Characterizing Cellular Responses During Oncolytic Maraba Virus Infection.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Role of Tribbles Pseudokinase 1 (TRIB1) in human hepatocyte metabolism.

Genome-wide association studies for plasma triglycerides and hepatic steatosis identified a risk locus on chromosome 8q24 close to the TRIB1 gene, encoding Tribbles Pseudokinase 1 (TRIB1). In previous studies conducted in murine models, hepatic over-expression of Trib1 was shown to increase fatty acid oxidation and decrease triglyceride synthesis whereas Trib1 knockdown mice exhibited hypertriglyceridemia. Here we have examined the impact of TRIB1 suppression in human and mouse hepatocytes. Examination of a panel of lipid regulator transcripts revealed species-specific effects, prompting us to focus on human models for the remainder of the study. Acute knockdown of TRIB1 in human primary hepatocytes resulted in decreased expression of MTTP and APOB, required for very low density lipoprotein (VLDL) assembly although particle secretion was not significantly affected. A parallel analysis performed in HepG2 revealed reduced MTTP, but not APOB, protein as a result of TRIB1 suppression. Global gene expression changes of human primary hepatocytes upon TRIB1 suppression were analyzed by clustering algorithms and found to be consistent with dysregulation of several pathways fundamental to liver function, including altered CEBPA and B transcript levels and impaired glucose handling. Indeed, TRIB1 expression in HepG2 cells was found to be inversely proportional to glucose concentration. Lastly TRIB1 downregulation in primary hepatocytes was associated with suppression of the HNF4A axis. In HepG2 cells, TRIB1 suppression resulted in reduced HNF4A protein levels while HNF4A suppression increased TRIB1 expression. Taken together these studies reveal an important role for TRIB1 in human hepatocyte biology.

ERK1/2 regulates hepatocyte Trib1 in response to mitochondrial dysfunction.

The TRIB1 locus (8q24.13) is a novel locus identified and replicated by several genome-wide association studies for associations with plasma triglycerides, apolipoprotein B and coronary artery disease. The TRIB1 protein product, tribbles-like protein 1 (Trib1), regulates MAPK activity. MAP kinases transduce a large variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation and apoptosis. Importantly, Trib1 has been shown to regulate hepatic lipogenesis and very low density lipoprotein production. Despite the relevance of hepatocyte Trib1 to lipid metabolism and atherosclerosis, little is known about the mechanisms regulating Trib1 itself. Here, we identify the mitochondria axis as a regulator of Trib1. Treatment of HepG2 cells with a short pulse of a low oligomycin concentration led to a potent and prolonged increase in the Trib1 mRNA, an effect that was shared with other mitochondria stressors. HuH7 cells as well murine hepatocytes were also responsive albeit to a weaker extent. The upregulation appeared largely independent of reactive oxygen species generation or metabolic stress and was mainly under transcriptional control, with ERK1/2 playing an important regulating role in the process. While the presence of the Trib1 protein could be inferred, attempts to correlate the increased mRNA to changes in protein level were unsuccessful due to the lack of recognizable Trib1 signal. Our data enrich the current paradigm of Trib1 as an activator of the MAPK pathway by uncovering a role for MAPK in regulating Trib1.

Variability in Grading Diabetic Retinopathy Using Retinal Photography and Its Comparison with an Automated Deep Learning Diabetic Retinopathy Screening Software.

BACKGROUND: Diabetic retinopathy (DR) screening using colour retinal photographs is cost-effective and time-efficient. In real-world clinical settings, DR severity is frequently graded by individuals of different expertise levels. We aim to determine the agreement in DR severity grading between human graders of varying expertise and an automated deep learning DR screening software (ADLS). METHODS: Using the International Clinical DR Disease Severity Scale, two hundred macula-centred fundus photographs were graded by retinal specialists, ophthalmology residents, family medicine physicians, medical students, and the ADLS. Based on referral urgency, referral grading was divided into no referral, non-urgent referral, and urgent referral to an ophthalmologist. Inter-observer and intra-group variations were analysed using Gwet's agreement coefficient, and the performance of ADLS was evaluated using sensitivity and specificity. RESULTS: The agreement coefficient for inter-observer and intra-group variability ranged from fair to very good, and moderate to good, respectively. The ADLS showed a high area under curve of 0.879, 0.714, and 0.836 for non-referable DR, non-urgent referable DR, and urgent referable DR, respectively, with varying sensitivity and specificity values. CONCLUSION: Inter-observer and intra-group agreements among human graders vary widely, but ADLS is a reliable and reasonably sensitive tool for mass screening to detect referable DR and urgent referable DR.

ACUTE SYPHILITIC POSTERIOR PLACOID CHORIORETINITIS: MULTIMODAL IMAGING AND ELECTROPHYSIOLOGIC FINDINGS BEFORE AND AFTER TREATMENT.

PURPOSE: To describe the clinical course of acute syphilitic posterior placoid chorioretinitis (ASPPC) in the preplacoid stage, placoid stage, and after treatment with penicillin. METHOD: A retrospective case report of serial multimodal imaging and electrophysiology studies of a patient with ASPPC, with 18 months of follow-up. RESULTS: A 47-year-old man presented with bilateral panuveitis. The patient defaulted follow-up and returned when his vision deteriorated. Tests for neurosyphilis and retroviral disease were positive, and treatment was initiated. The earliest change on serial optical coherence tomography was loss of the signal from the reflective band corresponding to the ellipsoid zone. In the placoid stage, there was nodular thickening of the retinal pigment epithelium. The ellipsoid zone signals reappeared after treatment. Fundus fluorescein angiogram at presentation showed peripapillary vasculitis and disk leakage; indocyanine green angiography revealed multiple hypofluorescent spots in the peripapillary region and posterior pole that was not visible clinically. The angiographic abnormalities resolved after treatment. Electrophysiology demonstrated bilateral maculopathy and reduction of both a- and b-waves from dark-adapted and light-adapted responses at presentation. The b-waves (inner retina) recovered partially with treatment. CONCLUSION: To the best of our knowledge, this is the first case report of the multimodal imaging and electrophysiology findings in a patient with acute syphilitic posterior placoid chorioretinitis, before the development of the classic placoid lesion. Improvement of structural and functional pathology after systemic treatment is demonstrated.

Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus.

OBJECTIVE: We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression. METHODS AND RESULTS: We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele. CONCLUSIONS: These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways.

Systemic Factors Associated with Treatment Response in Diabetic Macular Edema.

PURPOSE: To identify systemic factors that may influence the response to anti-VEGF therapy in patients with diabetic macular edema (DME). METHODS: 35 patients undergoing anti-VEGF injections for centre-involving DME were studied in this prospective observational study. The primary outcome was change in macular thickness one month after treatment, measured using spectral-domain optical coherence tomography (OCT). At baseline, information on various systemic factors was collected including glycosylated hemoglobin (HbA1c), serum VEGF levels, lipid profile and markers of renal function, and blood pressure. Thirty-three of the 35 patients were included in this study. Nonparametric statistical tests were used for the analysis of the data in view of the nonnormal distribution of the outcome variables. Multivariate analysis was performed using logistic regression. Stata 12.1 software was used for the analysis. Main Outcome Measures. Reduction in macular central subfield thickness (on spectral-domain OCT) and change in logMAR visual acuity at one month after injection. RESULTS: Lower HbA1c levels (7% or less) were significantly associated with greater reduction in central macular subfield thickness at one month after injection of bevacizumab or ranibizumab on both univariate analysis (p=0.012) and multivariate analysis (p=0.012) and multivariate analysis (. CONCLUSIONS: Better glycemic control is associated with a greater reduction in central macular thickness after the first injection of bevacizumab or ranibizumab in diabetic macular edema. Patients with high levels of HbA1c and poor response to anti-VEGF may benefit from strict control of their blood glucose.

CHI study: protocol for an observational cohort study on ageing and mental health in community-dwelling older adults.

INTRODUCTION: Ageing is associated with a multitude of healthcare issues including dementia, depression, frailty, morbidity associated with chronic disease and high healthcare utilisation. With Singapore's population projected to age significantly over the next two decades, it has become increasingly important to understand the disease burden and etiological process among older adults. The Community Health and Intergenerational study aims to holistically examine ageing in place by investigating the resilience and vulnerability factors of the ageing process in the biological, psychological and social domains within the environment. METHODS AND ANALYSIS: Using a cohort multiple randomised controlled trial design, comprehensive health profiles of community-dwelling older adults will be collected. The objective is to recruit 1000 participants (aged 60-99 years) living in the western region of Singapore within a period of 3 years (2018-2020). Assessments include basic sociodemographic, physical health and function (cardiac, oral and blood profiles and visual function), cognitive functioning, daily functioning, physical fitness, emotional state, free-flowing speech, sleep quality, social connectedness, caregiver burden, intergenerational communication, quality of life, life satisfaction, attitudes to ageing and gratitude and compassion. Results from the cohort will enable future studies to identify at-risk groups and develop interventions to improve the physical and mental health and quality of life of older adults. ETHICS AND DISSEMINATION: Approval of the cohort study by the National University of Singapore Institutional Review Board (NUS-IRB Reference code: H-17-047) was obtained on 12 October 2017. Written consent will be obtained from all participants. Findings from the cohort study will be disseminated by publication of peer-reviewed manuscripts, presentations at scientific meetings and conferences with local stakeholders.

Unusual ocular manifestations following viper bite.

We report a case of a 70-year-old farmer admitted for viper bite who presented with bilateral hyphema and angle closure attack. He was managed conservatively with topical steroids and cycloplegics. He responded well and was discharged after 2 weeks.

Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast.

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker's yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation.

Oncolytic virus synergizes with Smac mimetic compounds to induce rhabdomyosarcoma cell death in a syngeneic murine model.

Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo. Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A.

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1-2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner.

Elevated levels of ribosomal proteins eL36 and eL42 control expression of Hsp90 in rhabdomyosarcoma.

Mammalian 90 kDa heat shock protein (Hsp90) is a ubiquitous molecular chaperone whose expression is selectively upregulated during stress, although the precise control mechanism of this increase is yet to be fully elucidated. We used polysome profiling to show that Hsp90α mRNA is selectively translated, while global translation is inhibited during heat stress. Furthermore, we have identified 2 ribosomal proteins, eL36 and eL42 that modulate Hsp90α expression under both normal and heat shock conditions. Importantly, we noted that expression of eL36 and eL42 is elevated in a panel of human rhabdomyosarcomas where it drives high expression of Hsp90 and modulates sensitivity of these cells to an Hsp90 inhibitor 17-AAG.

ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism.

Programmed cell death 4 (PDCD4) is a tumour suppressor implicated in cancer development and progression and was recently identified as a repressor of cap-independent translation of specific genes involved in the regulation of apoptosis. We show that the RNA-binding protein HuR binds to the PDCD4 3'UTR to protect it from miR-21-induced silencing. However, following H2O2 treatment, PDCD4 mRNA is degraded via miR-21 binding. Importantly, we identify HuR as a novel substrate of the ERK8 kinase pathway in response to H2O2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.

Functional analysis of the TRIB1 associated locus linked to plasma triglycerides and coronary artery disease.

BACKGROUND: The TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease in humans. The lipid-associated single nucleotide polymorphisms (SNPs), identified by genome-wide association studies, are located ≈30 kb downstream from TRIB1, suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits. METHODS AND RESULTS: Characterization of the risk locus reveals that it encompasses a gene, TRIB1-associated locus (TRIBAL), composed of a well-conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and resequencing identified a single SNP, rs2001844, within the promoter region that associates with increased plasma triglycerides and reduced high-density lipoprotein cholesterol and coronary artery disease risk. Further, correction for triglycerides as a covariate indicated that the genome-wide association studies association is largely dependent on triglycerides. In addition, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs, including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes. CONCLUSIONS: These studies demonstrate an interplay between a novel locus, TRIBAL, and TRIB1. TRIBAL is located in the genome-wide association studies identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression, and associates with plasma triglyceride concentrations.

A 680 kb duplication at the FTO locus in a kindred with obesity and a distinct body fat distribution.

Common intronic SNPs in the human fat mass and obesity associated (FTO) gene are strongly associated with body mass index (BMI). In mouse models, inactivation of the Fto gene results in a lean phenotype, whereas overexpression of Fto leads to increased food intake and obesity. The latter finding suggests that copy number variants at the FTO locus might be associated with extremes of adiposity. To address this question, we searched for rare, private or de novo copy number variation in a cohort of 985 obese and 869 lean subjects of European ancestry drawn from the extremes of the BMI distribution, genotyped on Affymetrix 6.0 arrays. A ∼680 kb duplication, confirmed by real-time PCR and G-to-FISH analyses, was observed between ∼rs11859825 and rs9932411 in a 68-year-old male with severe obesity. The duplicated region on chromosome 16 spans the entire genome-wide association studies risk locus for obesity, and further encompasses RBL2, AKTIP, RPGRIP1L and all but the last exon of the FTO gene. Affected family members exhibit a unique obesity phenotype, characterized by increased fat distribution in the shoulders and neck with a significantly increased neck circumference. This phenotype was accompanied by increased peripheral blood expression of RBL2 with no alteration in expression of FTO or other genes in the region. No other duplications or deletions in this region were identified in the cohort of obese and lean individuals or in a further survey of 4778 individuals, suggesting that large rare copy number variants surrounding the FTO gene are not a frequent cause of obesity.

Scavenger receptor class B type I localizes to a late endosomal compartment.

Scavenger receptor class B type I (SR-BI) has an established role in mediating the selective uptake of cholesterol from HDL in hepatocytes, steroidogenic cells, and other tissues. SR-BI is present on the plasma membrane but also localizes to stable intracellular compartments of unknown function. Using indirect immunofluorescence and subcellular fractionation, we have investigated the subcellular distribution of SR-BI. We report that red fluorescent protein-tagged mouse SR-BI (RFP-mSR-BI) colocalizes with the late endosomal and lysosomal markers, Rab7, LBPA, and Rab9. In addition, endogenous SR-BI is also found on lysosomes and colocalizes with LAMP-2 in primary hepatocytes. Furthermore, we demonstrate that the trafficking of SR-BI through these compartments is Rab7 dependent. Interestingly, filipin staining indicates accumulation of lysosomal cholesterol in SR-BI-deficient ((-/-)) as compared with wild-type hepatocytes. In addition to its role as a plasma membrane receptor, SR-BI may function in cholesterol trafficking from late endosomes/lysosomes.