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Although acute organophosphate (OP) pesticide poisoning is associated with the risk cardiovascular diseases (CVD), the association between chronic occupational OP pesticide exposure and CVD risk is limited. We investigated serum high-sensitivity C-reactive protein (hs-CRP) levels and QTc intervals in 45 agricultural workers who were chronically exposed to OP pesticides and 45 non-exposed subjects. Serum hs-CRP level was determined by enzyme-linked immunosorbent assay (ELISA) and a level >1.0 mg/l was regarded as CVD risk. The QT interval was measured by routine 12-lead Electrocardiogram (ECG) with Lead II rhythm, and QTc was calculated. The median value of the serum hs-CRP level [1.20 (0.67-2.38) vs 0.74 (0.41-1.17) mg/l, P

IntroductionAgeing 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 analysisUsing 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 disseminationApproval 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.

// Christine C. Dobson 1, 2 , Thet Naing 1 , Shawn T. Beug 1 , Mame D. Faye 1 , Janelle Chabot 1 , Martin St-Jean 1 , Danielle E. Walker 1 , Eric C. LaCasse 1 , David F. Stojdl 1, 3 , Robert G. Korneluk 1, 3 , Martin Holcik 1, 4 1 Molecular Biomedicine Program, Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada 2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada 3 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada 4 Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Correspondence to: Martin Holcik, email: martin@arc.cheo.ca Keywords: rhabdomyosarcoma, Smac mimetic, oncolytic virus, immunotherapy Received: June 20, 2016 Accepted: November 23, 2016 Published: December 10, 2016 ABSTRACT 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 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 eIF2&alpha, ) 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 eIF2&alpha, 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.

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.

// Urszula Liwak-Muir 1 , Christine C. Dobson 1 , Thet Naing 1 , Quinlan Wylie 1 , Lucia Chehade 1 , Stephen D. Baird 1 , Pranesh K. Chakraborty 2,3 and Martin Holcik 1,2 1 Molecular Biomedicine Program, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada 2 Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada 3 Newborn Screening Ontario, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, ON, Canada Correspondence to: Martin Holcik, email: // Keywords : tumour supressor, miRNA, kinase Received : September 14, 2015 Accepted : November 16, 2015 Published : November 22, 2015 Abstract 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 H 2 O 2 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 H 2 O 2 treatment. We show that phosphorylation of HuR by ERK8 prevents it from binding to PDCD4 mRNA and allows miR-21-mediated degradation of PDCD4.

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.

Background The TRIB 1 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 ( SNP s), identified by genome‐wide association studies, are located ≈30 kb downstream from TRIB 1, 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 SNP s at the TRIB 1 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.

Pathogenic bacteria secrete various virulence factors, including toxins, lipases and proteases that allow them to infect, breakdown and colonize host tissue. Among various modes of action that the pathogenic bacteria use to damage the host, pore formation (by pore forming toxins (PFTs)) and lipid hydrolysis (by phospholipases) modes are common in damaging the eukaryotic cell membrane. PFTs in their monomeric form are extracellular diffusible and able to form hydrophilic pores in cell membrane while phospholipases cleaves and hydrolyzes the ester bonds of most phospholipids in cell membrane. Both modes of action cause uncontrolled permeation of ions and molecules across cell membrane, leading to cell death by apoptosis or necrosis. In this work, the toxins secreted by two clinically important human pathogens, methicillin susceptible Staphylococcus aureus (MSSA476) and Pseudomonas aeruginosa (PAO1) were studied via their interaction with a planar tethered bilayer lipid membrane (pTBLM) using surface plasmon resonance spectroscopy (SPR) and electrochemical impedance spectroscopy (EIS). Detection and discrimination is based on lipid-loss (lipid hydrolysis by phospholipases) or non lipid-loss (pore formation by PFTs) from pTBLM upon interaction with supernatant of pathogenic bacteria. Using EIS and SPR, it is demonstrated that major toxins of S. auerus are PFTs while most of toxin associated with P. aeruginosa are more lipid damaging lipolytic enzymes. Such a format might have future utility as a simple assay for measuring the presence membrane lytic virulence factors in clinical samples.

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.

Objectives— 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. Conclusion— 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.

Scavenger receptor class B type I (SR-BI) has an established role in mediating the selective uptake of choles- terol 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 distribu- tion of SR-BI. We report that red fluorescent protein-tagged mouse SR-BI (RFP-mSR-BI) colocalizes with the late endo- somal and lysosomal markers, Rab7, LBPA, and Rab9. In ad- dition, endogenous SR-BI is also found on lysosomes and colocalizes with LAMP-2 in primary hepatocytes. Further- more, 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 ( 2/2 ) as compared with wild-type hepato- cytes. In addition to its role as a plasma membrane receptor, SR-BI may function in cholesterol trafficking from late endo- somes/lysosomes.—Ahras, M., T. Naing, and R. McPherson. Scavenger receptor class B type I localizes to a late endosomal compartment. J. Lipid Res. 2008. 49: 1569-1576.

Age-related macular degeneration (AMD) is a major cause of blindness, but presents differently in Europeans and Asians. Here, we perform a genome-wide and exome-wide association study on 2,119 patients with exudative AMD and 5,691 controls, with independent replication in 4,226 patients and 10,289 controls, all of East Asian descent, as part of The Genetics of AMD in Asians (GAMA) Consortium. We find a strong association between CETP Asp442Gly (rs2303790), an East Asian-specific mutation, and increased risk of AMD (odds ratio (OR)=1.70, P=5.60 × 10(-22)). The AMD risk allele (442Gly), known to protect from coronary heart disease, increases HDL cholesterol levels by 0.17 mmol l(-1) (P=5.82 × 10(-21)) in East Asians (n=7,102). We also identify three novel AMD loci: C6orf223 Ala231Ala (OR=0.78, P=6.19 × 10(-18)), SLC44A4 Asp47Val (OR=1.27, P=1.08 × 10(-11)) and FGD6 Gln257Arg (OR=0.87, P=2.85 × 10(-8)). Our findings suggest that some of the genetic loci conferring AMD susceptibility in East Asians are shared with Europeans, yet AMD in East Asians may also have a distinct genetic signature.

A new methodology for detecting the microbiological state of a wound dressing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we report how stabilized lipid vesicles containing self-quenched carboxyfluorescein dye are sensitive to lysis only by toxins/virulence factors from P. aeruginosa and S. aureus but not by a non-toxic Escherichia coli species. The development of the stabilized vesicles is discussed and their response to detergent (triton), bacterial toxin (α-hemolysin) and lipases (phospholipase A 2). Finally, fabrics with stabilized vesicles attached via plasma deposited maleic anhydride coupling are shown visibly responding to S. aureus (MSSA 476) and P. aeruginosa (PAO1) but not E. coli DH5α in a prototype dressing. Refereed/Peer-reviewed

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.

29 male patients (14 from Taunggyi, 6 from Rangoon and 9 from Tharrawaddy in Burma) were treated simultaneously with 200 mg artemether (Single intramuscular dose), 1500 mg sulfadoxine and 75 mg [corrected] pyrimethamine (orally). The mean parasite clearance time was 106.7 +/- 48.7 h. Side effects were few and self-limiting. 13 of 29 patients had recrudescences before day 28; as all the patients were living in towns, reinfection was unlikely. This parasite clearance time was longer than that in patients treated with artemether alone (600 mg total dose), and the recrudescence rate was higher. This drug combination is not recommended for patients in areas where sulfadoxine/pyrimethamine resistance is already established.

Fifty-two patients with falciparum malaria during pregnancy were studied in Taunggyi, Shan States, Burma, during the period of April 1985 through December 1986. Severely ill cases were all treated with quinine, but uncomplicated cases were randomised to receive either quinine or amodiaquine. Fifty-one age-matched non-pregnant female patients were also randomised to receive either quinine or amodiaquine. All clinical and laboratory parameters were comparable between pregnant and non-pregnant group of patients. Falciparum malaria was most frequent among primigravidae, and occurred most frequently in the second trimester for all parities. There were no differences in parasite density, fever clearance and parasite clearance between groups with different parity or gestational period. Quinine and amodiaquine treatment were equally effective. The outcome of pregnancy with and without anti-malarial prophylaxis is discussed.