Biology subjects

Alexander, J.

Publications and source records attributed to Alexander, J..

29 records · Page 2Linked to original sources

bioRxiv2022

Identification and characterization of four bacteriome- and mycobiome-derived subtypes in tumour and adjacent mucosa tissue of colorectal cancer patients

ObjectiveHere, we systematically investigated alterations in the bacteriome and mycobiome of CRC patients in tumours and matched adjacent mucosa resulting in the identification of microbiome-based subtypes associated with host clinico-pathological and molecular characteristics. DesignDiversity and composition of bacteriome and mycobiome of tumour and adjacent mucosa, resulting subtypes were computationally deconvoluted from RNA sequencing, using >10000 samples from in-house and publically available patient cohorts. ResultsThe bacteriome of tumours had higher dominance and lower -diversity compared to matched adjacent local and distant mucosa. Tumours were enriched with Proteobacteria (Gammaproteobacteria class), Fusobacteria (including Fusobacterium Nucleatum species) and Basidiomycota fungi (Malasseziaceae family). Tumours were depleted of Bacteroidetes (Bacteroidia class), Firmicutes (Clostridia class) and Ascomycota (Sordariomycetes and Saccharomycotina). Tumours and adjacent mucosa samples were classified into 4 microbial subtypes, termed C1 to C4, based on the bacteriome and mycobiome composition. The bacterial Propionibacteriaceae, Enterobacteriaceae, Fusobacteriaceae, Bacteroidaceae and Ruminococcaceae and the fungal Malasseziaceae, Saccharomycetaceae and Aspergillaceae were among the key families driving the microbial subtyping. Microbial subtypes were associated with distinct tumour histology and patient phenotypes and served as an independent prognostic marker for disease-free survival. Key associations between microbial subtypes and alterations in host immune response and signalling pathways were validated in the TCGA pan-cancer cohort. The microbial subtyping demonstrated stratification value in the pan-cancer settings beyond merely representing differences in survival by cancer type. ConclusionsThis study demonstrates the translational potential of microbial subtyping in CRC patient stratification, and provides avenues to design tailored microbiota modulation therapy to further precision oncology. Statement of significanceO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIThe microbiome has been implicated in the pathogenesis, progression and therapeutic response in patients diagnosed with CRC and other cancers. C_LIO_LIThe vast majority of studies to date has focussed on investigating the bacteriome while the critical role played by the mycobiome in shaping cancer has begun to be explored more recently. C_LIO_LIThe bacterial and fungal composition and diversity in on-tumour tissue compared to matched local and distal off-tumour mucosa is largely unexplored. C_LIO_LITumorigenesis may be promoted via alterations of the microbiome ecosystem that may be better recapitulated by a multi-kingdom microbial signature rather than by the abundance of individual bacterial or fungal microorganisms. C_LI What are the new findings?O_LIOn-tumour tissue was enriched with Fusobacteria, Proteobacteria, Basidyomicota and depleted of Bacteroidetes, Firmicutes, Ascomycota compared with adjacent off-tumour mucosa. C_LIO_LIWe stratified >600 CRC patients into four distinct microbial-based subtypes (C1-C4) according to their bacteriome and mycobiome composition. The microbial subtypes were associated with distinct prognosis, clinical phenotypes such as staging, tumour location, history, lymphovascular invasion, TP53 status and clinical outcome. C_LIO_LIFurthermore, the majority of matched adjacent mucosa samples were classified as C1 and paired tumour-matched normal samples demonstrated a robust shift towards the C1 subtype in off-tumour tissue, suggesting that the C1 subtype may recapitulate a healthier-like microbiome. This hypothesis was supported by the microbial subtyping of colon samples from healthy subjects that were categorised as C1 almost exclusively. C_LIO_LIThe identified microbial subtyping demonstrated stratification value in the pan-cancer settings (n=28 additional solid cancer indications spanning >9000 samples) beyond merely representing differences in survival by cancer type, providing the strongest stratification in liver cancer. C_LI How might it impact on clinical practise in the foreseeable future?O_LIThis study laids the foundation to develop a microbial signature as biomarker to clinically manage CRC and other solid cancers and potentially lead to microbiome-based companion diagnostics to design microbiota modulation therapy tailored to specific patients subgroups with distinct bacteriomes and mycobiomes. C_LI

cancer biology↗

bioRxiv2022

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells

The PSMC3IP-MND1 heterodimer promotes RAD51 and DMC1-dependent D-loop formation during meiosis in yeast and mammalian organisms. For this purpose, it catalyzes the DNA strand exchange activities of the recombinases. Interestingly, in a panel of genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, we found that depletion of either PSMC3IP or MND1 caused sensitivity to clinical Poly (ADP-Ribose) Polymerase inhibitors (PARPi). A retroviral mutagenesis screen in mitotic cells also identified PSMC3IP and MND1 as genetic determinants of ionizing radiation sensitivity. The role PSMC3IP and MND1 play in preventing PARPi sensitivity in mitotic cells appears to be independent of a previously described role in alternative lengthening of telomeres (ALT). PSMC3IP or MND1 depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Although replication fork reversal is also affected, the epistatic relationship between PSMC3IP-MND1 and BRCA1/BRCA2 suggests that the abrogated D-loop formation is the major cause of PARPi sensitivity. This is corroborated by the fact that a PSMC3IP p.Glu201del D-loop formation mutant associated with ovarian dysgenesis fails to reverse PARPi sensitivity. These observations suggest that meiotic proteins such as MND1 and PSMC3IP could have a greater role in mitotic cells in determining the response to therapeutic DNA damage.

cancer biology↗

bioRxiv2022

Case-only rare variant analysis of severe alcohol dependence (AD) using a multivariate hierarchical gene clustering approach.

BackgroundVariation in genes involved in ethanol metabolism has been shown to influence risk for alcohol dependence (AD) including protective loss of function alleles in ethanol metabolizing genes. We therefore hypothesized that people with severe AD would exhibit different patterns of rare functional variation in genes with strong prior evidence for influencing ethanol metabolism and response when compared to genes not meeting these criteria. ObjectiveLeverage a novel case only design and Whole Exome Sequencing (WES) of severe AD cases from the island of Ireland to quantify differences in functional variation between genes associated with ethanol metabolism and/or response and their matched control genes. MethodsFirst, three sets of ethanol related genes were identified including those a) involved in alcohol metabolism in humans b) showing altered expression in mouse brain after alcohol exposure, and altering ethanol behavioral responses in invertebrate models. These genes of interest (GOI) sets were matched to control gene sets using multivariate hierarchical clustering of gene-level summary features from gnomAD. Using WES data from 190 individuals with severe AD, GOI were compared to matched control genes using logistic regression to detect aggregate differences in abundance of loss of function, missense, and synonymous variants, respectively. ResultsThree non-independent sets of 10, 117, and 359 genes were queried against control gene sets of 139, 1522, and 3360 matched genes, respectively. Significant differences were not detected in the number of functional variants in the primary set of ethanol-metabolizing genes. In both the mouse expression and invertebrate sets, we observed an increased number of synonymous variants in GOI over matched control genes. Post-hoc simulations showed the estimated effects sizes observed are unlikely to be under-estimated. ConclusionThe proposed method demonstrates a computationally viable and statistically appropriate approach for genetic analysis of case-only data for hypothesized gene sets supported by empirical evidence.

genetics↗

bioRxiv2022

Using machine learning to understand microgeographic determinants of the Zika vector, Aedes aegypti

There are limited data on why the 2016 Zika outbreak in Miami-Dade County, Florida was confined to certain neighborhoods. In this research, Aedes aegypti, the primary vector of Zika virus, are studied to examine neighborhood-level differences in their population dynamics and underlying processes. Weekly mosquito data were acquired from the Miami-Dade County Mosquito Control Division from 2016 to 2020 from 172 traps deployed around Miami-Dade County. Using Random Forest, a machine learning method, predictive models of spatiotemporal dynamics of Ae. aegypti in response to meteorological conditions and neighborhood-specific socio-demographic and physical characteristics, such as land-use and land-cover type and income level, were created. The study area was divided into two groups: areas affected by local transmission of Zika during the 2016 outbreak and unaffected areas. Aedes aegypti populations in areas affected by Zika were more strongly influenced by 14- and 21-day lagged weather conditions. In the unaffected areas, mosquito populations were more strongly influenced by land-use and day-of-collection weather conditions. There are neighborhood-scale differences in Ae. aegypti population dynamics. These differences in turn influence vector-borne disease diffusion in a region. These results have implications for vector control experts to lead neighborhood-specific vector control strategies and for epidemiologists to guide vector-borne disease risk preparations, especially for containing the spread of vector-borne disease in response to ongoing climate change.

ecology↗

bioRxiv2021

A global map of the Zika virus phosphoproteome reveals host-driven regulation of viral budding

Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within the Flavivirus genus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation. Both appear to regulate viral budding, while one also impacts ZIKV cytopathogenicity. Additionally, we discovered host kinases that interact with ZIKV proteins and determined that Bosutinib--an FDA-approved tyrosine kinase inhibitor that targets some of these host kinases--impairs ZIKV growth, in part by blocking phosphorylation of a tyrosine residue on the envelope protein. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered novel mechanisms of host driven-regulation of ZIKV budding and cytopathogenicity, and identified an FDA-approved inhibitor of ZIKV growth.

microbiology↗

bioRxiv2020

Towards establishing no observed adverse effect levels (NOAEL) for different sources 1 of dietary phosphorus in feline adult diets: results from a 7 month feeding study

High dietary phosphorus (P), particularly soluble salts, may contribute to chronic kidney disease development in cats. The aim of this study was to assess the safety of P supplied at 1g/1000kcal from a highly soluble P salt in P-rich dry format feline diets. Seventy-five healthy adult cats (n=25/group) were fed either a low P control (1.4g/1000 kcal; calcium:phosphorus ratio, Ca:P 0.97) or one of two test diets with 4g/1000 kcal (4184kJ); Ca:P 1.04 or 5g/1000kcal; Ca:P 1.27, both incorporating 1g/1000kcal (4184kJ) sodium tripolyphosphate (STPP) - for a period of 30 weeks in a randomised parallel-group study. Health markers in blood and urine, glomerular filtration rate, renal ultrasound and bone density were assessed at baseline and at regular time points. At the end of the test period, responses following transition to a commercial diet (total P - 2.34g/1000kcal, Ca:P 1.3) for a 4-week washout period were also assessed. No adverse effects on general, kidney or bone (skeletal) function and health were observed. P and Ca balance, some serum biochemistry parameters and regulatory hormones were increased in cats fed test diets from week 2 onwards (p[≤]0.05). Data from the washout period suggest that increased serum creatinine and urea values observed in the two test diet groups were influenced by dietary differences during the test period, and not indicative of changes in renal function. The present data suggest NOAELs for feline diets containing 1g P/1000kcal (4184kJ) from STPP and total phosphorus level of up to 5g/1000kcal (4184kJ) when fed for 30 weeks.

pharmacology and toxicology↗

bioRxiv2020

Impairment of a distinct cancer-associated fibroblast population limits tumour growth and metastasis

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 (MRC2) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability which, coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation.

cancer biology↗

bioRxiv2019

G-Graph: An interactive genomic graph viewer

MotivationEffective and efficient exploration of numeric data and annotations as a function of genomic position requires specialized software. ResultsWe present G-Graph, an interactive genomic scatter plot viewer. G-Graph stacks or tiles multiple data series in one graph using different colors and markers. It displays gene annotation and other metadata, allows easy changes to the appearance of data series, implements stack-based undo functionality, and saves user-selected application views as image and pdf files. G-Graph delivers smooth and rapid scrolling and zooming even for datasets with millions of points and line segments. The primary target user is a researcher examining many copy number profiles to identify potentially deleterious variants. G-Graph runs under Linux, Mac OSX and Windows. Availabilityhttps://github.com/docpaa/mumdex/ or https://mumdex.com/ggraph/ Contactandrewsp@cshl.edu (or paa@drpa.us)

genomics↗

bioRxiv2019

Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double stranded break (DSB), followed by NHEJ or HDR mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, i.e. to drive replacement of the DNA sequence intervening the dual cuts via NHEJ. In a proof-of-concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event, and opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

genomics↗

bioRxiv2018

Consensus interpretation of the Met34Thr and Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel

PURPOSEPathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants.\n\nMETHODSThe ClinGen Hearing Loss Expert Panel (HL-EP) collected published data and shared unpublished information from participating laboratories regarding the two variants. Functional, computational, allelic, and segregation data were also obtained.\n\nRESULTSThe panel reviewed the synthesized information, and classified the Met34Thr and Val37Ile variants according to professional variant interpretation guidelines. We found that Met34Thr and Val37Ile are significantly overrepresented in hearing loss patients, compared to the general population. Met34Thr or Val37Ile homozygotes or compound heterozygotes typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for those two variants.\n\nCONCLUSIONResolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification rules, review evidence, and reach a consensus. The ClinGen HL-EP concluded that Met34Thr and Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and age-dependent penetrance.

genetics↗

bioRxiv2017

TECPR2 a positive regulator of autophagy is implicated in healthy brain ageing

Understanding the healthy brain aging process is key to uncovering the mechanisms leading to pathological age-related neurodegeneration, including progression to Alzheimers disease (AD). Here, we report the first deep whole genome sequencing study aiming to identify variants that are associated specifically to healthy brain aging defined on both clinical and neuropathological level, thus tacking the issue of pathological heterogeneity that often underlies a clinical AD diagnosis. We studied samples from the VITA brain bank and followed an extreme phenotypic ends study design comparing neuropathologically \"healthy\" aging individuals above 80 years of age with pure AD patients of the same age. Focusing on the extreme ends of the phenotypic distribution, and potentially functional variants, we discover a single variant (rs10149146) carried by 53.6% of the \"healthy\" brain elderly individuals in our study (15/28 individuals) and none of the 12 AD cases. This variant lies on the autophagy and cell cycle associated TECPR2 gene. Autophagy dysfunction has been previously implicated in multiple progressive neurodegenerative diseases. An additional non-synonymous variant on the CINP gene (encoding a cell-cycle checkpoint protein) is also found in 46% of healthy controls and absent from all the AD cases. TECPR2 and CINP appear to be \"partner\" genes in terms of regulation and their associated transcription factors have been previously implicated in AD and neurodegeneration. Our study is the first to support the hypothesis that a TECPR2 non-synonymous variant carries a significant neuroprotective effect pointing to key molecules for the involvement of autophagy and cell cycle control in protection from neurodegeneration.

genomics↗