Biology subjects

Brooks, A.

Publications and source records attributed to Brooks, A..

11 recordsLinked to original sources

bioRxiv2026

Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes

We examined whether genes encoded by the mycobacteriophage Xavia disrupt growth of Mycobacterium smegmatis, a widely used mycobacterial model. Seventy-one Xavia genes were individually expressed using an inducible plasmid system and assessed for effects on colony formation. Two genes were lethal even without induction, indicating toxicity under basal expression. Induction of sixteen additional genes reduced bacterial growth, spanning structural proteins, lysogeny regulators, DNA-associated enzymes, a lysis protein, and several genes with no known function. These findings expand functional insights into mycobacteriophage gene repertoires and identify candidates for future mechanistic studies. AbstractBacteriophage genomes encode large numbers of genes with no known function, and many of these genes affect essential host processes when expressed in a heterologous system. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis provides a direct way to identify proteins that impair growth and to determine which mycobacterial pathways are sensitive to phage gene products. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, a lineage that has not undergone functional screening, we constructed an arrayed pExTra library containing 71 predicted Xavia genes under control of the anhydrotetracycline inducible promoter pTet. All constructs were sequence-verified and transformed into M. smegmatis, and induction allowed measurement of gene-specific effects on growth. Two genes prevented recovery of transformants, suggesting toxicity under basal promoter leakiness. Inducible expression of 16 additional genes impaired growth, and these inhibitory proteins include structural components, regulators of lysogeny, enzymes of DNA metabolism, a lysis factor, and several proteins with no known function. Four of the strongest inhibitors were genes with no known function. These results extend functional screening into the previously untested P3 branch of Actinobacteriophages and identify new proteins that require mechanistic analysis.

microbiology↗

bioRxiv2025

River pollution threatens preferred macroinvertebrate prey of endemic fishes in Aotearoa, New Zealand

O_LIMacroinvertebrates are a critical source of food for riverine fishes. Anthropogenic stressors can transform riverine macroinvertebrate communities, yet the consequences of such transformations to the fishes that feed on them are poorly understood. Changes in any combination of food abundance, accessibility or quality may be referred to as changes in the foodscape of fishes. Stressor-driven increases of inaccessible prey that are difficult for fish to detect, capture or ingest, may render foodscapes less profitable to fishes. C_LIO_LIWe examined how nutrient enrichment, fine sediment, acid mine drainage (AMD), flooding and drying reshape fish foodscapes by altering the accessibility of macroinvertebrate prey for two New Zealand river fishes, Galaxias vulgaris and Gobiomorphus breviceps. We determined relative accessibility of macroinvertebrate taxa and traits to these fishes by comparing the abundance of prey taxa and traits in the environment with their corresponding abundances in fish diets. To make these comparisons, invertebrate and fish gut samples were collected from the same river reaches, at the same times, over three years. Environmental DNA (eDNA) metabarcoding of fish gut contents facilitated efficient diet characterisation of over 700 fish guts. Finally, to determine how multiple stressors might affect fish foodscapes, we related invertebrate trait-specific accessibilities to published data defining how the composition of those macroinvertebrate traits varies along stressor gradients. C_LIO_LIMacroinvertebrate traits that were most accessible to fishes were those promoting detection (e.g., active foraging behaviours exhibited by crawlers and scrapers) and ingestion (e.g., moderate size, morphologies lacking defensive structures), typical of mayfly and free-living caddisfly larvae. In contrast, prey with traits inhibiting detection (e.g., small size and burrowing behaviour) and ingestion by gape-limited fishes (e.g., large size), were less accessible to the fishes we studied. C_LIO_LITrait abundance-stressor relationships revealed that that increasing fine sediment, nutrients, AMD and drying, and decreased flooding frequency, favoured macroinvertebrates with traits that erode accessibility to fishes. These results show that multiple stressors may decrease the profitability of fish foodscapes by filtering out macroinvertebrate prey with traits promoting accessibility to fishes. C_LIO_LIFood web approaches to understanding multi-stressor impacts are rare, but are necessary to understand how the effects of multiple stressors on primary consumers then go on to affect predators of higher trophic position. A major barrier to fish foodscape research is the difficulty and cost of estimating trophic networks. We discuss how eDNA metabarcoding, coupled with trait-based approaches may help overcome these barriers, enabling generalisable assessments of foodscape changes caused by multiple stressors. C_LI

ecology↗

bioRxiv2025

Paralogs with distinct phase behaviors broaden the in vivo stress response range of condensates

Paralogs are widespread, but their physiological roles are often masked by redundancy. H-NS, a nucleoid-associated protein in Gram-negative bacteria, typically coexists with paralogs such as StpA, whose functions have remained obscured due to the lack of strong phenotypes. We demonstrate here that the interaction between H-NS and StpA fine-tunes the physico-chemical properties of heterochromatin-forming condensates. H-NS forms dynamic condensates, while StpA assembles into stable insoluble fibrils. Yet together, the two proteins form heterotypic condensates, whose fluidity and stability are tunable by their relative stoichiometry. By increasing the levels of StpA over H-NS, bacteria increase heterochromatin-associated condensate stability, thus preserving gene repression and optimizing bacterial growth under stress conditions. Structural differences at these proteins dimerization sites help explain their distinct phase behaviors. Our findings reveal a novel paradigm in which paralogs that are positioned at the opposite ends of the phase spectrum can fine-tune condensates to promote survival in fluctuating environments.

biochemistry↗

bioRxiv2025

Epicardial contributions to fibro-inflammatory signaling in a Pkp2-deficient arrhythmogenic cardiomyopathy model

BackgroundArrhythmogenic Cardiomyopathy (ACM) is an inherited disease that is characterized by lethal ventricular arrhythmias stemming from myocyte dysfunction. ACM is associated with considerable subepicardial fibrosis and inflammation with right ventricle predominance. Most cases of gene positive ACM are caused by a desmosome protein mutation, with plakophilin-2 (Pkp2) mutations being most common. We hypothesized Pkp2-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. MethodsWe developed transgenic mice that lack Pkp2 in cardiomyocytes (Pkp2-cKO) or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Non-myocyte populations were isolated 21 days post-tamoxifen injection for single cell RNA-sequencing (scRNA-seq). Immunohistochemistry, flow cytometry, qRT-PCR, and echocardiography were used to interrogate cardiac physiology and cellular composition. ResultsWe identified a population of epicardium-derived fibroblasts characterized by the expression of Ccl2, Ccl7, Thbs1, and Ptx3 that accumulated upon Pkp2 deletion. While pro-inflammatory EPDCs are found in Pkp2-cKO mice, they become significantly enriched in Pkp2-ceKO mice. Pro-inflammatory fibroblasts acquired the senescence-associated secretory phenotype (SASP), correlating with elevated Senescence Associated (SA)-{beta}gal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, that progresses from right to bi-ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared to controls. Antibody-mediated B cell depletion delays the early inflammatory and fibrosis response but did not significantly alter end-stage cardiac physiology. ConclusionPkp2 deletion in EPDC facilitates the emergence of a fibro-inflammatory phenotype that may contribute to ACM pathogenesis.

cell biology↗

bioRxiv2025

Development and validation of five native Aotearoa New Zealand genera-specific large brown macroalgal eDNA ddPCR assays

Environmental DNA is a rapidly growing methodology for ecological studies, leading to an increase in innovative techniques, such as the droplet-digital PCR (ddPCR), that can quantify and target DNA at a high rate of sensitivity for eDNA studies. Large brown macroalgae, Phaeophyceae, are an ecologically significant, diverse, and abundant benthic group in temperate-reef coastal environments. Members of Phaeophyceae, such as Laminariales and Fucales, are becoming recognized as potential significant contributors to Blue Carbon, although measurement of their contribution is challenging. The goal of the present study was to track the fate of specific large, brown macroalgae (kelp) genera detritus, by designing and validating five assays targeting the eDNA of kelp genera that are abundant in the coastal environments of Aotearoa New Zealand: Carpophyllum, Cystophora, Ecklonia, Lessonia, and Macrocystis. Short, diagnostic sequences of the mitochondrial cytochrome c oxidase subunit III gene (cox3) were targeted using ddPCR analysis. Specificity and sensitivity testing were conducted to validate the design of each assay for future use. All five assays produced no cross-amplification and had limits of detection (LoD) that were less than 1 copy/{micro}l. These novel, highly sensitive assays will be beneficial for future experimental and in situ eDNA studies that focus on any of these indigenous genera.

molecular biology↗

bioRxiv2025

RIG-I Stimulation Enhances Effector Function and Proliferation of Primary Human CD8 T Cells

Cytotoxic CD8 T lymphocytes (CTL) are crucial in antiviral immune responses. However, their recruitment to infection sites renders them at risk of viral infection that could modulate their effector activity. CTL express RIG-I that detects cytosolic viral RNA and subsequently induce antiviral gene expression. Here, we investigated how influenza A virus (IAV) infection influence TCR-dependent effector responses. IAV infection of CTL stimulated RIG-I, and activated downstream pathways including TBK1 and NF-B, resulting in type-I interferon secretion. Transfection of CTL with a pure RIG-I ligand, tri-phosphorylated double stranded RNA(3p-dsRNA), not only stimulated these pathways but also enhanced CTL proliferation in vitro and protected them from IAV infection. Analogous with a positive effect on CD8 effector function, both IAV infection and RIG-I ligand transfection enhanced CTL degranulation and cytokine secretion. Conversely, activation of CTL via CD3/CD28-crosslinking increased their susceptibility to IAV infection. Altogether, RIG-I stimulation either by IAV infection or 3p-dsRNA transfection promoted cell intrinsic antiviral pathways and enhanced CD8 effector functions. These findings suggest that RIG-I agonists could enhance and prolong CTL effector function in immunotherapy.

immunology↗

bioRxiv2024

Mitochondrial membrane potential regulates nuclear DNA methylation and gene expression through phospholipid remodeling

Maintenance of the mitochondrial inner membrane potential ({Delta}{Psi}M) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While {Delta}{Psi}M loss and its consequences are well studied, little is known about the effects of increased {Delta}{Psi}M. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic {Delta}{Psi}M increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the {Delta}{Psi}M to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the {Delta}{Psi}M, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the {Delta}{Psi}M as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction. HighlightsO_LIMitochondria hyperpolarization leads to nuclear DNA hypermethylation C_LIO_LIDNA methylation regulates expression of mitochondrial and lipid metabolism genes C_LIO_LIPhospholipid remodeling mediates the epigenetic effects of mitochondrial hyperpolarization C_LI

genomics↗

bioRxiv2023

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in the C. elegans zygote that the feedback relies on aPKC phosphorylation of Cdc42 at serine 71. The turnover of CDC-42 phosphorylation ensures optimal aPKC asymmetry and activity throughout polarisation by tuning Par6/aPKC association with Par3 and Cdc42. Moreover, turnover of Cdc42 phosphorylation regulates actomyosin cortex dynamics that are known to drive aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust control of polarisation in many cell types. Key findings/graphical abstract- Phosphorylation of CDC-42 by aPKC accelerates aPKC dissociation from CDC-42, limiting aPKC activity - CDC-42/aPKC dissociation promotes aPKC association with PAR-3 and, thereby, aPKC asymmetry due to actomyosin flow - Cycling of CDC-42 phosphorylation fuels the exchange of aPKC between anteriorly transported PAR-3 and aPKC-active CDC-42 complexes - Turnover of CDC-42 phosphorylation alternates its association with effectors, aPKC and MRCK-1, ensuring proper actomyosin dynamics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/563985v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@e0ec65org.highwire.dtl.DTLVardef@c00b8dorg.highwire.dtl.DTLVardef@36565dorg.highwire.dtl.DTLVardef@160149a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Sustainable reference points for multispecies coral reef fisheries

Sustainably managing fisheries requires regular and reliable evaluation of stock status. However, most multispecies reef fisheries around the globe tend to be data-poor and lack research and monitoring capacity (e.g., long-term fishery data), preventing the estimation of sustainable reference points against which stocks can be assessed. Here, combining fish biomass data for more than 2000 coral reefs with catch estimates from 99 jurisdictions, we estimate site-specific sustainable reference points for coral reef fisheries and use these to assess the status of coral reef fish stocks. We reveal that more than half of jurisdictions with available information have stocks of conservation concern, having failed at least one fisheries sustainability benchmark. We quantify the trade-offs between biodiversity, mean fish length, and ecosystem functions relative to key benchmarks and highlight the ecological benefits of increasing sustainability. Our approach yields multispecies sustainable reference points for coral reef fisheries using environmental conditions, a promising means for enhancing the sustainability of the worlds coral reef fisheries. TeaserA global assessment of the sustainability of multispecies reef fisheries indicates that more than half of jurisdictions have failed at least one of two key sustainability benchmarks.

ecology↗

bioRxiv2021

Representing the sediment retention ecosystem service within an ecosystem account of the Mitchell catchment

If soil resources and the benefits derived from water quality are to be maintained, the on- and off-site effects of soil erosion must be adequately represented and communicated to decision makers so that appropriate management responses can be identified. The System of Environmental-Economic Accounting - Ecosystem Accounting (SEEA-EA), is one approach to quantify both the contributions that ecosystems make to the economy, and the impacts of economic activity on ecosystems. However, due to the difficulty of obtaining empirical data on ecosystem service flows, in many cases such quantification is informed by ecosystem service models. Previous research on the Mitchell catchment, Queensland Australia allowed us to explore the implications of using a model of hillslope erosion and sediment delivery in isolation (as represented in one of the most frequently used ecosystem service models - InVEST) by comparing such estimates against multiple lines of local empirical data, and a more comprehensive representation of locally important erosion and deposition processes through a sediment budget model. Estimates of the magnitude of hillslope erosion modelled using an approach similar to InVEST and the calibrated sediment budget differed by an order of magnitude. If an uncalibrated InVEST-type model was used to inform the relative distribution of erosion magnitude, findings suggest the incorrect erosion process would be identified as the dominant contributor to suspended sediment loads. However, the sediment budget model could only be calibrated using data on sediment sources and sinks that had been collected in the catchment through a sustained research effort. A comparable level of research investment may not be available to inform ecosystem service assessments elsewhere. The results summarised here for the Mitchell catchment demonstrate that practitioners must exercise caution when using estimates of the sediment retention ecosystem service flow which have not been calibrated and validated against locally collected empirical data.

ecology↗

bioRxiv2017

A pan cancer analysis of promoter activity highlights the regulatory role of alternative transcription start sites and their association with noncoding mutations

Most human protein-coding genes are regulated by multiple, distinct promoters, suggesting that the choice of promoter is as important as its level of transcriptional activity. While the role of promoters as driver elements in cancer has been recognized, the contribution of alternative promoters to regulation of the cancer transcriptome remains largely unexplored. Here we infer active promoters using RNA-Seq data from 1,188 cancer samples with matched whole genome sequencing data. We find that alternative promoters are a major contributor to context-specific regulation of isoform expression and that alternative promoters are frequently deregulated in cancer, affecting known cancer-genes and novel candidates. Our study suggests that a highly dynamic landscape of active promoters shapes the cancer transcriptome, opening many opportunities to further explore the interplay of regulatory mechanism and noncoding somatic mutations with transcriptional aberrations in cancer.

genomics↗