Biology subjects

Clarke, J.

Publications and source records attributed to Clarke, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Faecal glucocorticoid metabolite levels of intensive captive, free-range captive, and wild Tasmanian devils

Captivity can alter the stress physiology and behaviour of an animal in both the short- and long-term through repetitive exposure to novel stressors and, subsequently, may reduce the success of conservation efforts such as translocation and reintroduction. The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction from a fatal facial tumour disease which has led to the establishment of an insurance meta-population designed for future reintroductions of disease-free devils. The meta-population is comprised of intensive captive and free-range captive environments; however, no study has yet examined the long-term physiological implications of captivity on devils. We used non-invasive faecal glucocorticoid metabolite (FGM) monitoring to determine if there were any differences in adrenal activity between intensive captive, free-range captive, and wild devils. FGMs were not age- or sexdependent, and we found that all population-types had similar intra-population variability and mean FGMs. In conclusion, both types of captive environment maintain stress profiles similar to wild devils.

zoology↗

bioRxiv2022

Survival of mineral-bound peptides into the Miocene

The oldest authenticated peptide sequences to date were reported in 2016 from 3.8 Ma old ostrich eggshell (OES) from the site of Laetoli, Tanzania (Demarchi et al., 2016). Here we demonstrate survival of the same sequences in 6.5-9 Ma OES recovered from a palaeosteppe setting in northwestern China. The eggshell is thicker than those observed in extant species and consistent with the Liushu Struthio sp. ootaxon. These findings push the preservation of ancient proteins back to the Miocene and highlight their potential for paleontology, paleoecology and evolutionary biology.

paleontology↗

bioRxiv2022

Diversity, abundance, and host specificity of the human skin associated circular and single stranded DNA virome

The human skin is our point of contact with the microbial world, yet little is known about the diversity of the skin virome. Studies of the human skin virome have focused on bacteriophage and double-stranded DNA viral genomes, however, there have been few efforts to characterize circular single-stranded DNA viruses that populate human skin. Here, we evaluate the diversity of the circular single-stranded DNA virome collected across three anatomical skin locations from 60 human individuals with five time-point collections spanning six-months. Our analyses resulted in the identification of 272 novel and unique Rep-encoded single-stranded DNA viruses associated with human skin. Sequence similarity networks and maximum likelihood estimations of the Rep and Capsid protein amino acid sequences from our sequencing and public database references, reveal family level stability of the Cressdnaviricota across the study participants and a larger host-range than previously thought for these putative multi-host pathogens.

microbiology↗

bioRxiv2022

A wheat kinase and immune receptor form the host-specificity barrier against the blast fungus

Since emerging in Brazil in 1985, wheat blast has spread throughout South America and recently appeared in Bangladesh and Zambia. We show that two wheat resistance genes, Rwt3 and Rwt4, acting as host-specificity barriers against non-Triticum blast pathotypes encode a nucleotide-binding leucine-rich repeat immune receptor and a tandem kinase, respectively. Molecular isolation of these genes allowed us to develop assays that will ensure the inclusion of these two genes in the wheat cultivars to forestall the recurrence of blast host jumps.

plant biology↗

bioRxiv2021

Novel Feline Papillomavirus Isolate P20 Assembled from Metagenomic Data Isolated from Human Skin of a House Cat Owner

A feline papillomavirus was assembled from metagenomic sequencing data collected from the human skin of a house cat owner. This circular papillomavirus strain P20 is 8069 bp in length, has a GC content of 54.38%, and displays genome organization typical of feline papillomaviruses with six annotated protein coding regions. The genome exhibits approximately 75% sequence similarity to other feline papillomavirus genomes.

microbiology↗

bioRxiv2021

The Application of The Skin Virome for Human Identification

The use of skin virome for human identification purposes offers a unique approach to instances where a viable and statistically relevant human DNA profile is unavailable. The human skin virome may act as an alternative DNA profile and/or an additional form of probative genetic material. To date, no study has attempted to investigate the human virome over a time series across various physical locations of the body to identify its potential as a tool for human identification. For this study, we set out to evaluate the stability, diversity, and individualization of the human skin virome. An additional goal was to identify viral signatures that can be used in conjunction with traditional forensic STR loci. In order to accomplish this, human virome metagenomes were collected and sequenced from 42 individuals at three anatomical locations (left hand, right hand, and scalp) across multiple collections periods over a 6-month window of time. Assembly dependent and independent bioinformatic approaches were employed, along with a database-based assessment, which resulted in three sets of stable putative viral markers. In total, with the three sets combined, 59 viral species and uncharacterized viral genome assemblies were identified as being significantly stable (P=5.3x10-15). Viral diversity, based on presence or absence, is significantly different across subjects (P<0.001). Here we demonstrate that not only is the human virome applicable to be used for human identification, but we have identified many viral signatures that can be used for forensic applications, thus providing a foundation to the novel field of forensic virology. HighlightsO_LIHere we provide the largest human skin virome study, to date. Our study revealed novel diversity findings of high abundance for certain viral taxa, for example, the Cress-like DNA phages, that have not previously been characterized in human skin viral ecology studies. C_LIO_LIThere were 59 putative human skin viral biomarkers suitable for human identification from the core stable human skin virome of 42 subjects. C_LIO_LIThe putative markers we identified were significantly stable over a 6-month period of time within individuals and across three autosomal locations of left hand, right hand, and scalp. C_LIO_LIDiversity of profiles, based on the presence and absence of our putative marker data set, were significantly different across test subjects. C_LI

microbiology↗

bioRxiv2020

Pollen source richness may be a poor predictor of bumblebee colony growth

O_LIAgricultural intensification has drastically altered foraging landscapes for bees, with large-scale crop monocultures associated with floral diversity loss. C_LIO_LIResearch on bumblebees and honeybees has shown individuals feeding on pollen from a low richness of floral sources can experience negative impacts on health and longevity relative to higher pollen source richness of similar protein concentrations. Florally rich landscapes are thus generally assumed to better support social bees. Yet, little is known about whether the effects of reduced pollen source richness can be mitigated by feeding on pollen with higher crude protein concentration, and importantly how variation in diet affects whole colony growth, rearing decisions and sexual production. C_LIO_LIStudying queen-right bumblebee (Bombus terrestris) colonies, we monitored colony development under polyfloral pollen diet or monofloral pollen diet with 1.5-1.8 times higher crude protein concentration. C_LIO_LIOver six weeks, we found monofloral colonies performed better for all measures, with no apparent long-term effects on colony mass or worker production, and a higher number of pupae in monofloral colonies at the end of the experiment. Unexpectedly, polyfloral colonies showed higher mortality, and little evidence of any strategy to counteract the effects of reduced protein; with fewer and lower mass workers being reared, and males showing a similar trend. C_LIO_LIOur findings i) provide well-needed daily growth dynamics of queenright colonies under varied diets, and ii) support the view that pollen protein content in the foraging landscape rather than floral species richness per se is likely a key driver of colony success. C_LI

ecology↗

bioRxiv2020

The folding and unfolding behavior of ribonuclease H on the ribosome

The health of a cell depends on accurate translation and proper protein folding; misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the energy landscape of the nascent chain. However, quantifying these environmental effects is challenging due to the ribosomal proteins and rRNA, which preclude most spectroscopic measurements of protein energetics. We have applied two gel-based approaches, pulse proteolysis and force-peptide arrest assays, to probe the folding and unfolding pathways of RNase H ribosome-stalled nascent chains. We find that ribosome-stalled RNase H has an increased unfolding rate compared to free RNase H, which completely accounts for observed changes in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNase H on the ribosome. Surprisingly, we find that population of the RNase H folding intermediate is required to generate sufficient force to release the SecM stall and that readthrough of the stall sequence directly correlates with the stability of the folding intermediate. Together, these data imply that the folding pathway of RNase H is unchanged on the ribosome. Furthermore, our data indicate that the ribosome promotes unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of misfolding and assist in folding fidelity.

biophysics↗

bioRxiv2020

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that NOK/Pals1/Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Nok and Rab11a mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose these roles for both Nok and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction remodelling role of Nok is not specific to dividing cells.

neuroscience↗

bioRxiv2020

Vitamin D regulates MerTK-dependent phagocytosis in human myeloid cells

Vitamin D deficiency is a major environmental risk factor for the development of multiple sclerosis (MS). The major circulating metabolite of vitamin D (25OHD) is converted to the active form (calcitriol) by the hydroxylase enzyme CYP27B1. In MS lesions the tyrosine kinase MerTK expressed by microglia and macrophages regulates phagocytosis of myelin debris and apoptotic cells that can accumulate and inhibit tissue repair and remyelination. We show that calcitriol downregulates MerTK mRNA and protein expression in adult human microglia and monocyte-derived macrophages, thereby inhibiting myelin phagocytosis and apoptotic cell clearance. Proinflammatory myeloid cells express high levels of CYP27B1 compared to homeostatic (TGF{beta}-treated) myeloid cells. Only proinflammatory cells in the presence of TNF- generate calcitriol from 25OHD, resulting in repression of MerTK expression and function. The selective production of calcitriol in proinflammatory myeloid cells leading to downregulation of MerTK-mediated phagocytosis has the potential to reduce the risk for auto-antigen presentation while retaining the phagocytic ability of homeostatic myeloid cells, thereby contributing to inflammation reduction and enhanced tissue repair.

immunology↗

bioRxiv2020

Trophectoderm Potency is Retained Exclusively in Human Naïve Cells

Classical mouse embryology has established a paradigm of early development driven by sequential lineage bifurcations. Accordingly, mouse embryonic stem cells derived from early epiblast have lost the potency to produce extraembryonic trophectoderm. We show in contrast that human naive epiblast cells readily make trophectoderm. Inhibition of ERK signalling, instrumental in naive stem cell propagation, unexpectedly potentiates trophectoderm formation, an effect enhanced by Nodal inhibition. Transcriptome analyses authenticate conversion into trophectoderm with subsequent production of syncitiotrophoblast, cytotrophoblast and trophoblast stem cells. Genetic perturbations indicate that NANOG suppresses and TFAP2C enables trophectoderm induction. Consistent with post-implantation progression, trophectoderm potential is extinguished in conventional human pluripotent stem cells, which instead make amnion. Finally, human embryo epiblasts from late blastocysts efficiently generate trophectoderm and differentiated trophoblast. Thus, pluripotent cells in the human embryo retain extraembryonic lineage plasticity and regenerative potential until implantation. Harnessing this unanticipated regulative capacity may be beneficial for assisted reproduction technology.

developmental biology↗

bioRxiv2020

CD4+ follicular helper-like T cells are key players in anti-tumor immunity

To determine the nature of CD4+ T cells that provide help for generating robust anti-tumor CD8+ cytotoxic T cell (CTL) responses, we profiled the transcriptomes of patient-matched CD4+ and CD8+ T cells present in the tumor micro-environment (TME) and analyzed them jointly using integrated weighted gene correlation network analysis. We found the follicular helper T cell (TFH) program in CD4+ T cells was strongly associated with proliferation and tissue-residency in CD8+ CTLs. Single-cell analysis demonstrated the presence of TFH-like cells and features linked to cytotoxic function and their provision of CD8+ T cell help. Tumor-infiltrating TFH-like cells expressed PD-1 and were enriched in tumors following checkpoint blockade, suggesting that they may respond to anti-PD-1 therapy. Adoptive transfer or induction of TFH cells in mouse models resulted in augmented CD8+ CTL responses and impairment of tumor growth, indicating an important role of TFH-like CD4+ T cells in anti-tumor immunity.

immunology↗

bioRxiv2019

Establishing the phenotypic basis of adherent-invasive Escherichia coli (AIEC) pathogenicity in intestinal inflammation

Background & AimsAdherent-invasive Escherichia coli (AIEC) are enriched in ileal Crohns disease patients and implicated in disease etiology. However, AIEC pathogenesis is poorly understood, and it is unclear if the expansion of these organisms contributes to inflammatory bowel disease (IBD). Questions also remain as to what extent the various in vitro phenotypes used to classify AIEC are pathologically relevant.\n\nMethodsWe utilized a combination of in vitro phenotyping and a murine model of intestinal inflammation to systematically relate AIEC phenotypes to pathogenicity for 30 mucosa-associated human-derived E. coli strains. In vitro assays used included survival/replication in and TNF- production by J774 macrophages as well as invasion/replication in Caco2 intestinal epithelial cells.\n\nResultsAIEC do not form a phenotypic group that is clearly separated from non-AIEC. However, E. coli strains displaying in vitro AIEC phenotypes caused, on average, more severe intestinal inflammation. Survival/replication of strains in J774 and Caco2 cells were positively correlated with disease in vivo, while adherence to Caco2 cells and TNF- production by J774 cells were not. Importantly, co-colonization with adherent non-AIEC strains ameliorated AIEC-mediated disease.\n\nConclusionOur findings do not support the existence of an AIEC pathovar that can be clearly separated from commensal E. coli. However, intracellular survival/replication phenotypes do contribute to murine intestinal inflammation, suggesting that the AIEC overgrowth observed in human IBD makes a causal contribution to disease. The ability to differentiate pathologically-relevant AIEC phenotypes from those that are not provides an important foundation for developing strategies to predict, diagnose and treat human IBD through characterizing and modulating patient E. coli populations.

microbiology↗

bioRxiv2019

Spontaneous oligomerization of BAK/BAX is suppressed by hetero-dimerization with MCL-1

BCL-2 proteins control the intrinsic pathway of programmed cell death. Composed of anti- and pro-apoptotic members, their network of interactions forms a molecular switch that controls mitochondrial outer-membrane permeability. Apoptotic stimulation leads to BAK/BAX oligomerization and pore formation, yet the molecular details of this pivotal step remain poorly understood, and controversy persists regarding the activation mechanism. Here we use native mass spectrometry and kinetics to show that the homo-oligomerization of BAK and BAX is spontaneous in hydrophobic environments. This process is abrogated by hetero-dimerization of both BAK and BAX with the anti-apoptotic BCL-2 protein MCL-1. Pro-apoptotic BH3-only proteins disrupt these hetero-dimers by binding competitively to MCL-1, releasing BAK/BAX for homo-oligomerization. Thus, we infer that their oligomeric states are thermodynamically favored at the membrane. Our approach provides the framework for future quantitative biophysical characterizations of the BCL-2 network, and advances our molecular understanding of apoptosis.

biophysics↗

bioRxiv2019

Streptolysin production and activity is central to in vivo pathotype and disease outcome in GAS infections

Streptococcus pyogenes (GAS) is among the most diverse of all human pathogens, responsible for a range of clinical manifestations, from mild superficial infections such as pharyngitis to serious invasive infections such as necrotising fasciitis and sepsis. The drivers of these different disease phenotypes are not known. The GAS cholesterol-dependent cytolysin, streptolysin O (SLO), has well established cell and tissue destructive activity. We investigated the role of SLO in determining disease outcome in vivo, by using two different clinical lineages; the recently emerged hypervirulent outbreak emm type 32.2 strains, which result in sepsis, and the emm type 1.0 strains which cause septic arthritis. Using clinically relevant in vivo mouse models of sepsis and a novel septic arthritis model, we demonstrated that the amount and activity of SLO is vital in determining the pathotype of infection. The emm32.2 strain produced large quantities of highly haemolytic SLO that resulted in rapid development of sepsis. By contrast, the lower levels and haemolytic activity of emm1.0 SLO led to translocation of bacteria to joints. Importantly, sepsis associated strains that were attenuated by deletion or inhibition of SLO also translocated to the joint, confirming the key role of SLO in determining infection niche. Our findings demonstrate that SLO is key to in vivo pathotype and disease outcome. Careful consideration should be given to novel therapy or vaccination strategies that target SLO. Whilst neutralising SLO activity may reduce severe invasive disease, it has the potential to promote chronic inflammatory conditions such as septic arthritis.

microbiology↗

bioRxiv2019

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-of-concept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications.

biochemistry↗

bioRxiv2019

Efficient RNA-mediated reprogramming of human somatic cells to naive pluripotency facilitated by tankyrase inhibition

In contrast to conventional human pluripotent stem cells (hPSC) that are related to post-implantation embryo stages, naive hPSC exhibit features of pre-implantation epiblast. Naive hPSC are established by resetting conventional hPSC, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that tankyrase inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSC can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene free naive hPSC offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSC.

developmental biology↗

bioRxiv2019

An alanine permease mutation or exposure to D-cycloserine re-sensitizes MRSA to beta-lactam antibiotics

Prolonging the clinical effectiveness of {beta}-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted D-cycloserine (DCS) transporter gene cycA re-sensitized MRSA to {beta}-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and D-alanine ligase required for D-alanine incorporation into cell wall peptidoglycan (PG). Alanine transport was impaired in the cycA mutant and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal D-ala-D-ala and reduced PG crosslinking, prompting us to investigate synergism between {beta}-lactams and DCS. DCS re-sensitised MRSA to {beta}-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteraemia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to {beta}-lactam antibiotics.

microbiology↗