Anatomy and Neuroscience - Research Publications
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ItemStructure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator(PORTLAND PRESS LTD, 2022-06-01)The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.
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ItemRepeated eccentric contractions positively regulate muscle oxidative metabolism and protein synthesis during cancer cachexia in mice(AMER PHYSIOLOGICAL SOC, 2020-06-01)Cancer-induced wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover that have been associated with systemic inflammation, whereas exercise and stimulated muscle contractions can positively regulate muscle protein synthesis and mitochondrial homeostasis. In preclinical cancer cachexia models, a single bout of eccentric contractions (ECCs) can induce protein synthesis and repeated ECC bouts prevent myofiber atrophy. The cellular mechanisms providing this protection from atrophy have not been resolved. Therefore, the purpose of this study was to determine whether repeated stimulated ECC bouts affect basal muscle oxidative metabolism and protein synthesis during cancer cachexia, and if these changes were associated with plasma IL-6 levels. Male ApcMin/+ (MIN; n = 10) mice initiating cachexia and healthy C57BL/6 (B6; n = 11) control mice performed repeated ECC bouts over 2 wk. MIN mice exhibited body weight loss and elevated plasma IL-6 before and during repeated ECC bouts. Control MIN muscle demonstrated disrupted signaling related to inflammation, oxidative capacity, and protein synthesis regulation, which were all improved by repeated ECC bouts. With cachexia, plasma IL-6 levels were negatively correlated with myofiber cross-sectional area, oxidative capacity, and protein synthesis. Interestingly, ECC improvements in these outcomes were positively correlated with plasma IL-6 levels in MIN mice. There was also a positive relationship between muscle oxidative capacity and protein synthesis after repeated ECC bouts in MIN mice. Collectively, repeated ECC bouts altered the cachectic muscle phenotype independent of systemic wasting, and there was a strong association between muscle oxidative capacity and protein synthesis in this adaptive response.NEW & NOTEWORTHY Cancer-induced muscle wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover regulation, whereas exercise is a potent stimulator of muscle protein synthesis and mitochondrial homeostasis. In a preclinical model of cancer cachexia, we report that cachectic muscle retains anabolic and metabolic plasticity to repeated eccentric contraction bouts despite an overall systemic wasting environment. The attenuation of muscle atrophy is linked to improved oxidative capacity and protein synthesis during cancer cachexia progression.
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ItemDystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice(AMER PHYSIOLOGICAL SOC, 2021-08-01)Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of patients with Duchenne muscular dystrophy (DMD) and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n = 18) and mdx mice (n = 18) were examined every 4 h beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; P < 0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; P < 0.05), fission (increased; Dnm1l; P < 0.01), fusion (decreased; Opa1, Mfn1; P < 0.05), and autophagy/mitophagy (decreased: Bnip3; P < 0.05; increased: Becn1; P < 0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (P < 0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b, and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (P < 0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, P < 0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; P < 0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.
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ItemMuscular contraction's therapeutic potential for cancer-induced wasting.(American Physiological Society, 2022-08-01)Skeletal muscle atrophy and dysfunction contribute to morbidity and mortality in patients with cancer. Cachexia pathophysiology is highly complex, given that perturbations to the systemic cancer environment and the interaction with diverse tissues can contribute to wasting processes. Systemic interleukin 6 (IL-6) and glycoprotein 130 (gp130) receptors signaling have established roles in some types of cancer-induced muscle wasting through disruptions to protein turnover and oxidative capacity. Although exercise has documented benefits for cancer prevention and patient survival, there are significant gaps in our understanding of muscle adaptation and plasticity during severe cachexia. Preclinical models have provided valuable insight into the adaptive potential of muscle contraction within the cancer environment. We summarize the current understanding of how resistance-type exercise impacts mechanisms involved in cancer-induced muscle atrophy and dysfunction. Specifically, the role of IL-6 and gp130 receptors in the pathophysiology of muscle wasting and the adaptive response to exercise is explained. The discussion includes current knowledge gaps and future research directions needed to improve preclinical research and accelerate clinical translation in human patients with cancer.
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ItemLong-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection( 2021-12-15)Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2 , which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
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ItemSurgical applied anatomy: alive and kicking.(Wiley, 2021-05)BACKGROUND: Contention exists amongst anatomists, clinicians and surgeons about how much anatomical knowledge medical students need, although what is taught should be aligned with current surgical practice. The aim of this study was to explore the scope of recent advances in applied anatomy as highlighted in the ANZ Journal of Surgery in each of the surgical specialties. METHODS: The 2018 volume of the ANZ Journal of Surgery was narrowed to 254 articles by applying the search term 'anatomy'. The main topic was extracted from each paper. The content of the paper was assessed for 'novel description' or 'novel application' of anatomical knowledge and classified accordingly. RESULTS: Most papers with an anatomical focus were from general surgery, which focused on surgical techniques, outcomes and management. Vascular surgery had the highest percentage of papers with a novel description and application of anatomy. Although cardiothoracic and paediatric surgery had no papers with a novel description of anatomy, novel applications of anatomy were a focus in each speciality. CONCLUSION: The trend towards novel applications of anatomical knowledge in all surgical specialties should encourage medical schools to shape their anatomy curricula in tandem with such advances as they evolve. The high proportion of novel applications and descriptions of anatomy in general surgery indicates continued growth as a benchmark of anatomical understanding. Vascular surgery's proportion of novel application and description of anatomy may change the way students will learn vascular anatomy to incorporate endovascular, radiologically based approaches.
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ItemNo Preview AvailableUsing long-read RNA sequencing to decipher the role of RNA isoforms in disease(Elsevier BV, 2022-03)Accurate quantification of genes and their mRNA products is essential to understanding health and disease. In humans, processes such as alternative splicing cause almost all genes to express multiple mRNA products (isoforms), which can have different functions. In addition, aberrant splicing is a common cause of disease. Standard short-read RNA sequencing (RNA-seq) methodologies have limitations in identifying isoforms. In contrast, long-read RNA-seq can address this challenge by covering the entire mRNA sequence in a single read and so identify and quantify the isoforms present. We have utilised long-read RNA-seq to characterise isoforms involved in disease risk, genetic disease and viral infection. Investigation of >30 neuropsychiatric disease risk genes in human brain identified hundreds of novel isoforms, including many genes where most expression was from previously undiscovered isoforms. Exemplifying this, the calcium channel CACNA1C expressed >200 novel isoforms with abundant splice variants modifying channel regions regulating activation voltage and channel conductance. The accurate characterisation of RNA isoforms enabled by long-read RNA-seq enables the translation of genomic findings into a pathophysiological understanding of disease.
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ItemNanoSplicer: accurate identification of splice junctions using Oxford Nanopore sequencing(OXFORD UNIV PRESS, 2022-05-27)MOTIVATION: Long read sequencing methods have considerable advantages for characterising RNA isoforms. Oxford nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilising matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages. RESULTS: We developed "NanoSplicer" to identify splice junctions using raw nanopore signal (squiggles). For each splice junction the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using 1. synthetic mRNAs with known splice junctions 2. biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated. AVAILABILITY AND IMPLEMENTATION: NanoSplicer is freely available at https://github.com/shimlab/NanoSplicer and has been deposited in archived format at https://doi.org/10.5281/zenodo.6403849. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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ItemNo Preview AvailableImpact of COVID-19 Pandemic on Cardiovascular Testing in Asia(Elsevier BV, 2021-09)
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ItemNo Preview AvailableMicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback(WILEY, 2021-04-01)Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription-driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA-mRNA reaction circuits that differ from known feedback loop-like structures. Strikingly, we show that a wide range of biologically plausible post-transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA-mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning.