Biology subjects

Mason, S.

Publications and source records attributed to Mason, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Development and Validation of an LC-MS Method for Quantification of Sex Steroid Hormones in Skeletal Muscle

1Sex steroid hormones are not exclusively localised in the circulation and can be found in numerous extragonadal tissues, in concentrations unrelated to the circulating fraction. Existing methodology to measure intramuscular steroid hormone concentrations includes both immune-based assays and liquid chromatography-mass spectrometry (LC-MS), the gold standard for hormone measurements. To date, no LC-MS based methods validation has been published on the measurement of intramuscular sex steroid hormones, despite clear biological relevance. Here, we describe the development and validation of a simple, high-throughput LC-MS Orbitrap method for the measurement of 10 intramuscular sex steroid hormones, including pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, epitestosterone, dihydrotestosterone, oestrone, oestradiol, and oestriol. In brief, isotope labelled standards were added to 5-6 milligrams of lyophilised muscle tissue, homogenised and extracted with ethyl acetate. The extracts were dried down and sequentially derivatised with 1-methylimidazole-2-sulfonyl chloride and hydroxylamine hydrochloride to target both the phenolic hydroxyl groups and ketone groups. The limit of detection was 1.0 {+/-} 1.0 pg/mg (range 0.36 - 3.26 pg/mg), with a R2 > 0.99 for all analytes. Matrix effects were 90-110% for all analytes except for dihydrotestosterone (143.6%), and precision was <10 CV% for all analytes in the presence of a muscle matrix. Our method allows for 20-40 samples to be prepared in [~]4 h, with a sample data acquisition time of 13 minutes. Moreover, our method provides the opportunity for specific analysis of steroid hormone concentrations in skeletal muscle, allowing target tissue specificity instead of relying on proxy measures from the circulation.

biochemistry↗

bioRxiv2026

Combining Anion Exchange and Size Exclusion Chromatography for Extracellular Vesicle Enrichment from Small Volumes of Human and Mouse Plasma for Quantitative Proteomics

Extracellular vesicles (EVs) are promising biomarkers, yet their proteomic analysis from plasma is hampered by low abundance and co-purification of contaminants (e.g., lipoproteins, platelets) and technical variability, particularly in small-volume animal models. We developed and validated a modular protocol integrating Size Exclusion Chromatography (SEC) with Strong Anion Exchange (SEC-SAX) specifically tailored for quantitative LC-MS proteomics from small starting volumes (150 l of plasma). SEC alone successfully removed 99% of Albumin, and the SAX step significantly enriched EVs over contaminating lipoproteins. Downstream single pot solid phase enhanced (SP3) sample prep and STAGE tip solid phase extraction ensured maximum proteome depth. Critical confounding factors were objectively assessed: Platelet Factor 4 (PF4) was confirmed as a highly sensitive platelet marker, confirming the necessity of meticulous plasma preparation. Sample hemolysis impacted the plasma EV proteome data. As such, an objective measure (nanodrop spectrophotometer) of hemolysis and exclusion of hemolysed samples (heme >0.3 mg/ml) is recommended. The protocol is applicable to both human and mouse plasma as demonstrated by EV enrichment and quantification of biomarker proteins associated with neurodegenerative diseases from eight individual mouse plasma samples. Manuscript HighlightsO_LIDevelopmental workflow for a quantitative SEC-SAX protocol for EV proteomics from small plasma volumes (150 l). C_LIO_LIA range of variables tested including SAX beads amount, digestion buffer, digestion time, STAGE tip solid phase extraction, SAX elution buffer and sample filtration. C_LIO_LIThe SAX step significantly enhances EV proteome depth by increasing EV purity in relation to ApoB lipoproteins. C_LIO_LIShows the impact of the major confounding factors of sample hemolysis and platelet contamination on the EV proteome. C_LIO_LIPlatelet contamination increases the number and abundance of proteins detected including known disease biomarkers and sample hemolysis is associated with proteins derived from platelet and red blood cell derived EVs. C_LIO_LIPlatelet Factor 4 (PF4) is identified and confirmed as a sensitive marker for platelet contamination. C_LIO_LIApplicable to both human and mouse plasma. C_LI

molecular biology↗

bioRxiv2025

A Novel Technique to Characterize Klebsiella pneumoniae Populations Indicates that Mono-Colonization is Associated with Risk of Infection

Klebsiella pneumoniae and related species are a common cause of healthcare-associated infections. The gut is a major Klebsiella reservoir and gut colonization is a risk factor for developing an extraintestinal Klebsiella infection. Patients can be colonized by multiple Klebsiella strains or even species in the gut simultaneously, and there is high concordance between the gut colonizing- and infection causing-strains. The detection and characterization of colonizing strains is critical for a better understanding of the progression to infection and for developing interventions for colonized patients. However, the association between mixed or mono-colonization and subsequent infection is unknown. In this study, we developed an amplicon-based sequencing method called wzi-Seq that enables the detection and quantification of Klebsiella strains from complex samples and mixtures using the conserved capsule gene wzi as a molecular barcode. This method is highly accurate and precise with a sensitivity of 93% and specificity of 99.8% in mixtures containing as many as 58 unique wzi types. The assay was validated analytically and applied to an established case and control cohort. We determined that 63.2% (108/171) patients were mono-colonized with a single Klebsiella strain while 36.8% (63/171) had mixed colonization with multiple Klebsiella strains. Controlling for patient variables in multivariate analysis, we determined that mono-colonization was significantly (p = 0.034) associated with infection. Characterization of Klebsiella colonizing populations could improve the accuracy of assessing infection risk and enable targeted interventions to prevent these healthcare-associated infections. ImportanceKlebsiella gut colonization is a major risk factor the development of extraintestinal Klebsiella infections in hospitalized settings. However, it is unknown if patients are colonized by one or multiple strains of Klebsiella and how the population structure of Klebsiella in the gut impacts infection risk. Here we describe the development of a novel technique, wzi-Seq, to detect and quantify multiple Klebsiella strains from complex samples using standard techniques and rapid DNA sequencing. We applied wzi-Seq to rectal swabs from a well-characterized patient cohort and found that Klebsiella colonization with a single strain was more prevalent than colonization with multiple strains. Furthermore, mono-colonized patients had a significantly higher risk of developing a Klebsiella infection than mixed colonized patients. This work validates a new tool to study Klebsiella populations and reveals that population structure in the gut influences the risk of healthcare associated infections.

microbiology↗

bioRxiv2024

A novel method for integrating genomic and Tn-Seq data to identify common in vivo fitness mechanisms across multiple bacterial species

Sepsis is life-threatening organ dysfunction due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of the Enterobacterales cause nearly half of bacteremia cases annually. While previous Tn-Seq studies to identify novel bacteremia-fitness genes have provided valuable insight into virulence mechanisms, evidence for common pathways across species is lacking. To identify common fitness pathways in five bacteremia- caused Enterobacterales species, we utilized the JCVI pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four out of the five species. Integration of Tn-Seq fitness data enabled identification of 373 protein clusters that were conserved in all five species. A scoring rubric and filter was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data, which reduced the number of bacteremia-fitness genes and identified seven common fitness mechanisms. Independent mutational validation of one prioritized fitness gene, tatC, showed reduced fitness in vivo and increased susceptibility to beta- lactams that were restored following tatC complementation in trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms that are essential for colonization of, or survival in, the mammalian bloodstream. Our prediction and validation of tatC as a common bacteremia fitness factor and contributor of antibiotic resistance supports the utility of this bioinformatic approach. This study represents a major step forward to prioritize potentially novel targets for therapy against these deadly widespread sepsis infections. Author SummaryBacteremia is a leading cause of sepsis, a life-threatening condition where an unregulated immune response to infection causes systemic organ failure. Nearly half of bacteremia cases are caused by members of the Gram-negative bacterial taxonomic order Enterobacterales. Given the public health impact of bacteremia and the reduction of existing antibiotic treatment options, novel strategies are needed to combat these infections. Pan-genome software was used to predict seven shared fitness pathways in these bacteria that may serve as novel targets for treatment of bacteremia. Briefly, a scoring rubric was applied to shared pan-genome clusters, which incorporated multiple data types, including Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data to rank and prioritize fitness genes. To validate one of our predictions, mutations were constructed in tatC, which showed both reduced fitness in mice and increased susceptibility to beta-lactam antibiotics; complementation restored fitness and antibiotic susceptibility to wild type levels. This study takes a novel bioinformatics approach to build a core pan-genome across multiple distantly related bacteria to integrate computational and experimental data to predict important shared fitness genes and represents a major step forward toward identifying novel targets of therapy against these deadly widespread life-threatening infections.

bioinformatics↗

bioRxiv2024

Proteomic analysis of the TDP-43-associated insoluble fraction from NEFH-TDP-43 mouse brain suggests sustained stress granule formation, CLUH granule recruitment and impaired mitochondrial metabolism

Cytoplasmic accumulation and aggregation of TDP-43 is a hallmark of [~]97% of ALS cases. Formation of TDP-43 insoluble aggregates is suggested to either directly or indirectly cause motor neuron loss and progressive neuromuscular degeneration, although how this occurs is not precisely understood. Cytoplasmic TDP-43 is observed in stress granules (SG). SGs are ribonucleoprotein (RNP) complexes formed during stress conditions, consisting of mRNAs and RNA-binding proteins (RNPs). Chronic TDP-43/SG formation may play a role in neuromuscular degeneration in ALS. The composition of in vivo TDP-43-asscociated SGs in ALS not known. This knowledge may provide insights into the molecular pathways impaired by TDP-43-associated SGs and suggest disease modifying mechanisms. The aim of this study was to isolate and analyse the proteome of the insoluble TDP-43-associated SG fraction from brain tissue of end-stage TDP-43{Delta}NLS mice. Proteomic analysis identified 134 enriched and 17 depleted proteins in the TDP-43{Delta}NLS mice, when compared to the control mice. Bioinformatics analyses of the impacted proteins from the SG preparation suggested that brain tissue from end-stage NEFH-TDP-43{Delta}NLS mice have sustained SG formation, CLUH granule recruitment and impaired mitochondrial metabolism. This is the first time that CLUH granule recruitment has been demonstrated in ALS and the known role of CLUH suggests that cell starvation is a potential mechanism of motor neuron loss that could be targeted in ALS. HighlightsO_LIWe present a detailed a protocol for the extraction of cross-linked TDP containing stress granules from brain tissue. C_LIO_LIWe present proteomics data from the insoluble fraction from brain tissue of an ALS mouse model. C_LIO_LIWe identify the mitochondrial mRNA transport protein CLUH and CLUH targets trapped in insoluble SG fraction of brain. C_LIO_LIReanalysing proteomics data from axonal soluble fraction supports a link between proteins trapped in the brain and depleted from the axons. C_LIO_LIPropose a model where metabolic mitochondrial enzymes trapped in the insoluble fraction from the brain via a CLUH dependent mechanism results in motor neuron death by starvation in ALS. C_LI

neuroscience↗

bioRxiv2024

Patterns of Klebsiella pneumoniae bacteremic dissemination from the lung

Bacteremia, a leading cause of death, generally arises after bacteria establish infection in a particular tissue and transit to secondary sites. Studying dissemination from primary sites by solely measuring bacterial burdens does not capture the movement of individual clones. Here, by barcoding Klebsiella pneumoniae, a leading cause of bacteremia, we tracked pathogen dissemination following pneumonia. Variability in organ bacterial burdens was attributable to two distinct dissemination patterns distinguished by the extent of clonal expansion in the lungs. In "metastatic" dissemination, bacterial clones underwent heterogeneous clonal expansion within the lung and the dominant clones spread to secondary organs. In "direct" dissemination, bacterial clones exited the lungs without clonal expansion, leading to lower burdens in systemic sites. We uncover bacterial and host factors that govern these two modes of dissemination. Our data reveal unexpected heterogeneity in the dynamics of Klebsiella bacteremia and define a new paradigm for understanding within-host bacterial dissemination.

microbiology↗

bioRxiv2024

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from an unregulated immune response to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Each mutant in each species was used to cochallenge C57BL/6 mice via tail vein injection along with the respective wild-type strain to determine competitive indices for each fitness gene or operon. Among the five species, we found three fitness factor genes, that when mutated, attenuated the mutant for all species in the spleen and liver (tatC, ruvA, gmhB). Nine additional fitness factor genes or operons were validated as outcompeted by wild-type in three or four bacterial species in the spleen (xerC, wzxE, arcA, prc, apaGH, atpG, lpdA, ubiH, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections. >Author SummaryFrequent cases of bacteremia plague our ICUs, bone marrow transplant units, and inpatient facilities. Nearly half of these infections are caused by Gram-negative bacteria. The Enterobacterales order including E. coli, K. pneumoniae, S. marcescens, C. freundii, and E. hormaechei are leading causes of bacteremia. An alarming proportion of these are due to antibiotic-resistant isolates, which are four times more likely to kill than antibiotic-susceptible isolates. Clearly, we need new therapeutic targets to treat cases of bacteremia and sepsis. Previously, it has been unclear what genes contribute to their ability to survive in this hostile host environment. We have previously undertaken unbiased genetic screens to identify 18 genes shared by all five bacterial genera that are required for survival in blood and blood-filtering organs. These include genes that encode proteins that maintain proton motive force, resist antimicrobial peptides and complement, mediate genome maintenance, transport key metabolites and proteins, avoid oxidative stress, acquire iron, and regulate key pathways. Mutants, constructed in these shared genes in the five species, were validated for a high proportion of genes as critical for infection in the mouse model of bacteremia.

microbiology↗

bioRxiv2024

Associations between regional blood-brain barrier disruption, aging, and Alzheimers disease biomarkers in cognitively normal older adults

BackgroundBlood-brain barrier disruption (BBBd) has been hypothesized as a feature of aging that may lead to the development of Alzheimers disease (AD). We sought to identify the brain regions most vulnerable to BBBd during aging and examine their regional relationship with neuroimaging biomarkers of AD. MethodsWe studied 31 cognitively normal older adults (OA) and 10 young adults (YA) from the Berkeley Aging Cohort Study (BACS). Both OA and YA received dynamic contrast-enhanced MRI (DCE-MRI) to quantify Ktrans values, as a measure of BBBd, in 37 brain regions across the cortex. The OA also received Pittsburgh compound B (PiB)-PET to create distribution volume ratios (DVR) images and flortaucipir (FTP)-PET to create partial volume corrected standardized uptake volume ratios (SUVR) images. Repeated measures ANOVA assessed the brain regions where OA showed greater BBBd than YA. In OA, Ktrans values were compared based on sex, A{beta} positivity status, and APOE4 carrier status within a composite region across the areas susceptible to aging. We used linear models and sparse canonical correlation analysis (SCCA) to examine the relationship between Ktrans and AD biomarkers. ResultsOA showed greater BBBd than YA predominately in the temporal lobe, with some involvement of parietal, occipital and frontal lobes. Within an averaged ROI of affected regions, there was no difference in Ktrans values based on sex or A{beta} positivity, but OA who were APOE4 carriers had significantly higher Ktrans values. There was no direct relationship between averaged Ktrans and global A{beta} pathology, but there was a trend for an A{beta} status by tau interaction on Ktrans in this region. SCCA showed increased Ktrans was associated with increased PiB DVR, mainly in temporal and parietal brain regions. There was not a significant relationship between Ktrans and FTP SUVR. DiscussionOur findings indicate that the BBB shows regional vulnerability during normal aging that overlaps considerably with the pattern of AD pathology. Greater BBBd in brain regions affected in aging is related to APOE genotype and may also be related to the pathological accumulation of A{beta}.

neuroscience↗

bioRxiv2023

Interactions between the gut microbiome, dietary restriction, and aging in genetically diverse mice

The intestinal microbiome changes with age, but the causes and consequences of microbiome aging remain unclear. Furthermore, the gut microbiome has been proposed to mediate the benefit of lifespan- extending interventions such as dietary restriction, but this hypothesis warrants further exploration. Here, by analyzing 2997 metagenomes collected longitudinally from 913 deeply phenotyped, genetically diverse mice, we provide new insights into the interplay between the microbiome, aging, dietary restriction, host genetics, and a wide range of health parameters. First, we find that microbiome uniqueness increases with age across datasets and species. Moreover, age-associated changes are better explained by cumulative exposure to stochastic events (neutral theory) than by the influence of an aging host (selection theory). Second, we unexpectedly find that the majority of microbiome features are significantly heritable and that the amount of variation explained by host genetics is as large as that of aging and dietary restriction. Third, we find that the intensity of dietary restriction parallels the extent of microbiome changes and that dietary restriction does not rejuvenate the microbiome. Lastly, we find that the microbiome is significantly associated with multiple health parameters -- including body composition, immune parameters, and frailty -- but not with lifespan. In summary, this large and multifaceted study sheds light on the factors influencing the microbiome and aspects of host physiology modulated by the microbiome.

microbiology↗

bioRxiv2023

Systematic characterisation of perturbations in complex developing cell populations reveals mechanisms underpinning development and disease

Perturbation studies using gene knockouts have become a key tool for understanding the roles of regulatory genes in development and disease. Here we systematically characterise the knockout effects of the key developmental regulators T and Mixl1 in chimeric mouse embryos during gastrulation and organogenesis. We present a comprehensive and effective suite of statistical tools for systematic characterisation of effects at the level of differential abundance of cell types, lineage development, and gene dysregulation. Applying our computational approach to a novel chimera data set with Mixl1 knockout reveals a disruption in Epicardium development in the absence of Mixl1, characterized by lack of upregulation of the key transcription factor Tbx18 and the Wnt regulator Sfrp5, and by dysregulation of the recently identified juxta-cardiac field. Finally, we demonstrate the wider utility of our framework by applying it to published acute myeloid leukemia (AML) patient data, and show how different responses to therapy are reflected in changes in gene expression along the myeloid trajectory between healthy and AML patients.

genomics↗

bioRxiv2023

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD largely enhanced liver fitness and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis. Author SummaryGram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia. Klebsiella pneumoniae is the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection. K. pneumoniae is highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis of K. pneumoniae bacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50 K. pneumoniae genes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically define K. pneumoniae factors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions during K. pneumoniae bacteremia that may be extended to additional Gram-negative species.

microbiology↗

bioRxiv2023

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we unveil a mechanism of cell-cell communication between CAFs with myofibroblast phenotype and endothelial cells (ECs) based on intercellular protein transfer through extracellular vesicles (EVs). CAFs transfer proteins to ECs, including plasma membrane receptors, which we have identified by using mass spectrometry- based proteomics. Using THY1 as an example of transferred plasma membrane-bound protein, we show that CAF-derived proteins can influence how ECs interact with other cell types. Here, we show that CAFs produce high amounts of matrix-bound EVs that have a key role in protein transfer. Hence, our work paves the way for further studies to understand how CAF-derived matrix-bound EVs influence tumor pathology by regulating functions of neighboring cancer, stromal and immune cells. One sentence summaryCAFs with a myofibroblastic-like phenotype transfer proteins to ECs, including plasma membrane receptors, through matrix-bound EVs

cancer biology↗

bioRxiv2022

The Klebsiella pneumoniae ter operon enhances stress tolerance

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to ROS-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify genes required to resist K2TeO3- and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as stress tolerance factor, therefore explaining its role in enhancing fitness in the gut and bladder.

microbiology↗

bioRxiv2022

Spatial regulation of the glycocalyx component Podocalyxin is a switch for pro-metastatic function

The glycocalyx component and sialomucin Podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumour types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for Galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high versus low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

cell biology↗

bioRxiv2022

The small GTPase ARF3 controls metastasis and invasion modality by regulating N-cadherin levels.

ARF GTPases are central regulators of membrane trafficking that act by controlling local membrane identity and remodelling to facilitate vesicle formation. Unravelling ARF GTPase function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. The extent to which redundancy is a major factor in ARF function or whether individual ARF GTPases make unique contributions to cellular behaviour remains unclear. Through a functional genomic screen of 3-Dimensional (3D) prostate cancer cell behaviour we explore the contribution of all known ARF GTPases, GEFs, GAPs, and a large selection of interactors to collective morphogenesis. This revealed that the ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of the junctional adhesion molecule N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumour transplants and ARF3:N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organise during invasion and metastasis.

cell biology↗

bioRxiv2022

Transcriptome analysis of alcohol dependence and stress interactions in the nucleus of the solitary tract

Stress exposure contributes to the development of drug and alcohol use disorders. In animal models, stress exacerbates escalations in alcohol consumption in alcohol-dependent animals. The nucleus of the solitary tract (NTS) is a critical brainstem region for integrating and relaying peripheral signals to regulate stress responses. To define the molecular adaptions within this brain region that may contribute to stress-induced alcohol drinking, we exposed animals to chronic intermittent bouts of ethanol vapor (CIE), forced swim stress (FSS), or both (CIE + FSS) and then transcriptionally profiled the NTS at three different timepoints after the last vapor exposure (0-hr, 72-hr, and 186-hr). We identified interferon (IFN) signaling as a critical gene network correlated with alcohol consumption levels. Using a likelihood ratio test, we identified genes that were differentially expressed across time and between groups. Clustering analysis of these genes to identify unique expression patterns identified a subset of genes that fail to normalize in the CIE + FSS group, but not the others. These genes were enriched for cell-to-cell interaction and cellular movement pointing to long-term structural and functional changes in this brain region caused by the unique interaction of alcohol dependence and stress. Specific genes of interest identified in this group include Aqp4, Il16, Reln, Grm4, Gabrd, and Gabra6. We also compared gene expression changes in the NTS to the PFC and found a significant overlap of genes between the two brain regions. Overlapping NTS/PFC genes in the CIE + FSS group were enriched for type I IFN signaling. Finally, we tested the hypothesis that activation of type I IFN signaling increases alcohol consumption based on the three lines of evidence identifying type I IFN signaling as critical for escalations in alcohol intake. Mice treated with recombinant IFN{beta} showed significantly elevated levels of alcohol intake in a two-bottle choice procedure compared to saline-treated controls. Overall, these results define the transcriptomic changes across time in the NTS that may be critical to the development of stress-induced increases in alcohol consumption and alcohol dependence.

molecular biology↗

bioRxiv2021

Adolescent ethanol drinking promotes hyperalgesia, neuroinflammation and serotonergic deficits in mice that persist into adulthood

Adolescent alcohol use can permanently alter brain function and lead to poor health outcomes in adulthood. Emerging evidence suggests that alcohol use predispose to pain disorders or exacerbate existing pain conditions, but the neural mechanisms are currently unknown. Here we report that mice exposed to adolescent intermittent access to ethanol (AIE) exhibit increased pain sensitivity and depressive-like behaviors that persist after alcohol cessation and are accompanied by elevated CD68 expression in microglia and reduced numbers of serotonin (5-HT)-expressing neurons in the dorsal raphe nucleus (DRN). 5-HT expression was also reduced in the thalamus, anterior cingulate cortex (ACC) and amygdala as well as the lumbar dorsal horn of the spinal cord. We then found that chronic minocycline administration after AIE alleviated hyperalgesia and social deficits, while chemogenetic activation of microglia in the DRN of Cx3cr1-cre-GFP mice reproduced the effects of AIE on pain and social interaction. Taken together, these results indicate that microglial activation in the DRN may be a primary driver of pain and negative affect after AIE.

neuroscience↗

bioRxiv2021

Dual G9A and EZH2 inhibition stimulates an anti-tumour immune response in ovarian high-grade serous carcinoma

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumour immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells whilst suppressing tumour-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/-null ID8 ovarian tumours and resulted in tumour burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumour growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/443282v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4c4323org.highwire.dtl.DTLVardef@cbd6fdorg.highwire.dtl.DTLVardef@8fb5e5org.highwire.dtl.DTLVardef@eef9c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗