ApoB siRNA-induced Liver Steatosis is Resistant to Clearance by the Loss of Fatty Acid Transport Protein 5 (Fatp5)

The association between hypercholesterolemia and elevated serum apolipoprotein B (APOB) has generated interest in APOB as a therapeutic target for patients at risk of developing cardiovascular disease. In the clinic, mipomersen, an antisense oligonucleotide (ASO) APOB inhibitor, was associated with a trend toward increased hepatic triglycerides, and liver steatosis remains a concern. We found that siRNA-mediated knockdown of ApoB led to elevated hepatic triglycerides and liver steatosis in mice engineered to exhibit a human-like lipid profile. Many genes required for fatty acid synthesis were reduced, suggesting that the observed elevation in hepatic triglycerides is maintained by the cell through fatty acid uptake as opposed to fatty acid synthesis. Fatty acid transport protein 5 (Fatp5/Slc27a5) is required for long chain fatty acid (LCFA) uptake and bile acid reconjugation by the liver. Fatp5 knockout mice exhibited lower levels of hepatic triglycerides due to decreased fatty acid uptake, and shRNA-mediated knockdown of Fatp5 protected mice from diet-induced liver steatosis. Here, we evaluated if siRNA-mediated knockdown of Fatp5 was sufficient to alleviate ApoB knockdown-induced steatosis. We determined that, although Fatp5 siRNA treatment was sufficient to increase the proportion of unconjugated bile acids 100-fold, consistent with FATP5's role in bile acid reconjugation, Fatp5 knockdown failed to influence the degree, zonal distribution, or composition of the hepatic triglycerides that accumulated following ApoB siRNA treatment

Insulin Concentration Modulates Hepatic Lipid Accumulation in Mice in Part via Transcriptional Regulation of Fatty Acid Transport Proteins

Fatty liver disease (FLD) is commonly associated with insulin resistance and obesity, but interestingly it is also observed at low insulin states, such as prolonged fasting. Thus, we asked whether insulin is an independent modulator of hepatic lipid accumulation.In mice we induced, hypo- and hyperinsulinemia associated FLD by diet induced obesity and streptozotocin treatment, respectively. The mechanism of free fatty acid induced steatosis was studied in cell culture with mouse liver cells under different insulin concentrations, pharmacological phosphoinositol-3-kinase (PI3K) inhibition and siRNA targeted gene knock-down. We found with in vivo and in vitro models that lipid storage is increased, as expected, in both hypo- and hyperinsulinemic states, and that it is mediated by signaling through either insulin receptor substrate (IRS) 1 or 2. As previously reported, IRS-1 was up-regulated at high insulin concentrations, while IRS-2 was increased at low levels of insulin concentration. Relative increase in either of these insulin substrates, was associated with an increase in liver-specific fatty acid transport proteins (FATP) 2&5, and increased lipid storage. Furthermore, utilizing pharmacological PI3K inhibition we found that the IRS-PI3K pathway was necessary for lipogenesis, while FATP responses were mediated via IRS signaling. Data from additional siRNA experiments showed that knock-down of IRSs impacted FATP levels.States of perturbed insulin signaling (low-insulin or high-insulin) both lead to increased hepatic lipid storage via FATP and IRS signaling. These novel findings offer a common mechanism of FLD pathogenesis in states of both inadequate (prolonged fasting) and ineffective (obesity) insulin signaling

Burden of waterpipe smoking and chewing tobacco use among women of reproductive age group using data from the 2012-13 Pakistan Demographic and Health Survey

Background: Despite the general decline in cigarette smoking, use of alternative forms of tobacco has increased particularly in developing countries. Waterpipe (WP) and Chewing Tobacco (CT) are two such alternative forms, finding their way into many populations. However, the burden of these alternative forms of tobacco and their socio demographic determinants are still unclear. We assessed the prevalence of WP and CT use among women of reproductive age group in Pakistan. Methods: Data from the most recent Pakistan Demographic and Health Survey 2012–13 (n = 13,558) was used for this analysis. Information obtained from ever married women, aged between 15 and 49 years were analyzed using two separate data subgroups; exclusive WP smokers (total n = 12,995) and exclusive CT users (total n = 12,771). Univariate and Multivariate logistic regression analyses were conducted and results were reported as crude and adjusted Odds Ratio with 95 % confidence intervals. Results: Prevalence of WP smoking and CT were 4 % and 2 %, respectively. After multivariate adjustments, ever married women who were: older than 35 years (OR; 4.68 95 % CI, 2.62–8.37), were poorest (OR = 4.03, 95 % CI 2.08–7.81), and had no education (OR = 9.19, 95 % CI 5.10–16.54), were more likely to be WP smokers. Similarly, ever married women who were: older than 35 years (OR = 3.19, 95 % CI 1.69–6.00), had no education (OR = 4.94, 95 % CI 2.62–9.33), were poor (OR = 1.64, 95 % CI 1.07–2.48) and had visited health facility in last 12 months (OR = 1.81, 95 % CI 1.22–2.70) were more likely to be CT users as well. Conclusion: Older women with lower socio-economic profile were more likely to use WP and CT. Focused policies aiming towards reducing the burden of alternate forms of tobacco use among women is urgently needed to control the tobacco epidemic in the country

Numerical analysis of different heating systems for warm sheet metal forming

The main goal of this study is to present an analysis of different heating methods frequently used in laboratory scale and in the industrial practice to heat blanks at warm temperatures. In this context, the blank can be heated inside the forming tools (internal method) or using a heating system (external method). In order to perform this analysis, a finite element model is firstly validated with the simulation of the direct resistance system used in a Gleeble testing machine. The predicted temperature was compared with the temperature distribution recorded experimentally and a good agreement was found. Afterwards, a finite element model is used to predict the temperature distribution in the blank during the heating process, when using different heating methods. The analysis also includes the evaluation of a cooling phase associated to the transport phase for the external heating methods. The results of this analysis show that neglecting the heating phase and a transport phase could lead to inaccuracies in the simulation of the forming phase.The authors gratefully acknowledge the financial support of the Portuguese Foundation for Science and Technology (FCT) under project PTDC/EMS-TEC/1805/2012 and by FEDER funds through the program COMPETE—Programa Operacional Factores de Competitividade, under the project CENTRO-07-0224-FEDER-002001 (MT4MOBI). The authors would like to thank Prof. A. Andrade-Campos for helpful contributions on the development of the finite element code presented in this work.info:eu-repo/semantics/publishedVersio

Glycosaminoglycan and Proteoglycan Biotherapeutics in Articular Cartilage Protection and Repair Strategies: Novel Approaches to Visco?supplementation in Orthobiologics

The aim of this study is to review developments in glycosaminoglycan and proteoglycan research relevant to cartilage repair biology and in particular the treatment of osteoarthritis (OA). Glycosaminoglycans decorate a diverse range of extracellular matrix and cell associated proteoglycans conveying structural organization and physico‐chemical properties to tissues. They play key roles mediating cellular interactions with bioactive growth factors, cytokines, and morphogenetic proteins, and structural fibrillar collagens, cell interactive and extracellular matrix proteoglycans, and glycoproteins which define tissue function. Proteoglycan degradation detrimentally affects tissue functional properties. Therapeutic strategies have been developed to counter these degenerative changes. Neo‐proteoglycans prepared from chondroitin sulfate or hyaluronan and hyaluronan or collagen‐binding peptides emulate the interactive, water imbibing, weight bearing, and surface lubricative properties of native proteoglycans. Many neo‐proteoglycans outperform native proteoglycans in terms of water imbibition, matrix stabilization, and resistance to proteolytic degradation. The biospecificity of recombinant proteoglycans however, provides precise attachment to native target molecules. Visco‐supplements augmented with growth factors/therapeutic cells, hyaluronan, and lubricin (orthobiologicals) have the capacity to lubricate and protect cartilage, control inflammation, and promote cartilage repair and regeneration of early cartilage lesions and may represent a more effective therapeutic approach to the treatment of mild to moderate OA and deserve further study

Polymorphisms in the Presumptive Promoter Region of the SLC2A9 Gene Are Associated with Gout in a Chinese Male Population

BACKGROUND: Glucose transporter 9 (GLUT9) is a high-capacity/low-affinity urate transporter. To date, several recent genome-wide association studies (GWAS) and follow-up studies have identified genetic variants of SLC2A9 associated with urate concentrations and susceptibility to gout. We therefore investigated associations between gout and polymorphisms and haplotypes in the presumptive promoter region of GLUT9 in Chinese males. METHODOLOGY/PRINCIPAL FINDINGS: The approximately 2000 bp presumptive promoter region upstream of the start site of exon 1 of GLUT9 was sequenced and subjected to genetic analysis. A genotype-phenotype correlation was performed and polymorphisms-induced changes in transcription factor binding sites were predicted. Of 21 SNPs identified in GLUT9, five had not been previously reported. Two of the SNPs (rs13124007 and rs6850166) were associated with susceptibility to gout (p = 0.009 and p = 0.042, respectively). The C allele of rs13124007 appeared to be the risk allele for predisposition to gout (p = 0.006, OR 1.709 [95% CI 1.162-2.514]). For rs6850166, an increased risk of gout was associated with the A allele (p = 0.029, OR 1.645 [95% CI 1.050-2.577]). After Bonferroni correction, there was statistically difference in rs13124007 allele frequencies between gout cases and controls (P = 0.042). Haplotype analyses showed that haplotype GG was a protective haplotype (p = 0.0053) and haplotype CA was associated with increased risk of gout (p = 0.0326). Genotype-phenotype analysis among gout patients revealed an association of rs13124007 with serum triglycerides levels (P = 0.001). The C to G substitution in polymorphism rs13124007 resulted in a loss of a binding site for transcription factor interferon regulatory factor 1 (IRF-1). CONCLUSIONS/SIGNIFICANCE: Polymorphisms rs13124007 and rs6850166 are associated with susceptibility to gout in Chinese males

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

<p>Abstract</p> <p>Background</p> <p>Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.</p> <p>Methods</p> <p>HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.</p> <p>Results</p> <p>17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.</p> <p>Conclusions</p> <p>Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in <it>in vivo </it>induction of HIF. <it>In vitro </it>data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.</p

Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

Background: Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. Methodology/Principal Findings: Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i) in all ex vivo in biopsies of patients suffering from skin infections, (ii) in vitro using cell culture infection models and (iii) in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs) of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. Conclusions/Significance: Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections