Biology subjects

James, P.

Publications and source records attributed to James, P..

14 recordsLinked to original sources

bioRxiv2026

Environmental Toxin Rotenone Drives LRRK2-Mediated Microtubule, Cilia and Proteostasis Disruption in Parkinson's Disease Model

Exposure to the environmental toxin rotenone increases risk for Parkinsons disease (PD). However, protein phosphorylation changes induced by rotenone in neural-derived cells have not been reported. We examined the effect of rotenone on the proteome and phosphoproteome of cortex-derived cultures from wild-type and Lrrk2-/- mouse brains. We also analyzed the phosphoproteome of the PD cadaver brain using a previously unanalyzed dataset. Rotenone alters phosphorylation at 904 sites, most of which are unchanged in Lrrk2-/- cultures. Common targets include proteins that control microtubule stability, vesicular transport, and protein clearance via the autophagosomal/lysosomal pathway. Analysis of phosphosites with known function indicate that rotenone activates histone deacetylase-1 and represses the pro-survival transcriptional regulators Mef2C and Mef2D, while inhibiting translation by phosphorylating Eif2b5. These effects do not occur in Lrrk2-/- cultures. This study shows that Lrrk2 is required the earliest rotenone-triggered phospho-signalling events.

systems biology↗

bioRxiv2025

JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

GABAA-type receptors (GABAARs) mediate fast and tonic inhibition and are essential for maintaining excitatory/inhibitory balance in neural circuits. Disrupted GABAergic signaling leads to maladaptive changes associated with neuropsychiatric disorders, yet the mechanisms that regulate GABAAR surface availability remain incompletely understood. We previously identified that the stress-activated kinase JNK1 drives anxiety-like behaviours in mice suggesting a potential link between stress signalling and GABAergic regulation. Here, we show that genetic deletion or inhibition of JNK1 increases surface expression of {beta}3-containing GABAARs at extrasynaptic sites and at excitatory synapses where they may participate in shunting inhibition. We identify the signalling scaffold GIT1 as a direct JNK1 substrate, phosphorylated at S371, and find that GIT1 accumulates in dendritic spines in Jnk1-/- neurons or following JNK inhibition, whereas the phosphomimetic GIT1-S371D is excluded. Moreover, GIT1 is required for JNK-dependent regulation of {beta}3-GABAAR trafficking. Accordingly, neurons from Jnk1-/- mice exhibit increased spontaneous inhibitory postsynaptic potentials and enhanced tonic inhibition. These results reveal a JNK1-GIT1 signalling axis that suppresses GABAAR stability and inhibitory tone, providing a mechanism by which the stress-activated JNK pathway modulates synaptic and extrasynaptic inhibition.

neuroscience↗

bioRxiv2024

SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.

Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high- efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.

synthetic biology↗

bioRxiv2022

The role of Staphylococcus agnetis and Staphylococcus hyicus in the pathogenesis of buffalo fly skin lesions in cattle

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas most commonly on the medial canthus of the eye, along the lateral and ventral neck and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus have been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilised homogenized flies. Bacterial identification was conducted by MALDI-TOF, strain typing by rep-PCR and DNA sequencing to determine species similarity and virulence factors. Of 49 bacterial isolates collected from lesions, 37 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas from normal skin four isolates were S. hyicus and one was Staphylococcus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions.

microbiology↗

bioRxiv2022

Regulators of proteostasis are translationally repressed in fibroblasts from sporadic and LRRK2-G2019S Parkinson's patients

Parkinsons disease (PD) is characterized by heterogeneous motor and non-motor symptoms. The majority of PD cases are sporadic, whereas the leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common monogenic cause of familial PD and symptomatically indistinguishable from the sporadic form. Deficits in protein synthesis have been reported in PD. However, it is not known which proteins are affected or if there are synthesis differences between patients with sporadic and LRRK2 G2019S PD. Here we used non-canonical amino acid labeling and mass-spectrometry based proteomics to perform a quantitative comparison of nascent proteins in fibroblasts from sporadic and G2019S mutation PD patients compared to healthy individuals. We found that several proteins were under-represented among nascent proteins in cells from PD patients. Among these were regulators endo-lysosomal sorting, mRNA processing and translation itself. We further validated which of these proteins were dysregulated at the static proteome level using targeted proteomics. Key downregulated proteins included the autophagy regulator ATG9A and translational stability regulator YTHDF3. Notably, 68% of the identified proteins were downregulated in both sporadic and LRRK2-G2019S forms of PD. We identify translational repression of key regulators of proteostasis in patient fibroblasts that are common to both sporadic and LRRK2-G2019S PD.

neuroscience↗

bioRxiv2022

Machine learning for exploring microbial inter-kingdom associations in Cystic Fibrosis and Bronchiectasis

BackgroundCystic fibrosis (CF) and non-CF bronchiectasis (BX) are characterised by severe chronic infections. Fungal and bacterial components of infection are both recognized. Little however is known about how fungal and bacterial organisms interact and whether these interactions impact on disease outcomes. MethodsQuantitative PCR and next-generation sequencing of ITS2 and 16S rRNA gene was carried out on 107 patients with CF or BX with clinically defined fungal infection status for all patients. The relationship between fungal and bacterial community composition was extensively explored using: random forest modelling, correlation network analysis, multi-omics factor analysis, and sample-wise clustering, to understand associations both within and between the microbial communities and their relationship to respiratory disease. ResultsRandom forest modelling demonstrated distinct fungal and bacterial communities within CF and BX patients. The inclusion of both kingdoms in the models did not improve discrimination between the two diseases. Within the CF patients, bacterial community composition was independent of clinical fungal disease status. Bacterial and fungal communities did not relate to the presence of CF pulmonary exacerbations (CFPE). Correlation network analysis found intra-kingdom interactions were predominant in the data. Multi-omics factor analysis (MOFA) revealed latent factors corresponding to single kingdoms. Thus, in the bacterial community we identified two distinct clusters characterised by the presence or absence of Pseudomonas-domination. This was independent of fungal community which was characterised by a second set of independent clusters dominated by Saccharomycetes. ConclusionsIn this study we were unable to detect clear evidence of clinically significant inter-kingdom interactions between the bacterial and fungal communities. While further work is required to fully understand microbial interaction within the lung, our data suggests that interkingdom interactions may not be the primary driver of patient outcomes, particularly in the context of fungal infection.

molecular biology↗

bioRxiv2021

PhosPiR: An automated phospho-proteomic pipeline in R

Large-scale phospho-proteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from this data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from largescale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phospho-site annotation and translation across species, multi-level enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.

bioinformatics↗

bioRxiv2020

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to {pound}10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

genomics↗

bioRxiv2020

Protein synthesis is suppressed in sporadic and familial Parkinson's Disease by LRRK2

Gain of function LRRK2-G2019S is the most common mutation associated with both familial and sporadic Parkinsons disease. It is expected therefore that understanding the cellular function of LRRK2 will provide much needed insight on the pathological mechanism of sporadic Parkinsons, which is the most common form. Here we show that constitutive LRRK2 activity represses nascent protein synthesis in rodent neurons. Specifically, enzymatic inhibition of LRRK2, gene silencing or gene knockout of Lrrk2 increase translation. In the rotenone model of Parkinsons, LRRK2 activity increases, leading to repression of translation and dopaminergic neuron atrophy both of which are prevented by LRRK2 inhibition. This is accompanied by altered phosphorylation of eIF2-S52({uparrow}), eIF2s2-S2({downarrow}) and eEF2-T57({uparrow}) in striatum/substantia nigra in a direction that signifies inhibition of protein synthesis. Significantly, LRRK2 is activated and translation is 40% reduced in Parkinsons patient fibroblasts (G2019S and sporadic) and LRRK2 inhibition restores normal translation. In contrast, translation is unchanged in cells from multiple system atrophy patients, implying disease specificity. These findings suggest that LRRK2-dependent repression of translation may be a proximal function of LRRK2 in Parkinsons pathology.

neuroscience↗

bioRxiv2019

Transinfection of buffalo flies (Haematobia exigua) with Wolbachia and effect on host biology

A widespread insect endosymbiont Wolbachia is currently of much interest for use in novel strategies for the control of insect pests and blocking transmission of insect-vectored diseases. Wolbachia-induced effects can vary from beneficial to detrimental depending on host biology and the genetic background of the infecting strains. As a first step towards investigating the potential of Wolbachia for use in the biocontrol of buffalo flies (BF), embryos, pupae, and adult female BF were injected with three different Wolbachia strains (wAlbB, wMel and wMelPop). BF eggs were not easily injected because of their tough outer chorion and embryos were frequently damaged resulting in less than 1% hatch rate of microinjected eggs. No Wolbachia infection was recorded in flies successfully reared from injected eggs. Adult and pupal injection gave a much higher survival rate and resulted in somatic infection and germinal tissue infection in surviving flies with transmission to the succeeding generations on a number of occasions. Investigations of infection dynamics in flies from injected pupae confirmed that Wolbachia were increasing in numbers in BF somatic tissues and ovarian infections were confirmed with wMel and wMelPop in some instances, though not with wAlbB. Measurement of fitness traits indicated reduced longevity, decreased and delayed adult emergence, and reduced fecundity in Wolbachia-infected flies in comparison to mock-injected flies. Furthermore, fitness effects varied according to the Wolbachia strain injected with most marked reductions seen in the wMelPop-injected flies and least severe effects seen with the wAlbB strain.

microbiology↗

bioRxiv2019

Wolbachia successfully replicate in a newly established horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae) cell line

BACKGROUNDHaematobia spp., horn flies (HF) and buffalo flies (BF), are economically important ectoparasites of dairy and beef cattle. Control of these flies relies mainly on the treatment of cattle with chemical insecticides. However, the development of resistance to commonly used compounds is compromising the effectiveness of these treatments and alternative methods of control are required. Wolbachia are maternally transmitted endosymbiotic bacteria of arthropods that cause various reproductive distortions and fitness effects, making them a potential candidate for use in the biological control of pests. RESULTSHere we report the successful establishment of a continuous HF cell line (HIE-18) from embryonic cells and its stable transinfection with Wolbachia strain wAlbB native to mosquitoes, and wMel and wMelPop native to Drosophila melanogaster. The established HIE-18 cells are typically round and diploid with ten chromosomes (2n = 10) or tetraploid with 20 chromosomes (4n=20) having a doubling time of 67.2 hours. Wolbachia density decreased significantly in the HIE-18 cells in the first 48 hours of infection, possibly due to overexpression of antimicrobial peptides through the Imd immune signalling pathway. However, density recovered after this time and stably Wolbachia-infected HIE-18 cell lines have now all been subcultured more than 50 times as persistently infected lines. CONCLUSIONThe amenability of HF to infection with different strains of Wolbachia suggests the potential for use of Wolbachia in novel approaches for the control of Haematobia spp. Further, the availability of the HIE-18 cell line will provide an important resource for the study of genetics, host-parasite interactions and chemical resistance in Haematobia populations.

microbiology↗

bioRxiv2019

Complete genome assembly of the Wolbachia endosymbiont of the horn fly Haematobia irritans irritans: a supergroup A strain with multiple horizontally acquired cytoplasmic incompatibility genes

The horn fly, Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between USD 1-2.5 billion annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, USA and Alberta, Canada, but has also been identified in wild-caught samples from Canada, USA, Mexico and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularised 1.3 Mb Wolbachia genome (wHae). Annotation of wHae yielded 1249 coding genes, 34 tRNAs, three rRNAs, and five prophage regions. Comparative genomics and whole genome Bayesian evolutionary analysis of wHae compared to published Wolbachia genomes suggests that wHae is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wHae and closely related genomes indicates that wHae has undergone convoluted genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wHae suggests two phylogenetically distinct CI loci and acquisition of another CifB homolog from phylogenetically distant supergroup A Wolbachia strains suggesting horizontal acquisition of these loci. The wHae genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies.\n\nImportanceHorn flies, Haematobia irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts on vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrickville, USA horn fly (wHae). Annotation of wHae suggests its unique features including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study will provide the foundation for future Wolbachia-induced biological effect studies for control of horn flies.

microbiology↗

bioRxiv2019

Evaluating environmental and ecological landscape characteristics relevant to urban resilience across gradients of land-sharing-sparing and urbanity

Within urban landscape planning, debate continues around the relative merits of land-sparing (compaction) and land-sharing (sprawl) scenarios. Using part of Greater Manchester (UK) as a case-study, we present a landscape approach to mapping green infrastructure and variation in social-ecological-environmental conditions as a function of land sparing and sharing. We do so for the landscape as a whole as well as for areas of high and low urbanity. Results imply potential trade-offs between land-sparing-sharing scenarios relevant to characteristics critical to urban resilience such as landscape connectivity and diversity, air quality, surface temperature, and access to green space. These trade-offs may be particularly complex due to the parallel influence of patch attributes such as land-cover and size and imply that both ecological restoration and spatial planning have a role to play in reconciling tensions between land-sparing and sharing strategies.

ecology↗

bioRxiv2019

Distinctive airway microbial ecology of smokers and asthmatics

BackgroundNormal airway microbial communities play a central role in respiratory health but are poorly characterized. Cigarette smoking is the dominant global environmental influence on lung function, and asthma has become the most prevalent chronic respiratory disease worldwide. Both conditions have major microbial components that are also poorly defined. MethodsWe investigated airway bacterial communities in a general population sample of 529 Australian adults. Posterior oropharyngeal swabs were analysed by sequencing of the 16S rRNA and methionine aminopeptidase genes. The microbiota were characterised according to their prevalence, abundance, and network memberships. FindingsMicrobial communities were similar across the population and were strongly organized into co-abundance networks. Smoking associated with diversity loss, negative effects on abundant taxa, profound alterations to network structure and expansion of Streptococcus spp. By contrast, the asthmatic microbiota were selectively affected by an increase in Neisseria spp. and by reduced numbers of low abundance but prevalent organisms. InterpretationOur study shows healthy airway microbiota are contained within a highly structured ecosystem, indicating balanced relationships between the microbiome and human host factors. The marked abnormalities in smokers may be pathogenic for chronic obstructive pulmonary disease (COPD) and lung cancer. The narrow spectrum of abnormalities in asthmatics encourages investigation of damaging and protective effects of specific bacteria. FundingThe study was funded by the Asmarley Trust and a Wellcome Senior Investigator Award to WOCC and MFM (P46009). The Busselton Healthy Ageing Study is supported by the Government of Western Australia (Office of Science, Department of Health) the City of Busselton, and private donations.

ecology↗