Biochemistry and Pharmacology - Research Publications
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ItemPathobiological mechanisms underlying metabolic syndrome (MetS) in chronic obstructive pulmonary disease (COPD): clinical significance and therapeutic strategies(PERGAMON-ELSEVIER SCIENCE LTD, 2019-06-01)Chronic obstructive pulmonary disease (COPD) is a major incurable global health burden and is currently the 4th largest cause of death in the world. Importantly, much of the disease burden and health care utilisation in COPD is associated with the management of its comorbidities (e.g. skeletal muscle wasting, ischemic heart disease, cognitive dysfunction) and infective viral and bacterial acute exacerbations (AECOPD). Current pharmacological treatments for COPD are relatively ineffective and the development of effective therapies has been severely hampered by the lack of understanding of the mechanisms and mediators underlying COPD. Since comorbidities have a tremendous impact on the prognosis and severity of COPD, the 2015 American Thoracic Society/European Respiratory Society (ATS/ERS) Research Statement on COPD urgently called for studies to elucidate the pathobiological mechanisms linking COPD to its comorbidities. It is now emerging that up to 50% of COPD patients have metabolic syndrome (MetS) as a comorbidity. It is currently not clear whether metabolic syndrome is an independent co-existing condition or a direct consequence of the progressive lung pathology in COPD patients. As MetS has important clinical implications on COPD outcomes, identification of disease mechanisms linking COPD to MetS is the key to effective therapy. In this comprehensive review, we discuss the potential mechanisms linking MetS to COPD and hence plausible therapeutic strategies to treat this debilitating comorbidity of COPD.
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ItemTadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2018-05-11)Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.
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ItemNo Preview AvailableThe Asian Biophysics Association-supporting biophysics in the greater Asia region.(Springer Science and Business Media LLC, 2019-06)
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ItemRibosomal Protein S3 Gene Silencing Protects Against Cigarette Smoke-Induced Acute Lung Injury(CELL PRESS, 2018-09-07)Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.
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ItemNrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence(PUBLIC LIBRARY SCIENCE, 2016-06-08)Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion.
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ItemDimensionality reduction of diffusion MRI measures for improved tractometry of the human brain.(Elsevier BV, 2019-10-15)Various diffusion MRI (dMRI) measures have been proposed for characterising tissue microstructure over the last 15 years. Despite the growing number of experiments using different dMRI measures in assessments of white matter, there has been limited work on: 1) examining their covariance along specific pathways; and on 2) combining these different measures to study tissue microstructure. Indeed, it quickly becomes intractable for existing analysis pipelines to process multiple measurements at each voxel and at each vertex forming a streamline, highlighting the need for new ways to visualise or analyse such high-dimensional data. In a sample of 36 typically developing children aged 8-18 years, we profiled various commonly used dMRI measures across 22 brain pathways. Using a data-reduction approach, we identified two biologically-interpretable components that capture 80% of the variance in these dMRI measures. The first derived component captures properties related to hindrance and restriction in tissue microstructure, while the second component reflects characteristics related to tissue complexity and orientational dispersion. We then demonstrate that the components generated by this approach preserve the biological relevance of the original measurements by showing age-related effects across developmentally sensitive pathways. In summary, our findings demonstrate that dMRI analyses can benefit from dimensionality reduction techniques, to help disentangling the neurobiological underpinnings of white matter organisation.
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ItemLosartan does not inhibit cigarette smoke-induced lung inflammation in mice(NATURE PUBLISHING GROUP, 2019-10-21)Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease largely caused by cigarette smoking (CS) and is characterized by lung inflammation and airflow limitation that is not fully reversible. Approximately 50% of people with COPD die of a cardiovascular comorbidity and current pharmacological strategies provide little benefit. Therefore, drugs that target the lung and the cardiovascular system concurrently may be an advantageous therapeutic strategy. The aim of this study was to see whether losartan, an angiotensin-II AT1a receptor antagonist widely used to treat hypertension associated with cardiovascular disease, protects against CS-induced lung inflammation in mice. Male BALB/c mice were exposed to CS for 8 weeks and treated with either losartan (30 mg/kg) or vehicle daily. Mice were euthanized and bronchoalveolar lavage fluid (BALF) inflammation, and whole lung cytokine, chemokine and protease mRNA expression assessed. CS caused significant increases in BALF total cells, macrophages, neutrophils and whole lung IL-6, TNF-α, CXCL-1, IL-17A and MMP12 mRNA expression compared to sham-exposed mice. However, losartan only reduced CS-induced increases in IL-6 mRNA expression. Angiotensin-II receptor expression was reduced in lung tissue from CS-exposed mice. In conclusion, losartan did not inhibit CS-induced BALF cellularity despite reducing whole lung IL-6 mRNA and Ang-II receptor expression.
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ItemCigarette smoke extract exacerbates hyperpermeability of cerebral endothelial cells after oxygen glucose deprivation and reoxygenation(NATURE RESEARCH, 2019-10-30)Cigarette smoking is a risk factor for stroke and is linked to stroke severity. Previous studies have shown that cigarette smoke extract (CSE) triggers endothelial dysfunction in vitro by initiating oxidative stress and/or an inflammatory response. In addition, cerebral endothelial dysfunction (particularly at the level of the blood-brain barrier [BBB]) contributes to stroke pathogenesis. Therefore, we hypothesized that cigarette smoking may influence stroke, at least in part, by exacerbating ischaemia-induced BBB disruption. To test this, we examined the effect of CSE on the permeability of cerebral endothelial cells exposed to oxygen glucose deprivation and reoxygenation (OGD + RO). We found that the loss of BBB integrity following ischaemic/reperfusion-like conditions was significantly worsened by CSE. Despite this being associated with increased mRNA expression of Nox catalytic subunits, reactive oxygen species (ROS) levels were however markedly lower. Furthermore, this occurred in association with elevated expression of antioxidant enzymes (SOD1, SOD2, and Gpx-1), suggesting an antioxidant defence response. Lastly, we found that CSE significantly upregulated mRNA expression of cytokines (IL-6 and TGF-β). Collectively, these results show that acute exposure to CSE worsens BBB disruption caused by OGD + RO, however, this is not linked to elevated ROS levels but may involve inflammatory mechanisms.
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ItemNovel endosomal NOX2 oxidase inhibitor ameliorates pandemic influenza A virus-induced lung inflammation in mice(WILEY, 2019-10-01)BACKGROUND AND OBJECTIVE: Influenza A viruses (IAV) cause respiratory tract infections that can be fatal when the virus spreads to the alveolar space (i.e. alveolitis), and this is mainly observed with highly pathogenic strains. Reactive oxygen species (ROS) production by the NOX2 NADPH oxidase in endosomes has been directly implicated in IAV pathology. Recently, we demonstrated that treatment with a novel endosome-targeted NOX2 oxidase inhibitor, cholestanol-conjugated gp91dsTAT (Cgp91ds-TAT), attenuated airway inflammation and viral replication to infection with a low pathogenic influenza A viral strain. Here, we determined whether suppression of endosome NOX2 oxidase prevents the lung inflammation following infection with a highly pathogenic IAV strain. METHODS: C57Bl/6 mice were intranasally treated with either DMSO vehicle (2%) or Cgp91ds-TAT (0.2 mg/kg/day) 1 day prior to infection with the high pathogenicity PR8 IAV strain (500 PFU/mouse). At Day 3 post-infection, mice were culled for the evaluation of airway and lung inflammation, viral titres and ROS generation. RESULTS: PR8 infection resulted in a marked degree of airway inflammation, epithelial denudation, alveolitis and inflammatory cell ROS production. Cgp91ds-TAT treatment significantly attenuated airway inflammation, including neutrophil influx, the degree of alveolitis and inflammatory cell ROS generation. Importantly, the anti-inflammatory phenotype affected by Cgp91ds-TAT significantly enhanced the clearance of lung viral mRNA following PR8 infection. CONCLUSION: Endosomal NOX2 oxidase promotes pathogenic lung inflammation to IAV infection. The localized delivery of endosomal NOX2 oxidase inhibitors is a novel therapeutic strategy against IAV, which has the potential to limit the pathogenesis caused during epidemics and pandemics.
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ItemComplement 3(+)-astrocytes are highly abundant in prion diseases, but their abolishment led to an accelerated disease course and early dysregulation of microglia(BMC, 2019-05-22)Astrogliosis and activation of microglia are hallmarks of prion diseases in humans and animals. Both were viewed to be rather independent events in disease pathophysiology, with proinflammatory microglia considered to be the potential neurotoxic species at late disease stages. Recent investigations have provided substantial evidence that a proinflammatory microglial cytokine cocktail containing TNF-α, IL-1α and C1qa reprograms a subset of astrocytes to change their expression profile and phenotype, thus becoming neurotoxic (designated as A1-astrocytes). Knockout or antibody blockage of the three cytokines abolish formation of A1-astrocytes, therefore, this pathway is of high therapeutic interest in neurodegenerative diseases. Since astrocyte polarization profiles have never been investigated in prion diseases, we performed several analyses and could show that C3+-PrPSc-reactive-astrocytes, which may represent a subtype of A1-astrocytes, are highly abundant in prion disease mouse models and human prion diseases. To investigate their impact on prion disease pathophysiology and to evaluate their potential therapeutic targeting, we infected TNF-α, IL-1α, and C1qa Triple-KO mice (TKO-mice), which do not transit astrocytes into A1, with prions. Although formation of C3+-astrocytes was significantly reduced in prion infected Triple-KO-mice, this did not affect the amount of PrPSc deposition or titers of infectious prions. Detailed characterization of the astrocyte activation signature in thalamus tissue showed that astrocytes in prion diseases are highly activated, showing a mixed phenotype that is distinct from other neurodegenerative diseases and were therefore termed C3+-PrPSc-reactive-astrocytes. Unexpectedly, Triple-KO led to a significant acceleration of prion disease course. While pan-astrocyte and -microglia marker upregulation was unchanged compared to WT-brains, microglial homeostatic markers were lost early in disease in TKO-mice, pointing towards important functions of different glia cell types in prion diseases.