Biology subjects

Murphy, A.

Publications and source records attributed to Murphy, A..

31 records · Page 2Linked to original sources

bioRxiv2021

Village in a dish: a model system for population-scale hiPSC studies

The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and under different conditions. Human induced pluripotent stem cells (hiPSCs) are uniquely suited to study these context-dependent effects, but to do so requires cell lines from hundreds or potentially thousands of individuals. Village cultures, where multiple hiPSC lines are cultured and differentiated together in a single dish, provide an elegant solution for scaling hiPSC experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned back to a donor line using single cell sequencing, and addressing whether line-specific signaling alters the transcriptional profiles of companion lines in a village culture. We generated single cell RNA sequence data from hiPSC lines cultured independently (uni-culture) and in villages at three independent sites. We show that the transcriptional profiles of hiPSC lines are highly consistent between uni- and village cultures for both fresh (0.46 < R < 0.88) and cryopreserved samples (0.46 < R < 0.62). Using a mixed linear model framework, we estimate that the proportion of transcriptional variation across cells is predominantly due to donor effects, with minimal evidence of variation due to culturing in a village system. We demonstrate that the genetic, epigenetic or hiPSC line-specific effects on gene expression are consistent whether the lines are uni- or village-cultured (0.82 < R < 0.94). Finally, we identify the consistency in the landscape of cell states between uni- and village-culture systems. Collectively, we demonstrate that village methods can be effectively used to detect hiPSC line-specific effects including sensitive dynamics of cell states.

genomics↗

bioRxiv2021

Rethinking habitat occupancy modeling and the role of diel activity in an anthropogenic world

Current methods to model species habitat use through space and diel time are limited. Development of such models is critical when considering rapidly changing habitats where species are forced to adapt to anthropogenic change, often by shifting their diel activity across space. We use an occupancy modeling framework to develop a new model, the multi-state diel occupancy model (MSDOM), which can evaluate species diel activity against continuous response variables which may impact diel activity within and across seasons or years. We used two case studies, fosa in Madagascar and coyote in Chicago, USA, to conceptualize the application of this model and to quantify the impacts of human activity on species spatial use in diel time. We found support that both species varied their habitat use by diel states--in and across years, and by human disturbance. Our results exemplify the importance of understanding animal diel activity patterns and how human disturbance can lead to temporal habitat loss. The MSDOM will allow more focused attention in ecology and evolution studies on the importance of the short temporal scale of diel time in animal-habitat relationships and lead to improved habitat conservation and management.

ecology↗

bioRxiv2021

Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic

Pseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus (P) limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglycosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of a cystic fibrosis patient. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients. ClassificationIntegrated genomics and post-genomics approaches in microbial ecology

microbiology↗

bioRxiv2021

The responsiveness of gait and balance outcomes to disease progression in Friedreich ataxia

ObjectiveTo identify gait and balance measures that are responsive to change during the timeline of a clinical trial in Friedreich ataxia (FRDA) we administered a battery of potential measures three times over a 12-month period. MethodsSixty-one ambulant individuals with FRDA underwent assessment of gait and balance at baseline, six months and 12 months. Outcomes included: GAITRite(R) spatiotemporal gait parameters; Biodex Balance System Postural Stability Test (PST) and Limits of Stability; Berg Balance Scale (BBS); Timed 25 Foot Walk Test; Dynamic Gait Index (DGI); SenseWear MF Armband step and energy activity; and the Friedreich Ataxia Rating Scale Upright Stability Subscale (FARS USS). The standardised response mean (SRM) or correlation coefficients were reported as effect size indices for comparison of internal responsiveness. Internal responsiveness was also analysed in subgroups. ResultsSenseWear Armband daily step count had the largest effect size of all the variables over six months (SRM=-0.615), while the PST medial-lateral index had the largest effect size (SRM=0.829) over 12 months. The FARS USS (SRM=0.824) and BBS (SRM=-0.720) were the only outcomes able to detect change over 12 months in all subgroups. The DGI was the most responsive outcome in children, detecting a mean change of -2.59 (95% CI -3.52 to -1.66, p<0.001, SRM=-1.429). ConclusionsThe FARS USS and BBS are highly responsive and can detect change in a wide range of ambulant individuals with FRDA. However, therapeutic effects in children may be best measured by the DGI.

neuroscience↗

bioRxiv2021

Modelling Human Gait using a Nonlinear Differential Equation

We introduce an innovative method for the investigation of human gait, which is based on the visualisation of the vertical component of the movement of the centre of mass during walking or running, in the space of the coordinates position, velocity, and acceleration of the centre of mass. Collected data has been numerically approximated by the best fitting curve for a non-linear model. The resulting equation for the best fitting plane or curve in this space is a differential equation of second order. The model that we suggest is a Duffing equation with coefficients that depend on the height of a walker or runner and on the angular frequency of the oscillation. Statistics about the distribution of the Duffing stiffness depending on the speed is presented. 1 Author SummaryWe study the human gait modelled by the movement of the centre of mass of the test person. This is an example of a biological process which can be considered as a periodical dynamic system. Roughly, this movement behaves in a similar way to a vibrating mass suspended on a spring, but it is more complex. The vertical component of the movement during walking or running can be visualised as an oscillogram: a graph of the position as a function of time. We suggest a visualisation of the data in 3D space, where the coordinates describe position, velocity, and acceleration. Our new visualisation method allows us to model the movement of a persons centre of mass by a nonlinear differential equation. The resulting curve for an ideal spring-mass movement, without viscosity or external force, is an ellipse in the suggested 3D space. The shape of the data curve shows at which position an additional force was applied, or the movement slowed down. Some deviations are common for all test persons and others are different. In the future we plan to investigate the reasons for these deviations, such as different running techniques or the presence of injuries.

bioengineering↗

bioRxiv2021

A novel class of sulfur-containing aminolipids widespread in marine roseobacters

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated SalA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modelling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. SalA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

microbiology↗

bioRxiv2020

Common genetic variation in humans impacts in vitro susceptibility to SARS-CoV-2 infection

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant inter-individual variability. In addition to showing more disease in males, the elderly, and individuals with underlying co-morbidities, SARS-CoV-2 can seemingly render healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants also impact vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR-engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single nucleotide polymorphism (rs4702), common in the population at large, and located in the 3UTR of the protease FURIN, impacts alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can impact viral infection, and thus contribute to clinical heterogeneity in SARS-CoV-2. Ongoing genetic studies will help to better identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs that might treat disease.

cell biology↗

bioRxiv2020

KDM6B promotes oncogenic CDK4/6-pRB-E2F pathway via maintaining enhancer activation in high-risk neuroblastoma

The H3K27me2/me3 histone demethylase KDM6B is over-expressed in neuroblastoma and is essential to neuroblastoma cell survival. While the KDM6B inhibitor, GSK-J4, has shown activity in in vitro and in vivo preclinical models, the mechanism of action remains poorly defined. We demonstrate that genetic and pharmacologic inhibition of KDM6B downregulates the pRB-E2F transcriptome and MYCN expression. Chemical genetic analyses show that high expression of the E2F transcriptome is positively correlated with sensitivity of cancer cells to GSK-J4. Mechanistically, inhibition of KDM6B activity 1) reduces the chromatin accessibility of E2F target genes and MYCN, 2) selectively leads to an increase of H3K27me3 but a decrease of the enhancer mark H3K4me1 at the CTCF and BORIS binding sites, which may, consequently, disrupt the long-range chromatin interaction of MYCN and E2F target genes, and 3) phenocopies the transcriptome induced by the specific CDK4/6 inhibitor palbociclib. Overexpression of CDK4/6 or Rb1 knockout confers neuroblastoma cell resistance to both palbociclib and GSK-J4. A gene signature targeted by KDM6B inhibition is associated with poor survival of patients with neuroblastoma regardless of the MYCN status. These data indicate that KDM6B activity promotes an oncogenic CDK4/6-pRB-E2F pathway in neuroblastoma cells via H3K27me3-dependent enhancer-promoter interactions, providing a rationale to target KDM6B for high-risk neuroblastoma.

cancer biology↗

bioRxiv2020

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: a useful tool for inferring transmission pathways

IntroductionMany arboviruses of public health significance are maintained in zoonotic cycles with complex transmission pathways. The presence of serum antibody against arboviruses in vertebrates provides evidence of their historical exposure but reveals nothing about the vector-reservoir relationship. Moreover, collecting blood or tissue samples from vertebrate hosts is ethically and logistically challenging. We developed a novel approach for screening the immune status of vertebrates against Ross River virus that allows us to implicate the vectors that form the transmission pathway for this commonly notified Australian arboviral disease. MethodsA micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using some convenient mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralizing antibodies using the micro-PRNT. ResultsThere was significant agreement of the plaque neutralization resulting from the micro-PRNT and standard PRNT techniques (R2=0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies neutralised RRV by [≥]50% until 60 hr post feeding. Similarly, mosquito blood meals from RRV seropositive human volunteers neutralised the virus by [≥]50% until 48 hr post-blood feeding. ConclusionsThe small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can clearly be adapted for similar studies on other mosquito borne zoonoses.

microbiology↗

bioRxiv2019

Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

Global food production is reliant on the application of finite phosphorus (P) fertilisers. Numerous negative consequences associated with intensive P fertilisation have resulted in a high demand to find alternative sustainable methods that will enhance crop P uptake. Bacteroidetes, primarily from the genus Flavobacterium, have recently been shown to be abundant members of the plant microbiome, but their general ecological role and potential to mobilise P in the rhizosphere remains very poorly characterised. Here, we sought to determine the P mobilisation potential of Flavobacterium strains isolated from the rhizosphere of oilseed rape (Brassica napus L.). We show that these Flavobacterium strains possess novel mechanisms for P mobilisation and subsequent acquisition. These include the constitutive and inducible expression of completely novel and phylogenetically distinct phosphatases, the phosphate starvation inducible expression of uncharacterised and hypothetical genes and gene clusters and, for the first time, the involvement of outer membrane SusCD transport complexes (usually associated with carbohydrate transport) in P acquisition. The genes encoding these unusual phosphate starvation inducible proteins were enriched in plant-associated Flavobacterium strains suggesting that this machinery represents niche-adaptive strategies for overcoming P scarcity in this genus. We propose that abundant rhizosphere-dwelling Flavobacterium spp. have evolved unique mechanisms for coping with Pi-stress which may provide novel solutions for future sustainable agricultural practices.

microbiology↗

bioRxiv2019

Spatial and epidemiologic features of dengue in Sabah, Malaysia

In South East Asia, dengue epidemics have increased in size and geographical distribution in recent years. Most studies investigating dengue transmission and control have had an urban focus, while less consideration is currently given to rural settings, or where urban and rural areas overlap. We examined the spatiotemporal distribution and epidemiological characteristics of reported dengue cases in the predominantly rural state of Sabah, in Malaysian Borneo - an area where sylvatic and urban circulation of pathogens are known to intersect. We found that annual dengue incidence rates were spatially variable over the 7-year study period from 2010-2016 (state-wide mean annual incidence of 21 cases/100,000 people; range 5-42/100,000), but were highest in rural localities in the western districts of the state (Kuala Penyu, Nabawan, Tenom and Kota Marudu). The eastern districts exhibited lower overall dengue rates; however, we noted a concentration of severe (haemorrhagic) dengue cases (44%) in Sandakan and Tawau districts. Dengue incidence was slightly higher for males than females, and was significantly higher for both genders aged between 10 and 29 years (24/100,000; p=0.029). The largest ever recorded outbreaks occurred during 2015-2016, with the vector Aedes albopictus found to be most prevalent in both urban and rural households (House Index of 64%), compared with Ae. Aegypti (15%). These findings suggest that dengue outbreaks in Sabah are driven by the sporadic expansion of dengue virus in both urban and rural settings. This may require tailoring of preventative strategies to suit different transmission ecologies across Sabah. Further studies to better understand the drivers of dengue in Sabah may aid dengue control efforts in Malaysia, and more broadly in South East Asia.\n\nAuthor summaryIn order to combat the rising regional incidence of dengue in South East Asia, the drivers of transmission must be better characterised across different environmental settings. We conducted the first retrospective analysis of dengue epidemiology in the predominantly rural state of Sabah, Malaysia, where both urban and sylvatic transmission cycles exist. Human notification data over a 7-year period were reviewed and spatiotemporal and demographic risk factors identified. We found: O_LIUrban habitats and population density are not the only determinants mediating the spread of epidemic dengue in Sabah. Case from both urban and rural localities contributed equally to dengue outbreaks.\nC_LIO_LIHuman demographic risk factors included being aged between 10 and 29 years, and being male.\nC_LIO_LIHigh incidence areas for dengue do not predict the occurrence of severe dengue. Severe dengue was largely localised to lower incidence districts in the east of the state.\nC_LIO_LIThe sole presence of Aedes albopictus in and around the majority of urban and rural case households suggests that this vector may play a major role in facilitating outbreaks.\nC_LI\n\nA complex interplay of risk factors likely mediates dengue transmission in Sabah, influenced by both regional climate trends and localised human and ecological influences. This study emphasises that the increasing spread of dengue in urban South East Asia is also mirrored in more rural areas, and suggests a need for control strategies that address both urban and rural dengue risk.

epidemiology↗

bioRxiv2019

Development and Application of a Tri-allelic PCR Assay for Screening Vgsc-L1014F Kdr Mutations Associated with Pyrethroid and Organochlorine Resistance in the Mosquito Culex quinquefasciatus

BackgroundCulex quinquefasciatus, has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda.\n\nResultsThis new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches - pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with 98.64% and 100% against pyrosequencing and TaqMan, respectively.\n\nConclusionsOur results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

genetics↗

bioRxiv2018

Interpreting mosquito feeding patterns in Australia through an ecological lens; an analysis of blood meal studies.

Mosquito-borne pathogens contribute significantly to the global burden of disease, infecting millions of people each year. Mosquito feeding is critical to the transmission dynamics of pathogens, and thus it is important to understanding and interpreting mosquito feeding patterns. In this paper we explore mosquito feeding patterns and their implications for disease ecology through a meta-analysis of published blood meal results collected across Australia from more than 12,000 blood meals from 22 species. To assess mosquito-vertebrate associations and identify mosquitoes on a spectrum of generalist or specialist feeders, we analysed blood meal data in two ways; first using a novel odds ratio analysis, and secondly by calculating Shannon diversity scores. We find that each mosquito species had a unique feeding association with different vertebrates, suggesting species-specific feeding patterns. Broadly, mosquito species could be grouped broadly into those that were primarily ornithophilic and those that fed more often on livestock. Aggregated feeding patterns observed across Australia were not explained by intrinsic variables such as mosquito genetics or larval habitats. We discuss the implications for disease transmission by vector mosquito species classified as generalist-feeders (such as Aedes vigilax and Culex annulirostris), or specialists (such as Aedes aegypti) in light of potential influences on mosquito host choice. Overall, we find that whilst existing blood meal studies in Australia are useful for investigating mosquito feeding patterns, standardisation of blood meal study methodologies and analyses, including the incorporation of vertebrate surveys, would improve predictions of the impact of vector-host interactions on disease ecology. Our analysis can also be used as a framework to explore mosquito-vertebrate associations, in which host availability data is unavailable, in other global systems.

ecology↗