Biology subjects

Perkins, A.

Publications and source records attributed to Perkins, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Refining mechanistic models to better predict larval and nymphal activity patterns of Ixodes scapularis

The black-legged tick (Ixodes scapularis), a key vector of Lyme disease, anaplasmosis, and babesiosis, exhibits regionally distinct patterns of seasonal activity driven by climate. Consequently, the relative timing of larval and nymphal activity varies across geographic locations, influencing pathogen transmission dynamics. Early-emerging nymphs may increase pathogen transmission, whereas early-emerging larvae may reduce transmission. In addition, synchrony between the two life-stages facilitates co-feeding transmission, which contributes to pathogen maintenance and coinfection risk. Temperature is thought to be an important driver of tick phenology, but existing mechanistic models that incorporate temperature fail to accurately capture the timing of larval and nymphal tick activity. To address this limitation, we developed a mechanistic model that includes two additional factors: humidity-dependent questing and low rates of overwinter development. To assess the value of these factors for explaining real-world patterns, we fitted alternative models to tick collection data from the National Ecological Observatory Network. In doing so, we found that explicitly incorporating humidity is necessary to reproduce observed tick phenology, with larval ticks being especially sensitive to relative humidity compared to other life stages. In addition, we found that accounting for humidity had a larger effect at Mid-Atlantic sites than at Northeastern sites, underscoring the importance of region-specific interactions between temperature and humidity in shaping I. scapularis phenology. By more accurately capturing tick seasonality compared to existing mechanistic models, our model illustrates the importance of accounting for factors beyond temperature for investigating how climate variability influences seasonal tick activity and pathogen transmission.

ecology↗

bioRxiv2026

Ablation and Seed-driven Restoration of an Alpha-Satellite Devoid Human Centromere Reveals Size Homeostasis Mechanisms

Human centromeres are epigenetically defined chromatin domains marked by nucleosomes containing the histone H3 variant CENP-A, which recruit the constitutive centromere-associated network (CCAN) to assemble functional kinetochores. Maintaining centromere function, including chromatin domain size and the ability to assemble the kinetochore, is essential for proper mitotic division across eukaryotes. In humans however, mechanistic studies of centromere establishment, maintenance, and size regulation have been hindered by the highly repetitive nature of canonical alphoid centromeres. Here, we develop a genetically tractable human neocentromere system which is devoid of repetitive DNA sequences. Using targeted genetic manipulation of a monoallelic naturally occurring neocentromere, we show that partial loss of centromeric chromatin triggers restoration of the residual CENP-A domain through seed-driven, sequence-independent expansion into adjacent naive chromatin. In contrast, creation of new domain boundaries without loss of centromeric chromatin mass results only in local boundary remodeling, without substantial domain expansion. These findings indicate that centromere formation proceeds through two mechanistically distinct steps, beginning with acquisition of a CENP-A seed and followed by regulated domain expansion to generate a mature centromere. More broadly, our results support a model in which centromeres transition between a maintenance state that preserves domain size and a restoration state that rebuilds centromeric chromatin following perturbation. Together, this study establishes a genetically tractable platform for dissecting the mechanisms governing human centromere formation, chromatin domain dynamics, and size homeostasis.

cell biology↗

bioRxiv2026

PathoBench: an open community-driven benchmark registry for pathogen bioinformatics tools

SummaryThe proliferation of pathogen bioinformatics pipelines has outpaced the communitys ability to compare them on common ground. Self-reported performance numbers, ad-hoc evaluation datasets, and inconsistent metrics make pipeline selection difficult for clinical and public-health researchers. We present PathoBench, an open web platform that addresses this gap through three coordinated mechanisms: (i) a curated registry of 26 standard benchmark datasets across 10 human pathogens, each with persistent identifiers and direct download links; (ii) pathogen-specific evaluation metrics that submissions must report, allowing direct head-to-head comparison only on the same dataset; and (iii) a credibility framework combining mandatory dataset attestation, ORCID-linked attribution, public peer comments, and administrator verification. As a case study, four published Mycobacterium tuberculosis drug-resistance pipelines were evaluated against the WHO TB mutation catalogue, demonstrating the frameworks discriminating power. PathoBench is open for community contributions across all ten supported pathogens. Availability and implementationPathoBench is freely available at https://pathobench.vercel.app. Source code is released under the MIT license at https://github.com/BPHL-Molecular/pathobench. The platform requires no installation for end users; programmatic access is available via a Supabase REST API. Contactyibo.dong@flhealth.gov Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2026

A modular Bayesian framework for inferring transmission networks from polyclonal infections, with application to Plasmodium falciparum

MotivationMolecular surveillance and infectious disease transmission network reconstruction can provide compelling evidence for estimating public-health quantities that are difficult to observe directly, including importation, source-sink structure, and differences in onward transmission across locations or intervention strata. These quantities can be expressed as functions of the underlying transmission network, but individual transmission events are rarely observed and many networks may be consistent with the same data. Existing transmission network reconstruction methods leveraging genetic data are often built for settings in which each infection has one dominant source, one representative haplotype, and mutation-driven genetic divergence along transmission chains. These assumptions are poorly matched to polyclonal infections, in which hosts carry multiple genetically distinct clones and recipient infections may reflect contributions from multiple sources. Such infections are common in malaria, tuberculosis, HIV, and many parasitic infections. Methods are needed that can accommodate these data. ResultsWe present a modular Bayesian framework for estimating directed transmission on sampled cases, where an infection may have no sampled parent, one parent, or several parents, including sources outside the observed panel. Pathogen-specific modules supply likelihoods over candidate parent sets and connect to shared inference that yields marginal directed edge probabilities, posterior mean out-degree, and inclusion probabilities for unobserved parents. We demonstrate our framework with Plasmotrack, a transmission network model for Plasmodium falciparum that uses targeted amplicon sequencing data. We implemented these components with a per-locus allele-mixture transmission likelihood, an amplicon genotyping error model, and data augmentation allowing for unobserved parents. Simulations from a biologically informed generative model, under which the inferential per-locus allele-mixture likelihood is misspecified, showed recovery of aggregate network summaries including mean outdegree and mean unobserved-source inclusion, alongside high precision and recall for detecting directed transmission. Other pathogens can reuse the same modular composition after substituting transmission and observation likelihoods. AvailabilityThe Plasmotrack software and documentation are available at https://github.com/eppicenter/plasmotrack. Source code and example datasets are provided under an open-source license. Contactmaxwell.murphy@ucsf.edu

bioinformatics↗

bioRxiv2026

Estimation of adult census size from close-kin dyads in the malaria mosquito Anopheles gambiae

Accurate estimates of adult mosquito abundance are central to the design and evaluation of vector control strategies, yet they are difficult to obtain from natural populations. Conventional mark-recapture methods for estimating adult mosquito census size pose logistical and other challenges. A recently developed close-kin mark-recapture (CKMR) approach is a promising alternative. However, application of CKMR has been largely confined to long-lived vertebrates. Validation on empirical data from short-lived, highly fecund insects is lacking. Here, we apply CKMR to a natural population of Anopheles gambiae, the primary African malaria mosquito, sampled from a small island in Lake Victoria, Uganda. Using a high-diversity amplicon panel of genome-wide markers, we genotyped 714 adult mosquitoes collected over a 20-day period. We classified pairs to close-kin categories by implementing probabilistic latent-kinship estimation rather than using a deterministic threshold-based framework. We observe numerous full-sibling pairs but no parent-offspring pairs, a pattern indicative of extreme variance in reproductive success owing to frequent failure of mosquito egg clutches to produce adults. To adapt the CKMR framework to mosquito life history, we explicitly modeled the mosquito life cycle including the possibility of clutch failure. By incorporating probabilities of both parent-offspring and full-sibling relationships, we estimate an adult female census size of 26,887 (95% credible interval: 6,979 - 146,011), and a clutch failure probability of 97.6% (CrI: 91.3 - 99.6%). We use these estimates to calculate the predicted variance in reproductive success and effective population size. Through individual-based simulations we confirm our estimates and the necessity of modeling reproductive variance to explain observed kinship patterns. Our results demonstrate that CKMR can be applied successfully to mosquitoes, provided that appropriate adjustments are made to account for their natural history.

ecology↗

bioRxiv2026

Assessing the impact of host density on vector abundance and transmission scaling of Culicoides-transmitted pathogens

The spread of any pathogen depends on the dynamics of its hosts, with transmission rates typically assumed to either scale linearly with host population density (density-dependent transmission) or to be independent of it (frequency-dependent transmission). For vector-transmitted pathogens, a key determinant of transmission scaling is whether vector abundance is constant or sensitive to host abundance, with the latter being consistent with density-dependent transmission. Here, we assess whether Culicoides vector abundance increases in a manner that indicates density-dependent transmission of Culicoides-transmitted pathogens. To test this, we conducted trapping of Culicoides midges on eight livestock operations across a wide range of cattle abundances, placing traps at varying distances from the host aggregation. We used hierarchical Bayesian models to estimate the effect of host abundance on the vector-to-host ratio while accounting for differences in trapping efficacy between locations. Our results indicate a positive linear effect of host abundance on the ratio of vectors to hosts, with a posterior probability of 0.83. Median posterior values of the effect of host abundance on vector density predict a 2.3% increase (95% credible interval: -0.8, 23.2%) in the vector-to-host ratio with every 1,000 additional hosts. The weight of evidence from our study suggests that Culicoides-transmitted pathogens are likely subject to density-dependent transmission, and that transmission may be amplified in high-concentration livestock environments.

ecology↗

bioRxiv2025

Single cell long-read genotyping of transcriptomes reveals discrete mechanisms of clonal evolution in post-myeloproliferative neoplasm acute myeloid leukemia.

Myeloproliferative neoplasms (MPNs) are caused by acquired mutations in hematopoietic stem and progenitor cells (HSPCs). The acquisition of additional mutations like TP53 and the overall mutational burden influence a patients risk of disease progression toward lethal post-MPN acute myeloid leukemia (AML). Recent technological advancements in linking single-cell gene expression with genotype have improved our understanding of tumor heterogeneity. However, current methodologies have limitations in simultaneously genotyping low-expression genes (such as JAK2) alongside other pathogenic loci. To address this, we developed a novel long read genotyping pipeline of cDNA transcripts called LOTR-Seq, which can genotype the full length of expressed transcripts of 30 genes at once. Using LOTR-Seq, we genotyped HSPCs at the JAK2V617 locus in 9,075 single cells from eight patients with chronic phase MPN (CP-MPN) and in 5,016 cells from four patients with post-MPN AML. We then linked the mutations to the single cell transcriptome of 29,712 JAK2V617F-driven CP-MPN cells and 16,895 post-MPN AML cells. In our analysis of post-MPN AMLs, we identified nine mutated loci across six genes (JAK2, IDH1/2, TP53, SRSF2, U2AF1) and linked these mutations to specific transcriptional phenotypes. Overall, LOTR-Seq provides novel insights into the evolution of post-MPN AML.

cancer biology↗

bioRxiv2025

Larval development habitats of Culicoides midges in the western United States

Culicoides midges are vectors of bluetongue virus (BTV), an arbovirus affecting wild and domestic ruminants. Bluetongue distribution generally overlaps with vector range, so understanding the vectors ecology is necessary for predicting BTV risk. Culicoides require moist substrate for oviposition and development, and C. sonorensis, the primary vector in the western United States, is classically associated with livestock wastewater ponds. However, it is well-known that BTV can be found outside of managed livestock areas, with transmission also occurring in natural settings. To better classify development habitat for Culicoides, we conducted a broad biweekly survey of moist and wet habitats from June to September of 2022 in northern Colorado at ten sites, including large livestock operations, non-commercial domestic operations, and natural spaces. Samples were maintained in the insectary for 11 weeks and monitored for emergence of adult midges. Standing water substrates displayed higher presence and abundance of midges than running or transient habitats, though all microhabitats showed emergence. Additionally, livestock sites did not produce more midges than other site types, and in fact more midges were observed in natural spaces per sample and overall. Livestock spaces did, however, show significantly higher proportions of C. sonorensis midges, which are thought to be the most competent vectors of BTV in this region. These results suggest that development sites in natural areas may play an important role in maintaining vector populations in the western U.S. outside of previously implicated livestock operations, and that differences in larval habitat lead to differences in vector species composition.

ecology↗

bioRxiv2025

Distinct origin and fate for fetal hematopoietic progenitors

It was proposed that two sequential sources of intraembryonic multipotent progenitors ensure blood cell production from late gestation into adulthood, with only the latter producing self-renewing hematopoietic stem cells (HSC). How these two populations differ and how they impact the establishment of the postnatal immune system, remains poorly understood. Using complementary lineage tracing models, we showed that the first emerging embryonic multipotent progenitors (eMPP) are responsible for late gestation hematopoiesis. They are distinct from HSC that do not significantly contribute to embryonic mature blood cells. eMPP are the predominant source of embryonic lymphocytes and lymphoid tissue inducer cells, some of which persist for life. Between E12.5 and E16.5 eMPP rapidly differentiate, whereas HSC expand 20-fold. Altogether, these results support the notion that eMPP establish the embryonic adaptive immune system and shape the lymphoid organs where later adaptive immune responses occur, while HSC expand to sustain blood cell production throughout life.

developmental biology↗

bioRxiv2024

Phosphorylation at serine 214 correlates with tau seeding activity in an age-dependent manner in two mouse models for tauopathies and is required for tau transsynaptic propagation.

Pathological aggregation and propagation of hyperphosphorylated and aberrant forms of tau are critical features of the clinical progression of Alzheimers disease and other tauopathies. To better understand the correlation between these pathological tau species and disease progression, we profiled the temporal progression of tau seeding activity and the levels of various phospho- and conformational tau species in the brains of two mouse models of human tauopathies. Our findings indicate that tau seeding is an early event that occurs well before the appearance of AT8-positive NFT. Specifically, we observed that tau phosphorylation in serine 214 (pTau-Ser214) positively correlates to tau seeding activity during disease progression in both mouse models. Furthermore, we found that the histopathology of pTau-Ser214 appears much earlier and has a distinct pattern and compartmentalization compared to the pathology of AT8, demonstrating the diversity of tau species within the same region of the brain. Importantly, we also observed that preventing the phosphorylation of tau at Ser214 significantly decreases tau propagation in mouse primary neurons, and seeding activity in a Drosophila model of tauopathy, suggesting a role for this tau phosphorylation in spreading pathological forms of tau. Together, these results suggest that the diverse spectrum of soluble pathological tau species could be responsible for the distinct pathological properties of tau and that it is critical to dissect the nature of the tau seed in the context of disease progression.

neuroscience↗

bioRxiv2024

Role of the antiparallel double-stranded filament form of FtsA in activating the Escherichia coli divisome

The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. Yet, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionallity of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis. IMPORTANCECell division is fundamental for cellular duplication. In simple cells like Escherichia coli bacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments help to tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division, but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, the E. coli divisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.

microbiology↗

bioRxiv2023

Prenatal alcohol exposure alters expression of genes involved in cell adhesion, immune response, and toxin metabolism in adolescent rat hippocampus

Prenatal alcohol exposure can result in mild to severe consequences for children throughout their lives, with this range of symptoms referred to as Fetal Alcohol Spectrum Disorders (FASD). These consequences are thought to be linked to changes in gene expression and transcriptional programming in the brain, but the identity of those changes, and how they persist into adolescence are unclear. In this study, we isolated RNA from the hippocampus of adolescent rats exposed to ethanol during prenatal development and compared gene expression to controls. Exposure to ethanol caused widespread downregulation of many genes as compared to control rats. Gene ontology analysis demonstrated that affected pathways included cell adhesion, toxin metabolism, and immune responses. Interestingly, these differences were not strongly affected by sex. Furthermore, these changes were consistent when comparing ethanol-exposed rats to pair-fed controls provided with a liquid diet and those fed ad libitum on a standard chow diet. We conclude from this study that changes in genetic architecture and the resulting neuronal connectivity after prenatal exposure to alcohol continue through adolescent development. Further research into the consequences of specific gene expression changes on neural and behavioral changes will be vital to our understanding of the FASD spectrum of diseases. AUTHOR SUMMARYAlcohol exposure during fetal development is associated with a wide range of behavioral and physical symptoms that can be observed from childhood throughout adolescence and beyond. It is believed that this exposure may alter gene expression patterns permanently by changing genomic architecture, but the actual changes themselves are still unclear. In this study we examined gene expression patterns in rats whose mothers were given ethanol during their pregnancies. These were compared to control rats whose mothers were fed a similar liquid diet without ethanol as well as rats fed a normal diet. We identified the top differentially expressed genes and performed gene ontology analysis to identify both genes and pathways important in the response to ethanol during fetal development. We focused on adolescent rats since prenatal alcohol exposure has been shown at this stage to influence behavior. We indeed found a number of significant changes in gene expression, suggesting that prenatal alcohol exposure has ongoing consequences throughout and likely beyond adolescence into adulthood. Pathways such as cell adhesion, immune response, and toxin response were all highlighted. Future work will focus on making connections between these gene expression changes and behavioral changes observed at this same life stage.

genetics↗

bioRxiv2022

Modeling cellular co-infection and reassortment of bluetongue virus in Culicoides midges

When related segmented RNA viruses co-infect a single cell, viral reassortment can occur, potentially leading to new strains with pandemic potential. One virus capable of reassortment is bluetongue virus, which causes substantial health impacts in ruminants and is transmitted via Culicoides midges. Because midges can become co-infected by feeding on multiple different host species and remain infected for their entire life-span, there is high potential for reassortment to occur. Once a midge is co-infected, additional barriers must be crossed for a reassortant virus to emerge, such as cellular co-infection and dissemination of reassortant viruses to the salivary glands. We developed three mathematical models of within-midge bluetongue virus dynamics of increasing complexity, allowing us to explore the conditions leading to the emergence of reassortment viruses. In confronting the simplest model with published data, we estimate the average lifespan of a bluetongue virion in the midge midgut is about six hours, a key determinant of establishing a successful infection. Examination of the full model, which permits cellular co-infection and reassortment, shows that small differences in fitness of the two infecting strains may have a large impact on the frequency with which reassortant virions are observed. This is consistent with experimental co-infection studies with BTV strains with different relative fitnesses that did not produce reassortant progeny. Our models also highlight several gaps in existing data which would allow us to elucidate these dynamics in more detail, in particular the times it takes the virus to disseminate to different tissues, and measurements of viral load and reassortant frequency at different temperatures.

microbiology↗

bioRxiv2022

Hematological and gene co-expression network analyses of high-risk beef cattle defines immunological mechanisms and biological complexes involved in bovine respiratory disease and weight gain

Bovine respiratory disease (BRD), the leading disease complex in beef cattle production systems, remains highly elusive regarding diagnostics and disease prediction. Previous research has employed cellular and molecular techniques to describe hematological and gene expression variation that coincides with BRD development. Here, we utilized weighted gene co-expression network analysis (WGCNA) to leverage total gene expression patterns from cattle at arrival and generate hematological and clinical trait associations to describe mechanisms that may predict BRD development. Gene expression counts of previously published RNA-Seq data from 23 cattle (2017; n=11 Healthy, n=12 BRD) were used to construct gene co-expression modules and correlation patterns with complete blood count (CBC) and clinical datasets. Modules were further evaluated for cross-populational preservation of expression with RNA-Seq data from 24 cattle in an independent population (2019; n=12 Healthy, n=12 BRD). Genes within well-preserved modules were subject to functional enrichment analysis for significant Gene Ontology terms and pathways. Genes which possessed high module membership and association with BRD development, regardless of module preservation ("hub genes"), were utilized for protein-protein physical interaction network and clustering analyses. Five well-preserved modules of co-expressed genes were identified. One module ("steelblue"), involved in alpha-beta T-cell complexes and Th2-type immunity, possessed significant correlation with increased erythrocytes, platelets, and BRD development. One module ("purple"), involved in mitochondrial metabolism and rRNA maturation, possessed significant correlation with increased eosinophils, fecal egg count per gram, and weight gain over time. Fifty-two interacting hub genes, stratified into 11 clusters, may possess transient function involved in BRD development not previously described in literature. This study identifies co-expressed genes and coordinated mechanisms associated with BRD, which necessitates further investigation in BRD-prediction research. Author SummaryBovine respiratory disease (BRD), the leading disease in beef cattle, is a highly dynamic disease complex. Through simultaneous sequencing of thousands of genes active in the blood of cattle at arrival, we pursued the co-expression patterns of these genes to evaluate associations with BRD development and severity overtime. This approach allows for a better understanding of gene expression active in cattle at arrival, and the discovery of new molecules and biological complexes that may predict BRD before the onset of clinical signs. Our work provides evidence that genes related to T-cells, a type of immune cell, are strongly co-expressed when cattle arrive to beef production system, and correlate with increased red blood cell (RBC) factors and BRD development. Further analysis shows that genes involved in cellular energy production and the respiratory electron transport are strongly co-expressed when cattle arrive to beef production system, and correlate with increased eosinophils, a type of immune cell, and weight gain overtime. Additionally, using genes which strongly correlate with BRD development and severity overtime, we identify a novel protein interaction complex that may drive future research for discovering new ways to manage and treat BRD in beef cattle.

systems biology↗

bioRxiv2020

A bacterial inflammation sensor regulates c-di-GMP signaling, adhesion, and biofilm formation.

The reactive oxygen species produced during inflammation through the neutrophilic respiratory burst play profound roles in combating bacterial pathogens and regulating the microbiota. Among these, the neutrophilic oxidant bleach, hypochlorous acid (HOCl), is the most prevalent and strongest oxidizer and kills bacteria through non-specific oxidation of proteins, lipids, and DNA. Thus, HOCl can be viewed as a host-specific cue that conveys important information about what bacterial physiology and lifestyle programs may be required for successful colonization. Nevertheless, bacteria that colonize animals face a molecular challenge in how to achieve highly selective detection of HOCl due to its reactive and transient nature and chemical similarity to more benign and non-host-specific oxidants like hydrogen peroxide (H2O2). Here, we report that in response to increasing HOCl levels E. coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We show the molecular mechanism of this activation to be specific oxidation of a conserved cysteine that coordinates the zinc of its regulatory chemoreceptor zinc-binding (CZB) domain, forming a zinc-cysteine redox switch 685-fold more sensitive to oxidation by HOCl over H2O2. Dissection of the signal transduction mechanism through quantum mechanics, molecular dynamics, and biochemical analyses reveal how the cysteine redox state alters the delicate equilibrium of competition for Zn++ between the CZB domain and other zinc binders to relay the presence of HOCl through activating the associated GGDEF domain to catalyze c-di-GMP. We find biofilm formation and HOCl-sensing in vivo to be regulated by the conserved cysteine, and point mutants that mimic oxidized CZB states increase production of the biofilm matrix polymer poly-N-acetylglucosamine and total biofilm. We observe CZB-regulated diguanylate cyclases and chemoreceptors in phyla in which host-associated bacteria are prevalent and are possessed by pathogens that manipulate host inflammation as part of their colonization strategy. A phylogenetic survey of all known CZB sequences shows these domains to be conserved and widespread across diverse phyla, suggesting CZB origin predates the bacterial last universal common ancestor. The ability of bacteria to use CZB protein domains to perceive and thwart the host neutrophilic respiratory burst has implications for understanding the mechanisms of diseases of chronic inflammation and gut dysbiosis.

microbiology↗

bioRxiv2020

Learning Peptide Recognition Rules for a Low-Specificity Protein

Many proteins interact with short linear regions of target proteins. For some proteins, however, it is difficult to identify a well-defined sequence motif that defines its target peptides. To overcome this difficulty, we used supervised machine learning to train a model that treats each peptide as a collection of easily-calculated biochemical features rather than as an amino acid sequence. As a test case, we dissected the peptide-recognition rules for human S100A5 (hA5), a low-specificity calcium binding protein. We trained a Random Forest model against a recently released, high-throughput phage display dataset collected for hA5. The model identifies hydrophobicity and shape complementarity, rather than polar contacts, as the primary determinants of peptide binding specificity in hA5. We tested this hypothesis by solving a crystal structure of hA5 and through computational docking studies of diverse peptides onto hA5. These structural studies revealed that peptides exhibit multiple binding modes at the hA5 peptide interface--all of which have few polar contacts with hA5. Finally, we used our trained model to predict new, plausible binding targets in the human proteome. This revealed a fragment of the protein -1-syntrophin binds to hA5. Our work helps better understand the biochemistry and biology of hA5, as well as demonstrating how high-throughput experiments coupled with machine learning of biochemical features can reveal the determinants of binding specificity in low-specificity proteins.

biophysics↗

bioRxiv2020

CONTAIN: An open-source shipping container laboratory optimisedfor automated COVID-19 diagnostics

The COVID-19 pandemic has challenged diagnostic systems globally. Expanding testing capabilities to conduct population-wide screening for COVID-19 requires innovation in diagnostic services at both the molecular and industrial scale. No report to-date has considered the complexity of laboratory infrastructure in conjunction with the available molecular assays to offer a standardised solution to testing. Here we present CONTAIN. A modular biosafety level 2+ laboratory optimised for automated RT-qPCR COVID-19 testing based on a standard 40ft shipping container. Using open-source liquid-handling robots and RNA extraction reagents we demonstrate a reproducible workflow for RT-qPCR COVID-19 testing. With five OT2 liquid handlers, a single CONTAIN unit reaches a maximum daily testing capacity of 2400 tests/day. We validate this workflow for automated RT-qPCR testing, using both synthetic SARS-CoV-2 samples and patient samples from a local NHS hospital. Finally, we discuss the suitability of CONTAIN and its flexibility in a range of diagnostic testing scenarios including high-density urban environments and mobile response units. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/106625v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@18acad6org.highwire.dtl.DTLVardef@10ae5f1org.highwire.dtl.DTLVardef@7e34d3org.highwire.dtl.DTLVardef@1be3815_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

Incipient resistance to an effective pesticide results from genetic adaptation and the canalization of gene expression

The resistance of bacteria, disease vectors, and pest species to chemical controls has vast ecological, economic, and societal costs. In most cases, resistance is only detected after spreading throughout an entire population. Detecting resistance in its incipient stages, by comparison, provides time to implement preventative strategies. Incipient resistance can be detected by coupling standard toxicology assays with large-scale gene expression experiments. We apply this approach to a system where an invasive parasite, sea lamprey (Petromyzon marinus), has been treated with the highly-effective pesticide 3-trifluoromethyl-4-nitrophenol (TFM) for 60 years. Toxicological experiments revealed that lamprey from treated populations did not have higher survival to TFM exposure than lamprey from their native range, demonstrating that full-fledged resistance has not yet evolved. In contrast, we find hundreds of genes differentially expressed in response to TFM in the population with the longest history of exposure, many of which relate to TFMs primary mode of action, the uncoupling of oxidative phosphorylation and subsequent depletion of ATP. Three genes critical to oxidative phosphorylation, ATP5PB, PLCB1, and NDUFA9, were nearly fixed for alternative alleles in comparisons of SNPs between native and treated populations (FST > 5 SD from the mean). ATP5PB encodes subunit b of ATP synthase and an additional subunit, ATP5F1B, was canalized for high expression in treated populations, but remained plastic in response to TFM treatment in individuals from the native range. These combined genomic and transcriptomic results demonstrate that an adaptive, genetic response to TFM is driving incipient resistance in a damaging pest species.

genetics↗