Biology subjects

Wood, H.

Publications and source records attributed to Wood, H..

10 recordsLinked to original sources

bioRxiv2025

Methicillin-Resistant Staphylococcus aureus has Phenotypic Variation in mecA Expression that Alters Antibiotic Sensitivity

Methicillin resistant Staphylococcus aureus (MRSA) sepsis has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA sepsis is treated with dual antibiotic therapy that includes a {beta}-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report a serendipitous observation revealed a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA {beta}-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We therefore conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression in the mRNA level and limited PBP2a protein level when cultured in FBS or synovial fluid as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in {beta}-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between {beta}-lactam resistant and sensitive MRSA phenotypes. Together, this suggests pathways for clpP, rpoB, and WTA glycosylation can be new potential targets for MRSA treatment. IMPORTANCEThis study demonstrates that MRSA has phenotypic variation in mecA expression, PBP2a protein levels, and patterns of wall teichoic acid glycosylation based on environmental conditions which can result in a phenotype of {beta}-lactam antibiotic sensitivity. The presence of an antibiotic resistance gene does not necessarily result in antibiotic resistance. This provides a possible explanation for the clinical observations of potential therapeutic benefit of {beta}-lactam antibiotics in dual therapy treatment of MRSA sepsis and suggests new therapeutic strategies to combat multidrug resistant bacteria. More importantly, identifying genes potentially responsible for this phenotype offers new potential therapeutic targets for the treatment of multidrug resistant infection.

microbiology↗

bioRxiv2024

alpha-Actinin-3 deficiency protects from the effects of acute cold exposure through altered skeletal muscle Ca2+ and OXPHOS signaling.

Summary / AbstractA nonsense polymorphism in the ACTN3 gene (R577X, rs1815739) results in the loss of the fast skeletal muscle fiber protein -actinin-3 in an estimated 1.5 billion humans worldwide. Homozygosity for this common polymorphism (ACTN3 577XX) does not cause disease but is detrimental to sprint performance in elite athletes. Recently, we reported that ACTN3 577XX humans and Actn3 knockout mice show improved cold tolerance. This was not due to an increase in brown adipose tissue activity or a greater muscle shivering response, but was associated with an increased abundance of slow myosin heavy chain muscle fibers in XX individuals, which in turn increased muscle tone to improve cold tolerance. Using the Actn3 knockout mouse we have now analyzed the molecular and physiological impacts of acute cold exposure (4{degrees}C, for 5 hours) on skeletal muscle. In addition, we used statistical inference of publicly available archaic (Neanderthal and Denisovan) and modern human DNA data to estimate the age of the ACTN3 X-allele and to identify a novel haplotype associated with this variant that provides support for genetic selection in some populations of modern humans. Our results indicate that skeletal muscle of Actn3 knockout mice exhibit higher mitochondrial oxidative phosphorylation abundance and activity than wild-type controls. Following acute cold exposure, we identified reductions in gene ontology pathways associated with metabolism and protein breakdown, which impact muscle mass loss and improved muscle fatigue resistance. Together this provides further evidence for improved cold tolerance in Actn3 knockout mice and ACTN3 577XX individuals. Furthermore, we confirm that the X-allele first appeared in modern humans [~]135,000 years ago, with this variant increasing in frequency in European and Asian populations from as early as 42,000 years ago, which aligns with modern human migration out of Africa. Taken together these data provide additional molecular and functional evidence for a protective advantage of -actinin-3 deficiency following cold exposure, highlighting the importance of skeletal muscle in mammalian thermogenesis and its potential role in the evolution of modern-day humans.

cell biology↗

bioRxiv2023

Omicron Spike Protein Is Vulnerable to Reduction

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.

biochemistry↗

bioRxiv2022

Whole genome sequencing of 2,023 colorectal cancers reveals mutational landscapes, new driver genes and immune interactions

To characterise the somatic alterations in colorectal cancer (CRC), we conducted whole-genome sequencing analysis of 2,023 tumours. We provide the most detailed high-resolution map to date of somatic mutations in CRC, and demonstrate associations with clinicopathological features, in particular location in the large bowel. We refined the mutational processes and signatures acting in colorectal tumorigenesis. In analyses across the sample set or restricted to molecular subtypes, we identified 185 CRC driver genes, of which 117 were previously unreported. New drivers acted in various molecular pathways, including Wnt (CTNND1, AXIN1, TCF3), TGF-{beta}/BMP (TGFBR1) and MAP kinase (RASGRF1, RASA1, RAF1, and several MAP2K and MAP3K loci). Non-coding drivers included intronic neo-splice site alterations in APC and SMAD4. Whilst there was evidence of an excess of mutations in functionally active regions of the non-coding genome, no specific drivers were called with high confidence. Novel recurrent copy number changes included deletions of PIK3R1 and PWRN1, as well as amplification of CCND3 and NEDD9. Putative driver structural variants included BRD4 and SOX9 regulatory elements, and ACVR2A and ANKRD11 hotspot deletions. The frequencies of many driver mutations, including somatic Wnt and Ras pathway variants, showed a gradient along the colorectum. The Pks-pathogenic E. coli signature and TP53 mutations were primarily associated with rectal cancer. A set of unreported immune escape driver genes was found, primarily in hypermutated CRCs, most of which showed evidence of genetic evasion of the anti-cancer immune response. About 25% of cancers had a potentially actionable mutation for a known therapy. Thirty-three of the new driver genes were predicted to be essential, 17 possessed a druggable structure, and nine had a bioactive compound available. Our findings provide further insight into the genetics and biology of CRC, especially tumour subtypes defined by genomic instability or clinicopathological features.

genomics↗

bioRxiv2022

Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test

Neutralization assays are important in understanding and quantifying neutralizing antibody responses towards SARS-CoV-2. The SARS-CoV-2 Lentivirus Surrogate Neutralization Assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable, alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and pre-screening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity produced acceptable results with 100% (95% CI: 94-100) specificity and 100% (95% CI: 94-100) sensitivity against ancestral Wuhan spike pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike pseudotyped lentivirus resulted in 88.3% (95% CI: 77.8 to 94.2) and 100% (95% CI: 94-100), respectively. Assay precision measuring intra-assay variability produced acceptable results for High (1:[≥] 640 PRNT50), Mid (1:160 PRNT50) and Low (1:40 PRNT50) antibody titer concentration ranges based on the PRNT50, with %CV of 14.21, 12.47, and 13.28 respectively. Intermediate precision indicated acceptable ranges for the High and Mid concentrations, with %CV of 15.52 and 16.09, respectively. However, the Low concentration did not meet the acceptance criteria with a %CV of 26.42. Acceptable ranges were found in the robustness evaluation for both intra-assay and inter-assay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT.

scientific communication and education↗

bioRxiv2021

Exposure route, sex, and age influence disease outcome in a golden Syrian hamster model of SARS-CoV-2 infection

The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.

microbiology↗

bioRxiv2020

Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation

The fast skeletal muscle protein -actinin-3 is absent in 1.5 billion people worldwide due to homozygosity for a nonsense polymorphism in the ACTN3 gene (R577X) 1. The prevalence of the 577X allele increased as modern humans moved to colder climates, suggesting a link between -actinin-3 deficiency and improved cold tolerance 1,2. Here, we show that humans lacking -actinin-3 (XX) are superior in maintaining core body temperature during cold-water immersion due to changes in skeletal muscle thermogenesis. Muscles of XX individuals displayed a shift towards more slow-twitch isoforms of myosin heavy chain (MyHC) and sarcoplasmic reticulum (SR) proteins, accompanied by altered neuronal muscle activation resulting in increased tone rather than overt shivering 3,4. Experiments on Actn3 knockout mice showed no alterations in brown adipose tissue (BAT) properties that could explain the improved cold tolerance in XX individuals. Thus, this study provides a clear mechanism for the positive selection of the ACTN3 X-allele in cold climates and supports a key thermogenic role of skeletal muscle during cold exposure in humans.

genetics↗

bioRxiv2020

North American deer mice are susceptible to SARS-CoV-2

The zoonotic spillover of the pandemic SARS-coronavirus 2 (SARS-CoV-2) from an animal reservoir, currently presumed to be the Chinese horseshoe bat, into a naive human population has rapidly resulted in a significant global public health emergency. Worldwide circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive non-domesticated animals, potentially seeding new host reservoir species and geographic regions in which bat SARS-like coronaviruses have not historically been endemic. Here we report that North American deer mice (Peromyscus maniculatus) and some closely related members of the Cricetidae family of rodents possess key amino acid residues within the angiotensin-converting enzyme 2 (ACE2) receptor known to confer SARS-CoV-2 spike protein binding. Peromyscus rodent species are widely distributed across North America and are the primary host reservoirs of several emerging pathogens that repeatedly spill over into humans including Borrelia burgdorferi, the causative agent of Lyme disease, deer tick virus, and Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome (HPS). We demonstrate that adult deer mice are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naive deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 is unknown. Nevertheless, efforts to monitor wild, peri-domestic Peromyscus rodent populations are likely warranted as the SARS-CoV-2 pandemic progresses.

microbiology↗

bioRxiv2020

PanGIA: A Metagenomics Analytical Framework for RoutineBiosurveillance and Clinical Pathogen Detection

Metagenomics is emerging as an important tool in biosurveillance, public health, and clinical applications. However, ease-of-use for execution and data analysis remains a barrier-of-entry to the adoption of metagenomics in applied health and forensics settings. In addition, these venues often have more stringent requirements for reporting, accuracy, and precision than the traditional ecological research role of the technology. Here, we present PanGIA (Pan-Genomics for Infectious Agents), a novel bioinformatics analysis platform for hosting, processing, analyzing, and reporting shotgun metagenomics data of complex samples suspected of containing one or more pathogens. PanGIA was developed to address gaps that often preclude clinicians, medical technicians, forensics personnel, or other non-expert end-users from the routine application of metagenomics for pathogen identification. Though primarily designed to detect pathogenic microorganisms within clinical and environmental metagenomics data, PanGIA also serves as an analytical framework for microbial community profiling and comparative metagenomics. To provide statistical confidence in PanGIAs taxonomic assignments, the system provides two independent estimations of probability for species and strain level detection. First, PanGIA integrates coverage data with uniqueness information mapped across each reference genome for a stand-alone determination of confidence for each query sequence at each taxonomy level. Second, if a negative-control sample is provided, PanGIA compares this sample with a corresponding experimental unknown sample and determines a measure of confidence associated with detection above background. An integrated graphical user interface allows interactive interrogation and enables users to summarize multiple sample results by confidence score, normalized read abundance, reference genome linear coverage, depth-of-coverage, RPKM, and other metrics to detect specific organisms-of-interest. Comparison testing of the PanGIA algorithm against a number of recent k-mer, read-mapping, and marker-gene based taxonomy classifiers across various real-world datasets with spiked targets shows superior mean positive predictive value, sensitivity, and specificity. PanGIA can process a five million paired-end read dataset in under 1 hour on commodity computational hardware. The source code and documentation are publicly available at https://github.com/LANL-Bioinformatics/PanGIA or https://github.com/mriglobal/PanGIA. The database for PanGIA can be downloaded from ftp://bioinformatics.mriglobal.org/. The full GUI-based PanGIA analysis environment is available in a Docker container and can be installed from https://hub.docker.com/r/poeli/pangia/.

bioinformatics↗

bioRxiv2017

HistMapR: Rapid digitization of historical land-use maps in R

1. Habitat destruction and degradation represent serious threats to biodiversity, and quantification of land-use change over time is important for understanding the consequences of these changes to organisms and ecosystem service provision.\n\n2. Comparing land use between maps from different time periods allows estimation of the magnitude of habitat change in an area. However, digitizing historical maps manually is time-consuming and analyses of change are usually carried out at small spatial extents or at low resolutions.\n\n3. We developed a method to semi-automatically digitize historical land-use maps using the R environment. We created a number of functions that use the existing raster package to classify land use according to a maps colours, as defined by the RGB channels of the raster image. The method was tested on three different types of historical land-use map and results were compared to manual digitisations.\n\n4. Our method is fast, and agreement with manually-digitised maps of around 80-92% meets common targets for image classification. We hope that the ability to quickly classify large areas of historical land-use will promote the inclusion of land-use change into analyses of biodiversity, species distributions and ecosystem services.

ecology↗