Veterinary Biosciences - Research Publications
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ItemProtease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products(WILEY, 2022-05)BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.
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ItemProteomic analysis of Sarcoptes scabiei reveals that proteins differentially expressed between eggs and female adult stages are involved predominantly in genetic information processing, metabolism and/or host-parasite interactions(PUBLIC LIBRARY SCIENCE, 2022-12)Presently, there is a dearth of proteomic data for parasitic mites and their relationship with the host animals. Here, using a high throughput LC-MS/MS-based approach, we undertook the first comprehensive, large-scale proteomic investigation of egg and adult female stages of the scabies mite, Sarcoptes scabiei-one of the most important parasitic mites of humans and other animals worldwide. In total, 1,761 S. scabiei proteins were identified and quantified with high confidence. Bioinformatic analyses revealed differentially expressed proteins to be involved predominantly in biological pathways or processes including genetic information processing, energy (oxidative phosphorylation), nucleotide, amino acid, carbohydrate and/or lipid metabolism, and some adaptive processes. Selected, constitutively and highly expressed proteins, such as peptidases, scabies mite inactivated protease paralogues (SMIPPs) and muscle proteins (myosin and troponin), are proposed to be involved in key biological processes within S. scabiei, host-parasite interactions and/or the pathogenesis of scabies. These proteomic data will enable future molecular, biochemical and physiological investigations of early developmental stages of S. scabiei and the discovery of novel interventions, targeting the egg stage, given its non-susceptibility to acaricides currently approved for the treatment of scabies in humans.
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ItemNo Preview AvailableSeverely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1(AMER SOC HEMATOLOGY, 2022-03-24)Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.
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ItemNo Preview AvailableUbiquitination pathway model for the barber's pole worm - Haemonchus contortus(ELSEVIER SCI LTD, 2022-08)The ubiquitin-mediated pathway has been comprehensively explored in the free-living nematode Caenorhabditis elegans, but very little is known about this pathway in parasitic nematodes. Here, we inferred the ubiquitination pathway for an economically significant and pathogenic nematode - Haemonchus contortus - using abundant resources available for C. elegans. We identified 215 genes encoding ubiquitin (Ub; n = 3 genes), ubiquitin-activating enzyme (E1; one), -conjugating enzymes (E2s; 21), ligases (E3s; 157) and deubiquitinating enzymes (DUBs; 33). With reference to C. elegans, Ub, E1 and E2 were relatively conserved in sequence and structure, and E3s and DUBs were divergent, likely reflecting functional and biological uniqueness in H. contortus. Most genes encoding ubiquitination pathway components exhibit high transcription in the egg compared with other stages, indicating marked protein homeostasis in this early developmental stage. The ubiquitination pathway model constructed for H. contortus provides a foundation to explore the ubiquitin-proteasome system, crosstalk between autophagy and the proteasome system, and the parasite-host interactions. Selected E3 and DUB proteins which are very divergent in sequence and structure from host homologues or entirely unique to H. contortus and related parasitic nematodes may represent possible anthelmintic targets.
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ItemThermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868(FRONTIERS MEDIA SA, 2022-10-14)Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber's pole worm, and in other socioeconomically important parasitic nematodes. The "hit-to-target" workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.
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ItemEvidence that Transcriptional Alterations in Sarcoptes scabiei Are under Tight Post-Transcriptional (microRNA) Control(MDPI, 2022-09)Here, we explored transcriptomic differences among early egg (Ee), late egg (Le) and adult female (Af) stages of the scabies mite, Sarcoptes scabiei, using an integrative bioinformatic approach. We recorded a high, negative correlation between miRNAs and genes with decreased mRNA transcription between the developmental stages, indicating substantial post-transcriptional repression; we also showed a positive correlation between miRNAs and genes with increased mRNA transcription, suggesting indirect post-transcriptional regulation. The alterations in mRNA transcription between the egg and adult female stages of S. scabiei were inferred to be linked to metabolism (including carbohydrate and lipid degradation, amino acid and energy metabolism), environmental information processing (e.g., signal transduction and signalling molecules), genetic information processing (e.g., transcription and translation) and/or organismal systems. Taken together, these results provide insight into the transcription of this socioeconomically important parasitic mite, with a particular focus on the egg stage. This work encourages further, detailed laboratory studies of miRNA regulation across all developmental stages of S. scabiei and might assist in discovering new intervention targets in the egg stage of S. scabiei.
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ItemRepurposing of a human antibody-based microarray to explore conserved components of the signalome of the parasitic nematode Haemonchus contortus(BMC, 2022-07-30)BACKGROUND: Gaining insight into molecular signalling pathways of socioeconomically important parasitic nematodes has implications for understanding their molecular biology and for developing novel anthelmintic interventions. METHODS: Here, we evaluated the use of a human antibody-based microarray to explore conserved elements of the signalome in the barber's pole worm Haemonchus contortus. To do this, we prepared extracts from mixed-sex (female and male) adult worms and third-stage larvae (L3s), incubated these extracts on the antibody microarray and then measured the amounts of antibody-bound proteins ('signal intensity'). RESULTS: In total, 878 signals were classified into two distinct categories: signals that were higher for adults than for larvae of H. contortus (n = 376), and signals that were higher for larvae than for adults of this species (n = 502). Following a data-filtering step, high confidence ('specific') signals were obtained for subsequent analyses. In total, 39 pan-specific signals (linked to antibodies that recognise target proteins irrespective of their phosphorylation status) and 65 phosphorylation-specific signals were higher in the adult stage, and 82 pan-specific signals and 183 phosphorylation-specific signals were higher in L3s. Thus, notably more signals were higher in L3s than in the adult worms. Using publicly available information, we then inferred H. contortus proteins that were detected (with high confidence) by specific antibodies directed against human homologues, and revealed relatively high structural conservation between the two species, with some variability for select proteins. We also in silico-matched 763 compound structures (listed in the DrugBank and Kinase SARfari public databases) to four H. contortus proteins (designated HCON_00005760, HCON_00079680, HCON_00013590 and HCON_00105100). CONCLUSIONS: We conclude that the present antibody-based microarray provides a useful tool for comparative analyses of signalling pathways between/among developmental stages and/or species, as well as opportunities to explore nematocidal target candidates in H. contortus and related parasites.
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ItemNKG7 Enhances CD8+T Cell Synapse Efficiency to Limit Inflammation(FRONTIERS MEDIA SA, 2022-07-06)Cytotoxic lymphocytes are essential for anti-tumor immunity, and for effective responses to cancer immunotherapy. Natural killer cell granule protein 7 (NKG7) is expressed at high levels in cytotoxic lymphocytes infiltrating tumors from patients treated with immunotherapy, but until recently, the role of this protein in cytotoxic lymphocyte function was largely unknown. Unexpectedly, we found that highly CD8+ T cell-immunogenic murine colon carcinoma (MC38-OVA) tumors grew at an equal rate in Nkg7+/+ and Nkg7-/- littermate mice, suggesting NKG7 may not be necessary for effective CD8+ T cell anti-tumor activity. Mechanistically, we found that deletion of NKG7 reduces the ability of CD8+ T cells to degranulate and kill target cells in vitro. However, as a result of inefficient cytotoxic activity, NKG7 deficient T cells form a prolonged immune synapse with tumor cells, resulting in increased secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF). By deleting the TNF receptor, TNFR1, from MC38-OVA tumors, we demonstrate that this hyper-secretion of TNF compensates for reduced synapse-mediated cytotoxic activity against MC38-OVA tumors in vivo, via increased TNF-mediated tumor cell death. Taken together, our results demonstrate that NKG7 enhances CD8+ T cell immune synapse efficiency, which may serve as a mechanism to accelerate direct cytotoxicity and limit potentially harmful inflammatory responses.
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ItemNo Preview AvailableChromosome-level genome of Schistosoma haematobium underpins genome-wide explorations of molecular variation(PUBLIC LIBRARY SCIENCE, 2022-02)Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.
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ItemNo Preview Available3D genomics across the tree of life reveals condensin II as a determinant of architecture type(AMER ASSOC ADVANCEMENT SCIENCE, 2021-05-28)We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.