and endothelial co-culture influence viral tropism, replication kinetics, and host responses across anatomically distinct regions of the respiratory tract in vitro. Dynamic flow promoted formation of more physiologically relevant tissue architecture, pseudostratified bronchial epithelium and alveolar sac-like structures, with enhanced epithelial differentiation and retention of region-specific cell phenotypes at the transcriptomic level. Both static and dynamic flow models demonstrated responsiveness to inflammatory stimuli (poly(I:C), LPS), producing distinct, tissue-specific cytokine profiles and supporting infection with multiple SARS-CoV- 2 variants. Differences in infection efficiency, viral replication, and host gene expression were observed between variants, with dynamic flow models offering enhanced sensitivity and resolution. In alveolar tissues, dynamic flow increased infection efficiency and reduced variability, enabling more robust and consistent transcriptional responses. This facilitated the identification of interferon signalling pathways as key targets of the host response. Among the variants tested, Delta induced the most extensive tissue damage and strongest transcriptional response, whereas Omicron BA.5 exhibited greater infectivity in alveolar models compared to earlier variants. Our findings demonstrate that dynamic flow MPS more closely replicate human lung tissue architecture and cellular diversity, while also enhancing the predictive power and clinical relevance of airway models for ex vivo studies of SARS-CoV-2 infection. These improvements strengthen the reliability of data generated for the study of host-pathogen interaction studies and support the use of dynamic systems for evaluating novel anti-infectives, immunomodulators, and functional characterisation of immune sera generated by next-generation vaccines. Collectively, our results highlight the value of integrating dynamic in vitro models into preclinical pipelines for emerging respiratory pathogens.

microbiology↗

bioRxiv2025

Missense mutations in intrinsically disordered protein regions link pathogenicity and phase separation

The impact of missense genetic variations on protein function is often enigmatic, especially for mutations that map to intrinsically disordered regions (IDRs). Given the functional importance of phase separation of IDRs, it has been proposed that mutations that modulate phase separation might preferentially lead to disease. To examine this idea, we used the robust predictability of phase-separating (PS) IDRs and annotation of disease-associated proteins and mutations to map the correlation between disease and phase separation. Consistent with previous work linking phase separation to cancer and autism spectrum disorder, we find a higher prevalence of predicted phase separation behavior in disease-associated proteins than typical for human proteins. We map the prevalence of phase separation across a wide range of diseases, finding that many, but not all, show an enrichment of phase separation in the proteins associated with them. Strikingly, the pathogenic mutation rate in predicted PS IDRs was elevated three-fold relative to IDRs not predicted to phase separate. Substitutions involving arginine and the aromatic types were among the most pathogenic for PS IDRs, while substitutions involving serine, threonine, and alanine the most benign. We applied these trends to mutations of uncertain clinical significance and predict that half found in PS IDRs are likely pathogenic. We find that phosphorylation sites were enriched in PS IDRs when compared to other protein regions, though mutations at such sites were mostly benign. Pathogenicity was highest for mutations in predicted PS IDRs when also found in a short linear motif, known mediators of protein-protein interactions.

biophysics↗

bioRxiv2024

Longitudinal Associations Between MicroRNAs and Weight in the Diabetes Prevention Program

OBJECTIVECirculating microRNAs show cross-sectional associations with overweight and obesity. Few studies provided data to differentiate between a snapshot perspective on these associations versus how microRNAs characterize prodromal risk from disease pathology and complications. This study assessed longitudinal relationships between circulating microRNAs and weight at multiple time-points in the Diabetes Prevention Program trial. RESEARCH DESIGN AND METHODSA subset of participants (n=150) from the Diabetes Prevention Program were included. MicroRNAs were measured from banked plasma using a Fireplex Assay. We used generalized linear mixed models to evaluate relationships between microRNAs and changes in weight at baseline, year-1, and year-2. Logistic regression was used to evaluate whether microRNAs at baseline were associated with weight change after 2 years. RESULTSIn fully adjusted models that included relevant covariates, seven miRs (i.e., miR-126, miR-15a, miR-192, miR-23a, and miR-27a) were statistically associated with weight over 2 years. MiR-197 and miR-320a remained significant after adjustment for multiple comparisons. Baseline levels of let-7f, miR-17, and miR-320c were significantly associated with 3% weight loss after 2 years in fully adjusted models. DISCUSSIONThis study provided evidence for longitudinal relationships between circulating microRNAs and weight. Because microRNAs characterize the combined effects of genetic determinants and responses to behavioral determinants, they may provide insights about the etiology of overweight and obesity in the context or risk for common, complex diseases. Additional studies are needed to validate the potential genes and biological pathways that might be targeted by these microRNA biomarkers and have mechanistic implications for weight loss and disease prevention.

genomics↗

bioRxiv2023

ParSe 2.0: A web application that enables proteome-scale searches for sequences that drive protein-mediated phase separation

We have developed an algorithm, ParSe, that accurately identifies from the primary sequence those protein regions likely to exhibit physiological phase separation behavior. Originally, ParSe was designed to test the hypothesis that, for flexible proteins, phase separation potential is correlated to hydrodynamic size. While our results were consistent with that idea, we also found that many different descriptors could successfully differentiate between three classes of protein regions: folded, intrinsically disordered, and phase-separating intrinsically disordered. Consequently, numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. Built from that finding, ParSe 2.0 uses an optimal set of property scales to predict domain-level organization and compute a sequence-based prediction of phase separation potential. The algorithm is fast enough to scan the whole of the human proteome in minutes on a single computer and is equally or more accurate than other published predictors in identifying proteins and regions within proteins that drive phase separation. Here, we describe a web application for ParSe 2.0 that may be accessed through a browser by visiting https://stevewhitten.github.io/Parse_v2_FASTA to quickly identify phase-separating proteins within large sequence sets, or by visiting https://stevewhitten.github.io/Parse_v2_web to evaluate individual protein sequences.

biophysics↗

bioRxiv2023

How individual P. aeruginosa cells with diverse stator distributions collectively form a heterogeneous macroscopic swarming population

Swarming is a macroscopic phenomenon in which surface bacteria organize into a motile population. The flagellar motor that drives swarming in Pseudomonas aeruginosa is powered by stators MotAB and MotCD. Deletion of the MotCD stator eliminates swarming, whereas deletion of the MotAB stator enhances swarming. Interestingly, we measured a strongly asymmetric stator availability in the WT strain, with MotAB stators produced [~]40-fold more than MotCD stators. However, recruitment of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily recruited at low viscosities. Importantly, we find that cells with MotCD stators are [~]10x more likely to have an active motor compared to cells without, so wild-type, WT, populations are intrinsically heterogeneous and not reducible to MotAB-dominant or MotCD-dominant behavior. The spectrum of motility intermittency can either cooperatively shut down or promote flagellum motility in WT populations. In P. aeruginosa, transition from a static solid-like biofilm to a dynamic liquid-like swarm is not achieved at a single critical value of flagellum torque or stator fraction but is collectively controlled by diverse combinations of flagellum activities and motor intermittencies via dynamic stator recruitment. Experimental and computational results indicate that the initiation or arrest of flagellum-driven swarming motility does not occur from individual fitness or motility performance but rather related to concepts from the jamming transition in active granular matter. ImportanceAfter extensive study, it is now known that there exist multifactorial influences on swarming motility in P. aeruginosa, but it is not clear precisely why stator selection in the flagellum motor is so important or how this process is collectively initiated or arrested. Here, we show that for P. aeruginosa PA14, MotAB stators are produced [~]40-fold more than MotCD stators, but recruitment of MotCD over MotAB stators requires higher liquid viscosities. Moreover, we find the unanticipated result that the two motor configurations have significantly different motor intermittencies, the fraction of flagellum-active cells in a population on average, with MotCD active [~]10x more often than MotAB. What emerges from this complex landscape of stator recruitment and resultant motor output is an intrinsically heterogeneous population of motile cells. We show how consequences of stator recruitment led to swarming motility, and how they potentially relate to surface sensing circuitry.

microbiology↗

bioRxiv2022

Intrinsically disordered regions that drive phase separation form a robustly distinct protein class

Liquid-liquid phase separation (LLPS) of proteins is thought to be a primary driving force for the formation of membraneless organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. LLPS of proteins in cells is primarily, though not exclusively, driven by intrinsically disordered (ID) domains. Accurate identification of ID regions (IDRs) that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and phase-separating (PS) ID sequences. We then used these sequence sets to examine how broadly existing amino acids scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of LLPS. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving LLPS.

biophysics↗

bioRxiv2021

Non-motile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pili- and Pel-dependent mechanism

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, swarming motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-swarming cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. In contrast, flagellated cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species. ImportanceOur study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of the larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and non-motile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

microbiology↗

bioRxiv2020

Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

In most eukaryotes, the enzyme telomerase maintains the termini of linear chromosomes through the addition of repetitive telomeric sequences. It is widely assumed that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of linear chromosomes. We show here that a second substrate, created by spontaneous replication fork collapse during duplex telomeric DNA replication in wild type budding yeast, is elongated by telomerase at a much higher frequency ([~]50%) than fully replicated chromosome termini. Furthermore, as much as [~]200 nucleotides can be added in a single cell division to these newly collapsed forks, indicating that spontaneous replication fork collapse and the subsequent response by telomerase is a major determinant of telomere length homeostasis. This challenges a long-standing model for telomere length regulation which posits a length-sensing mechanism that assesses individual telomeres to determine whether chromosome ends are in "telomerase-extendible" or "telomerase-non-extendible" states. We propose that these two states are instead structurally and temporally distinct substrates for telomerase, generated by two different processes (fork collapse vs. completion of DNA replication). We also show that replication fork collapse at telomeres is kept in check by a telomere-dedicated Cdc13/Stn1/Ten1 complex in collaboration with the canonical RPA complex, indicating that these two complexes bind single-stranded DNA exposed at the replication fork to facilitate replisome progression through duplex telomeric DNA. Although failures during DNA replication are often genotoxic events, this represents an opposing example in which fork collapse has been co-opted to promote genome stability. Significance StatementIn most eukaryotes, the termini of linear chromosomes are composed of arrays of short repeats that are continually replenished by the enzyme telomerase. If telomerase is unable to act, gradual loss of these terminal repeats results in an eventual block to cell division. Therefore, in cells that depend on continuous cell division, the mechanism(s) by which telomerase is directed to chromosome ends is tightly regulated. This study shows that in addition to the ends of fully replicated chromosomes, a second site of action for telomerase is generated when replication through duplex telomeric DNA is disrupted. These results suggest that the disparate response of telomerase to two temporally and structurally distinct substrates is a major determinant of telomere length homeostasis.

genetics↗

bioRxiv2019

Phenotypic differences in reversible attachment behavior reveal distinct P. aeruginosa surface colonization strategies

Despite possessing the machinery to sense, adhere to, and proliferate on surfaces, it is commonly observed that bacteria initially have a difficult time attaching to a surface. Before forming a bacterial biofilm, planktonic bacteria exhibit a random period of transient surface attachment known as \"reversible attachment\" which is poorly understood. Using community tracking methods at single-cell resolution, we examine how reversible attachment progresses during initial stages of surface sensing. Pseudomonas aeruginosa strains PAO1 and PA14, which exhibit similar exponential trends of surface cell population increase, show unanticipated differences when the behavior of each cell was considered at the full lineage level and interpreted using the unifying quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two complementary surface colonization strategies, which are roughly analogous to \"immediate-\" vs \"deferred-gratification\" in a prototypical cognitive-affective processing system. PAO1 lineages commit relatively quickly to a surface compared to PA14 lineages. PA14 lineages allow detaching cells to retain memory of the surface so that they are primed for improved subsequent surface attachment. In fact, it is possible to identify motility suppression events in PA14 lineages in the process of surface commitment. We hypothesize that these contrasting strategies are rooted in downstream differences between Wsp-based and Pil-Chp-based surface sensing systems.

microbiology↗

bioRxiv2019

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Here we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility (P. aureginosa only). We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. We conclude that, while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. We finally report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bioontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

bioinformatics↗

bioRxiv2018

Ethanol decreases Pseudomonas aeruginosa flagellar motility through a cyclic-di-GMP- and stator-dependent pathway

Pseudomonas aeruginosa frequently encounters microbes that produce bioactive metabolites including ethanol. At concentrations that do not affect growth, we found that ethanol reduces P. aeruginosa motility by 30% in a swim agar assay and this decrease is accompanied by a 2.5-fold increase in levels of cyclic diguanylate (c-di-GMP), a second messenger that represses motility, in planktonic cells. A screen of mutants lacking genes involved in c-di-GMP metabolism identified SadC and GcbA as diguanylate cyclases involved in swim zone reduction by ethanol and ethanol-induced c-di-GMP production. The reduction of swimming in response to ethanol also required the stator set, MotAB, two PilZ-domain proteins (FlgZ and PilZ), PilY1-a proposed surface-sensing protein, and PilMNOP, which comprises the pilus alignment complex and these proteins have been previously implicated in the control of motility on agar surfaces. Microscopic analysis of the fraction of quiescent cells in swim medium showed that ethanol decreased the portion of motile cells in the wild type, but had opposite effects in the {triangleup}pilY1, {triangleup}pilMNOP, {triangleup}motAB, and {triangleup}pilZ{triangleup}flgZ mutants. Together, these data indicate ethanol induces a regulated change in motility in planktonic cells at concentrations similar to those produced by other microbes. We propose that this ethanol-responsiveness may contribute to the co-localization of P. aeruginosa with ethanol-producing microbes.

microbiology↗