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Item Open Access The Oxidation of Oxygen and Sulfur-Containing Heterocycles by Cytochrome P450 Enzymes(Wiley-VCH GmbH, 2023) Podgorski, M.N.; Keto, A.B.; Coleman, T.; Bruning, J.B.; De Voss, J.J.; Krenske, E.H.; Bell, S.G.The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a g-keto-α,β-unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.Item Metadata only Shigella flexneri remodeling and consumption of host lipids during infection(American Society for Microbiology, 2023) Ascari, A.; Frölich, S.; Zang, M.; Tran, E.N.H.; Wilson, D.W.; Morona, R.; Eijkelkamp, B.A.; Galperin, M.Y.Shigella flexneri is a major cause of bacillary dysentery in the developing world, predominantly affecting the pediatric age group, with malnutrition being a common co-morbidity. Lipids are key nutritional components, and their abundance and composition are likely to influence the pathobiology of S. flexneri. S. flexneri expresses a plethora of polysaccharides on its cell surface, but how this hydrophilic surface layer influences S. flexneri interaction with hydrophobic host molecules, such as fatty acids and lipids, is not well understood. In this study, we sought to interrogate how this hydrophilic layer affects S. flexneri during its intracellular lifestyle and how lipid homeostasis changes in both the host and pathogen. We characterized changes in S. flexneri cell envelope composition and surface-associated glycolipids, in particular lipopolysaccharide (LPS), during different phases of infection. We found that a dynamic capacity in LPS expression is necessary for the pathogen to manage delicate interaction with host fatty acids and maintain optimum virulence. Additionally, through confocal immunofluorescent microscopy, coupled with transcriptional and lipid analyses, we demonstrate that S. flexneri induces major host lipid remodeling during infection, by hijacking host lipid homeostasis pathways to its own benefit. Finally, this study suggests that fatty acid supplementation can influence the persistence and magnitude of S. flexneri infection. This work provides novel insights into the potential roles of balanced and sufficient dietary fatty acid intake in protection against gastroenteric pathogen infection.Item Open Access Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy(eLife Sciences Publications, Ltd, 2023) Liffner, B.; Cepeda Diaz, A.K.; Blauwkamp, J.; Anaguano, D.; Frolich, S.; Muralidharan, V.; Wilson, D.W.; Dvorin, J.D.; Absalon, S.Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.Item Open Access Trends in use of medicines for opioid agonist treatment in Australia, 2013-2022(Elsevier BV, 2023) Bharat, C.; Chidwick, K.; Gisev, N.; Farrell, M.; Ali, R.; Degenhardt, L.Background: There are limited longitudinal data on national patterns of opioid agonist treatment (OAT). This study describes 10-year trends in the sales of OAT medicines in Australia. Methods: A descriptive and time-series analysis of methadone, sublingual (SL) buprenorphine (+/− naloxone), and long-acting injectable (LAI) buprenorphine sold in Australia between 2013 and 2022 was performed. Total units sold were converted into an estimate of the number of clients that could be treated over a 28-day period with that amount of medicine (‘client-months’). Results: Between January 2013 and December 2022, the estimated number of client-months on: any OAT increased by 50 % to 53,501, methadone decreased (− 8.5%), SL buprenorphine increased (+78%), and LAI buprenorphine increased substantially after September 2019. In January 2013, 78 % of OAT client-months received methadone. By December 2022, 48 % received methadone, 26 % SL buprenorphine, and 26 % LAI buprenorphine. Between 2013 to 2022, OAT client-months per capita were highest in the state of New South Wales. Over the study period, greater increases in OAT were observed in very remote areas (88%) compared to major cities (53%). The number of client-months in non-community pharmacy settings remained stable from 2013 to 2019/20, before increasing markedly. The introduction of LAI buprenorphine was associated with an immediate, sustained increase of 1,636 OAT client-months, and further increases of 190 OAT client-months each month. Conclusion: Patterns of OAT have shifted over the last 10-years with buprenorphine (SL/LAI) now the most common OAT used in Australia. The introduction of LAI buprenorphine has expanded OAT access, particularly in non-community pharmacy settings, and in remote areas.Item Metadata only Exercise and the gut microbiome: implications for supportive care in cancer(Springer Science and Business Media LLC, 2023) Hart, N.H.; Wallen, M.P.; Farley, M.J.; Haywood, D.; Boytar, A.N.; Secombe, K.; Joseph, R.; Chan, R.J.; Kenkhuis, M.F.; Buffart, L.M.; Skinner, T.L.; Wardill, H.R.Purpose: Growing recognition of the gut microbiome as an infuential modulator of cancer treatment efcacy and toxicity has led to the emergence of clinical interventions targeting the microbiome to enhance cancer and health outcomes. The highly modifable nature of microbiota to endogenous, exogenous, and environmental inputs enables interventions to promote resilience of the gut microbiome that have rapid efects on host health, or response to cancer treatment. While diet, probiotics, and faecal microbiota transplant are primary avenues of therapy focused on restoring or protecting gut function in people undergoing cancer treatment, the role of physical activity and exercise has scarcely been examined in this population. Methods: A narrative review was conducted to explore the nexus between cancer care and the gut microbiome in the context of physical activity and exercise as a widely available and clinically efective supportive care strategy used by cancer survivors. Results: Exercise can facilitate a more diverse gut microbiome and functional metabolome in humans; however, most physical activity and exercise studies have been conducted in healthy or athletic populations, primarily using aerobic exercise modalities. A scarcity of exercise and microbiome studies in cancer exists. Conclusions: Exercise remains an attractive avenue to promote microbiome health in cancer survivors. Future research should elucidate the various infuences of exercise modalities, intensities, frequencies, durations, and volumes to explore doseresponse relationships between exercise and the gut microbiome among cancer survivors, as well as multifaceted approaches (such as diet and probiotics), and examine the influences of exercise on the gut microbiome and associated symptom burden prior to, during, and following cancer treatment.Item Open Access The oxidation of steroid derivatives by the CYP125A6 and CYP125A7 enzymes from Mycobacterium marinum(Elsevier BV, 2023) Ghith, A.; Bell, S.G.The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.Item Open Access Proteoliposomes reconstituted with human aquaporin-1 reveal novel single ion channel properties(Elsevier BV, 2023) Henderson, S.W.; Nakayama, Y.; Whitelaw, M.L.; Bruning, J.B.; Anderson, P.A.; Tyerman, S.D.; Ramesh, S.A.; Martinac, B.; Yool, A.J.Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.Item Open Access Recurrent SPECC1L–NTRK fusions in pediatric sarcoma and brain tumors(Cold Spring Harbor Laboratory, 2020) Khuong-Quang, D.-A.; Brown, L.M.; Wong, M.; Mayoh, C.; Sexton-Oates, A.; Kumar, A.; Pinese, M.; Nagabushan, S.; Lau, L.; Ludlow, L.E.; Gifford, A.J.; Rodriguez, M.; Desai, J.; Fox, S.B.; Haber, M.; Ziegler, D.S.; Hansford, J.R.; Marshall, G.M.; Cowley, M.J.; Ekert, P.G.The identification of rearrangements driving expression of neurotrophic receptor tyrosine kinase (NTRK) family kinases in tumors has become critically important because of the availability of effective, specific inhibitor drugs. Whole-genome sequencing (WGS) combined with RNA sequencing (RNA-seq) can identify novel and recurrent expressed fusions. Here we describe three SPECC1L–NTRK fusions identified in two pediatric central nervous system cancers and an extracranial solid tumor using WGS and RNA-seq. These fusions arose either through a simple balanced rearrangement or in the context of a complex chromoplexy event. We cloned the SPECC1L–NTRK2 fusion directly from a patient sample and showed that enforced expression of this fusion is sufficient to promote cytokine-independent survival and proliferation. Cells transformed by SPECC1L–NTRK2 expression are sensitive to a TRK inhibitor drug. We report here that SPECC1L–NTRK fusions can arise in a range of pediatric cancers. Although WGS and RNA-seq are not required to detect NTRK fusions, these techniques may be of benefit when NTRK fusions are not suspected on clinical grounds or not identified by other methods.Item Open Access Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines(MDPI AG, 2023) Kutyna, M.M.; Loone, S.; Saunders, V.A.; White, D.L.; Kok, C.H.; Hiwase, D.K.Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.Item Open Access Sorting Out the Role of the Sortilin-Related Receptor 1 in Alzheimer's Disease(IOS Press, 2020) Barthelson, K.; Newman, M.; Lardelli, M.Sortilin-related receptor 1 (SORL1) encodes a large, multi-domain containing, membrane-bound receptor involved in endosomal sorting of proteins between the trans-Golgi network, endosomes and the plasma membrane. It is genetically associated with Alzheimer's disease (AD), the most common form of dementia. SORL1 is a unique gene in AD, as it appears to show strong associations with the common, late-onset, sporadic form of AD and the rare, early-onset familial form of AD. Here, we review the genetics of SORL1 in AD and discuss potential roles it could play in AD pathogenesis.Item Open Access The hospital admission profile of people presenting to specialist addiction services with problematic use of alcohol or opioids: A national retrospective cohort study in England(Elsevier, 2021) Roberts, E.; Hotopf, M.; Strang, J.; Marsden, J.; White, M.; Eastwood, B.; Drummond, C.Background: Over the past decade in England the rate of alcohol and opioid-related hospitalisation has increased alongside a simultaneous reduction in people accessing specialist addiction treatment. We aimed to determine the hospitalisation patterns of people presenting to addiction treatment with problematic use of alcohol or opioids, and estimate how individual sociodemographic characteristics and hospital admission diagnoses are associated with the rate of hospitalisation, death and successful completion of addiction treatment. Methods: A national record linkage between Hospital Episode Statistics (HES) and the National Drug Treatment Monitoring System (NDTMS) captured lifetime hospital admission profiles of people presenting to addiction services in England in 2018/19. Latent class analysis assigned individuals to clusters based on the ICD-10 diagnosis coded as primary reason for admission. Negative binomial, and multilevel logistic regression models determined if outcomes differed due to sociodemographic characteristics or assigned diagnostic clusters. Findings: Inpatient data were available for 64,840 alcohol patients, and 107,296 opioid patients. The most common reasons for admission were alcohol withdrawal (n = 20,024 (5.3% of alcohol-cohort admissions)), and unspecified illness (n = 11,387 (2.1% of opioid-cohort admissions)). Seven diagnostic clusters were identified for each substance cohort. People with admissions predominantly relating to mental and behavioural disorders, and injuries or poisonings had significantly higher hospitalisation rates (adjusted IRR 7.06 (95%CI 6.72 7.42);p < 0.001), higher odds of death during addiction treatment (adjusted OR 2.71 (95%CI 2.29 3.20);p < 0.001) and lower odds of successful treatment completion (adjusted OR 0.72 (95%CI 0.68 0.76);p < 0.001). Interpretation: This is the first study to interrogate national hospitalisation patterns within people presenting to addiction services with problematic use of alcohol or opioids. Having identified high-risk, high-cost individuals with increased hospital usage, and increased odds of death, future work should focus on targeting appropriate interventions, to improve their health outcomes and prevent unnecessary hospital readmission.Item Open Access "Chemobrain" in childhood cancer survivors - the impact on social, academic, and daily living skills: a qualitative systematic review(Springer, 2023) Semendric, I.; Pollock, D.; Haller, O.; George, R.; Collins-Praino, L.; Whittaker, A.Purpose: To examine children’s experiences of chemotherapy-induced cognitive impairment––colloquially “chemobrain”–– and the impact on children’s social, academic, and daily living skills via a qualitative systematic review. Experiencing chemotherapy as a child, when the brain is still developing, may cause lifelong detriment to survivors’ lives. There is a significant gap in understanding their lived experience, including the self-identified barriers that children face following treatment. Such a gap can only be fully bridged by listening to the child’s own voice and/or parent proxy report through an exploration of the qualitative research literature. Methods: A search of MEDLINE, Embase, PsycINFO, and CINAHL databases was conducted. Inclusion criteria were qualitative studies with a focus on children (0–18 years) during and/or following chemotherapy treatment and explored children’s experiences of chemobrain. Results: Two synthesized findings were identified from six studies. (1) Chemobrain has an academic and psychosocial impact, which may not be understood by education providers. (2) Children and their parents have concerns about their reintegration and adaptation to school, social lives, and their future selves as independent members of society. Children’s experiences primarily related to changes in their academic and social functioning. Conclusion: This review highlights two important considerations: (1) the lived experiences of pediatric childhood cancer survivors guiding where future interventions should be targeted, and (2) a need to perform more qualitative research studies in this area, as well as to improve the quality of reporting among the existing literature, given that this is a current gap in the field.Item Metadata only Prediction of Multiple Types of RNA Modifications via Biological Language Model(Institute of Electrical and Electronics Engineers (IEEE), 2023) Zhang, Y.; Ge, F.; Li, F.; Yang, X.; Song, J.; Yu, D.-J.It has been demonstrated that RNA modifications play essential roles in multiple biological processes. Accurate identification of RNA modifications in the transcriptome is critical for providing insights into the biological functions and mechanisms. Many tools have been developed for predicting RNA modifications at single-base resolution, which employ conventional feature engineering methods that focus on feature design and feature selection processes that require extensive biological expertise and may introduce redundant information. With the rapid development of artificial intelligence technologies, end-to-end methods are favorably received by researchers. Nevertheless, each well-trained model is only suitable for a specific RNA methylation modification type for nearly all of these approaches. In this study, we present MRM-BERT by feeding task-specific sequences into the powerful BERT (Bidirectional Encoder Representations from Transformers) model and implementing fine-tuning, which exhibits competitive performance to the state-of-the-art methods. MRM-BERT avoids repeated de novo training of the model and can predict multipleRNAmodifications such as pseudouridine, m6A, m5C, and m1A in Mus musculus, Arabidopsis thaliana, and Saccharomyces cerevisiae. In addition, we analyse the attention heads to provide high attention regions for the prediction, and conduct saturated in silico mutagenesis of the input sequences to discover potential changes of RNA modifications, which can better assist researchers in their follow-up research.Item Open Access Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection(Springer Nature, 2023) Ullah, T.R.; Johansen, M.D.; Balka, K.R.; Ambrose, R.L.; Gearing, L.J.; Roest, J.; Vivian, J.P.; Sapkota, S.; Jayasekara, W.S.N.; Wenholz, D.S.; Aldilla, V.R.; Zeng, J.; Miemczyk, S.; Nguyen, D.H.; Hansbro, N.G.; Venkatraman, R.; Kang, J.H.; Pang, E.S.; Thomas, B.J.; Alharbi, A.S.; et al.TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.Item Open Access Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo(Nature Research (part of Springer Nature), 2023) Hawdon, A.; Geoghegan, N.D.; Mohenska, M.; Elsenhans, A.; Ferguson, C.; Polo, J.M.; Parton, R.G.; Zenker, J.The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.Item Open Access Comparative localization of colorectal sensory afferent central projections in the mouse spinal cord dorsal horn and caudal medulla dorsal vagal complex(Wiley, 2024) Wang, Q.Q.; Caraballo, S.G.; Rychkov, G.; McGovern, A.E.; Mazzone, S.B.; Brierley, S.M.; Harrington, A.M.The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)-evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)-projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD-activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN-projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD-activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD-activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses.Item Open Access O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss(Public Library of Science (PLoS), 2023) Qin, J.; Hong, Y.; Morona, R.; Totsika, M.; Hughes, D.Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.Item Metadata only Influence of Exercise Training on the Left Atrium : Implications for Atrial Fibrillation, Heart Failure and Stroke(American Physiological Society, 2023) Elliott, A.; Ariyaratnam, J.; Howden, E.J.; La Gerche, A.; Sanders, P.The left atrium (LA) plays a critical role in receiving pulmonary venous return and modulating left ventricular (LV) filling. With the onset of exercise, LA function contributes to the augmentation in stroke volume. Due to the growing focus on atrial imaging, there is now evidence that structural remodelling and dysfunction of the LA is associated with adverse outcomes including incident cardiovascular disease. In patients with established disease, pathological changes in atrial structure and function are associated with exercise intolerance, increased hospital admissions and mortality, independent of left ventricular function. Exercise training is widely recommended in patients with cardiovascular disease to improve patient outcomes and maintain functional capacity. There are widely documented changes in LV function with exercise, yet less attention has been given to the LA. In this review, we first describe LA physiology at rest and during exercise, before exploring its association with cardiac disease outcomes including atrial fibrillation, heart failure and stroke. The adaptation of the LA to short- and longer-term exercise training is evaluated through review of longitudinal studies of exercise training in healthy participants free of cardiovascular disease and athletes. We then consider the changes in LA structure and function amongst patients with established disease, where adverse atrial remodelling may be implicated in the disease process. Finally, we consider important future directions for assessment of atrial structure and function using novel imaging modalities, in response to acute and chronic exercise.Item Metadata only Critical assessment of computational tools for prokaryotic and eukaryotic promoter prediction(Oxford University Press (OUP), 2022) Zhang, M.; Jia, C.; Li, F.; Li, C.; Zhu, Y.; Akutsu, T.; Webb, G.I.; Zou, Q.; Coin, L.J.M.; Song, J.Promoters are crucial regulatory DNA regions for gene transcriptional activation. Rapid advances in next-generation sequencing technologies have accelerated the accumulation of genome sequences, providing increased training data to inform computational approaches for both prokaryotic and eukaryotic promoter prediction. However, it remains a significant challenge to accurately identify species-specific promoter sequences using computational approaches. To advance computational support for promoter prediction, in this study, we curated 58 comprehensive, up-to-date, benchmark datasets for 7 different species (i.e. Escherichia coli, Bacillus subtilis, Homo sapiens, Mus musculus, Arabidopsis thaliana, Zea mays and Drosophila melanogaster) to assist the research community to assess the relative functionality of alternative approaches and support future research on both prokaryotic and eukaryotic promoters. We revisited 106 predictors published since 2000 for promoter identification (40 for prokaryotic promoter, 61 for eukaryotic promoter, and 5 for both). We systematically evaluated their training datasets, computational methodologies, calculated features, performance and software usability. On the basis of these benchmark datasets, we benchmarked 19 predictors with functioning webservers/local tools and assessed their prediction performance. We found that deep learning and traditional machine learning-based approaches generally outperformed scoring function-based approaches. Taken together, the curated benchmark dataset repository and the benchmarking analysis in this study serve to inform the design and implementation of computational approaches for promoter prediction and facilitate more rigorous comparison of new techniques in the future.Item Metadata only Assessing the performance of computational predictors for estimating protein stability changes upon missense mutations(Oxford University Press (OUP), 2021) Iqbal, S.; Li, F.; Akutsu, T.; Ascher, D.B.; Webb, G.I.; Song, J.Understanding how a mutation might affect protein stability is of significant importance to protein engineering and for understanding protein evolution genetic diseases. While a number of computational tools have been developed to predict the effect of missense mutations on protein stability protein stability upon mutations, they are known to exhibit large biases imparted in part by the data used to train and evaluate them. Here, we provide a comprehensive overview of predictive tools, which has provided an evolving insight into the importance and relevance of features that can discern the effects of mutations on protein stability. A diverse selection of these freely available tools was benchmarked using a large mutation-level blind dataset of 1342 experimentally characterised mutations across 130 proteins from ThermoMutDB, a second test dataset encompassing 630 experimentally characterised mutations across 39 proteins from iStable2.0 and a third blind test dataset consisting of 268 mutations in 27 proteins from the newly published ProThermDB. The performance of the methods was further evaluated with respect to the site of mutation, type of mutant residue and by ranging the pH and temperature. Additionally, the classification performance was also evaluated by classifying the mutations as stabilizing (∆∆G ≥ 0) or destabilizing (∆∆G < 0). The results reveal that the performance of the predictors is affected by the site of mutation and the type of mutant residue. Further, the results show very low performance for pH values 6-8 and temperature higher than 65 for all predictors except iStable2.0 on the S630 dataset. To illustrate how stability and structure change upon single point mutation, we considered four stabilizing, two destabilizing and two stabilizing mutations from two proteins, namely the toxin protein and bovine liver cytochrome. Overall, the results on S268, S630 and S1342 datasets show that the performance of the integrated predictors is better than the mechanistic or individual machine learning predictors. We expect that this paper will provide useful guidance for the design and development of next-generation bioinformatic tools for predicting protein stability changes upon mutations.