Analyzing human knockouts to validate GPR151 as a therapeutic target for reduction of body mass index.

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we further investigate GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify eleven GPR151 putative loss-of-function (plof) variants, three of which are present at homozygosity (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7), with a cumulative allele frequency of 2.2%. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits and find that GPR151 deficiency in complete human knockouts is not associated with clinically significant differences in these traits. Relative to Gpr151+/+ mice, Gpr151-/- animals exhibit no difference in body weight on normal chow and higher body weight on a high-fat diet. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach to treatment of obesity.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

Genome-wide analysis of blood lipid metabolites in over 5000 South Asians reveals biological insights at cardiometabolic disease loci.

BACKGROUND: Genetic, lifestyle, and environmental factors can lead to perturbations in circulating lipid levels and increase the risk of cardiovascular and metabolic diseases. However, how changes in individual lipid species contribute to disease risk is often unclear. Moreover, little is known about the role of lipids on cardiovascular disease in Pakistan, a population historically underrepresented in cardiovascular studies. METHODS: We characterised the genetic architecture of the human blood lipidome in 5662 hospital controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS) and 13,814 healthy British blood donors from the INTERVAL study. We applied a candidate causal gene prioritisation tool to link the genetic variants associated with each lipid to the most likely causal genes, and Gaussian Graphical Modelling network analysis to identify and illustrate relationships between lipids and genetic loci. RESULTS: We identified 253 genetic associations with 181 lipids measured using direct infusion high-resolution mass spectrometry in PROMIS, and 502 genetic associations with 244 lipids in INTERVAL. Our analyses revealed new biological insights at genetic loci associated with cardiometabolic diseases, including novel lipid associations at the LPL, MBOAT7, LIPC, APOE-C1-C2-C4, SGPP1, and SPTLC3 loci. CONCLUSIONS: Our findings, generated using a distinctive lipidomics platform in an understudied South Asian population, strengthen and expand the knowledge base of the genetic determinants of lipids and their association with cardiometabolic disease-related loci.

May Measurement Month 2017: an analysis of the blood pressure screening campaign results in Pakistan-South Asia.

Elevated blood pressure (BP) is a growing burden worldwide, leading to over 10 million deaths each year. May Measurement Month (MMM) is a global initiative aimed at raising awareness of high BP and to act as a temporary solution to the lack of screening programs worldwide. Hypertension is a global health concern for developing countries. In Pakistan, apart from few population-based studies which evaluated the prevalence of hypertension, there is no current nationally representative study (the latest nationwide survey was conducted more than two decades ago). Pakistan Hypertension League, in accordance with the International Society of Hypertension directive under the banner of the May Measurement Month 2017 (MMM17) campaign, carried out a nationwide cross-sectional survey of volunteers aged ≥18 in May 2017 through its 14 regional chapters. Blood pressure measurement recorded through digital apparatus, the definition of hypertension (≥140/90 mmHg or being on BP-lowering treatment) and statistical analysis followed the standard MMM protocol. A total of 5333 individuals were screened during the MMM17 campaign with mean age 45.0 (11.6). Males had a higher rate (66.3%, n = 3536) in those screened than females (33.0%, n = 1757). A total of 55.2% (n = 2943) people had hypertension. This result shows very high rates of hypertension in Pakistani people. Therefore, there is an urgent need for federal implementation of BP screening as well as awareness programs across the nation.

Entropy Generation on Nanofluid Thin Film Flow of Eyring-Powell Fluid with Thermal Radiation and MHD Effect on an Unsteady Porous Stretching Sheet.

This research paper investigates entropy generation analysis on two-dimensional nanofluid film flow of Eyring-Powell fluid with heat amd mass transmission over an unsteady porous stretching sheet in the existence of uniform magnetic field (MHD). The flow of liquid films are taken under the impact of thermal radiation. The basic time dependent equations of heat transfer, momentum and mass transfer are modeled and converted to a system of differential equations by employing appropriate similarity transformation with unsteady dimensionless parameters. Entropy analysis is the main focus in this work and the impact of physical parameters on the entropy profile are discussed in detail. The influence of thermophoresis and Brownian motion has been taken in the nanofluids model. An optima approach has been applied to acquire the solution of modeled problem. The convergence of the HAM (Homotopy Analysis Method) has been presented numerically. The disparity of the Nusslet number, Skin friction, Sherwood number and their influence on the velocity, heat and concentration fields has been scrutinized. Moreover, for comprehension, the physical presentation of the embedded parameters are explored analytically for entropy generation and discussed.

Functional inhibition of Hsp70 by Pifithrin-µ switches Gambogic acid induced caspase dependent cell death to caspase independent cell death in human bladder cancer cells.

Heat shock protein-70kDa (Hsp70) is a member of molecular chaperone family, involved in the proper folding of various proteins. Hsp70 is important for tumor cell survival and is also reported to be involved in enhancing the drug resistance of various cancer types. Hsp70 controls apoptosis both upstream and downstream of the mitochondria by regulating the mitochondrial membrane permeabilization (MMP) and apoptosome formation respectively. In the present study, we have elucidated the role of Hsp70 in Gambogic acid (GA) induced apoptosis in bladder cancer cells. We observed that functional inhibition of Hsp70 by Pifithrin-µ switches GA induced caspase dependent (apoptotic) cell death to caspase independent cell death. However, this cell death was not essentially necrotic in nature, as shown by the observations like intact plasma membranes, cytochrome-c release and no significant effect on nuclear condensation/fragmentation. Inhibition of Hsp70 by Pifithrin-µ shows differential effect on MMP. GA induced MMP and cytochrome-c release was inhibited by Pifithrin-µ at 12h but enhanced at 24h. Pifithrin-µ also reverted back GA inhibited autophagy which resulted in the degradation of accumulated ubiquitinated proteins. Our results demonstrate that Hsp70 plays an important role in GA induced apoptosis by regulating caspase activation. Therefore, inhibition of Hsp70 may hamper with the caspase dependent apoptotic pathways induced by most anti-cancer drugs and reduce their efficacy. However, the combination therapy with Pifithrin-µ may be particularly useful in targeting apoptotic resistant cancer cells as Pifithrin-µ may initiate alternative cell death program in these resistant cells.

Epidemiology and outcomes of out-of-hospital cardiac arrest in a developing country-a multicenter cohort study.

BACKGROUND: Out-of-hospital cardiac arrest (OHCA) is one of the leading causes of death and disability worldwide. Overall survival after an OHCA has been reported to be poor and limited studies have been conducted in developing countries. We aimed to investigate the rates of survival from OHCA and explore components of the chain of survival in a developing country. METHODS: We conducted a multicenter prospective cohort study in the emergency departments (ED) of five major public and private sector hospitals of Karachi, Pakistan from January 2013 to April 2013. Twenty-four hour data collection was performed by trained data collectors, using a structured questionnaire. All patients ≥18 years of age, presenting with OHCA of cardiac origin, were included. Patients with do-not-resuscitate status or referred from other hospitals were excluded. Our primary outcome was survival of OHCA patients at the end of ED stay. RESULTS: During the three month period, data was obtained from 310 OHCA patients. The overall survival to ED discharge was 1.6 % which decreased to 0 % at 2-months after discharge. More than half (58.3 %) of these OHCA patients were brought to the hospital in a non-EMS (emergency medical service) vehicle i.e. public or private transportation. Patients utilizing non-EMS transportation reached the hospital earlier with a median time of 23 min compared to patients utilizing any type of ambulances which had a delay of 7 min hospital reaching time (median time 30 min). However, patients utilizing ambulances with life-support facilities, as compared to all other types of pre-hospital transportation, had the shortest time to first life-support intervention (15 min). Most of the patients (92.9 %) had a witnessed cardiac arrest out of which only a small percentage (2.3 %) received bystander CPR (cardio pulmonary resuscitation). Median time from arrest to receiving first CPR was 20 min. Only 1 % of patients were found to have a shockable rhythm on first assessment. CONCLUSION: This study showed that the overall survival of OHCA is null in this population. Lack of bystander CPR and weaker emergency medical services (EMS) leading to a delay in receiving life-support interventions were some of the important observations. Poor survival emphasizes the need to standardize EMS systems, initiate public awareness programs and strengthen links in the chain of survival.

Gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (NF-κB, Beclin-1, p62 and NBR1) in human bladder cancer cells.

BACKGROUND: Gambogic acid is a potent anticancer agent and has been found effective against various types of cancer cells. The present study was addressed to explore the cytotoxic potential of Gambogic acid and the modulation of autophagy and apoptosis in bladder cancer cells T24 and UMUC3. METHODS: Bladder cancer cell lines T24 and UMUC3 were treated with Gambogic acid, apoptosis was checked by flow-cytometry and expression of various autophagy and apoptosis related proteins was monitored by Western blotting. Confocal microscope was used for colocalization of p62 and Beclin-1. RESULTS: Gambogic acid induces reactive oxygen species, and elicits a strong autophagic response by activating JNK at earlier time points, which is inhibited at later time points with the activation of caspases. Reactive oxygen species mediated caspase activation causes degradation of autophagic proteins, cleavage of molecular chaperones (Hsp90 and GRP-78) and adaptor proteins (p62 and NBR1). Gambogic acid treatment results in mitochondrial hyperpolarization and cytochrome c release and activates caspases involved in both extrinsic and intrinsic apoptotic pathways. Gambogic acid abrogates NF-κB activation by ROS mediated inhibition of IκB-α phosphorylation. Functionally Gambogic acid induced autophagy acts as a strong cell survival response and delays caspase activation. CONCLUSION: Our study provides the new insights about the mechanism of Gambogic acid induced modulation of autophagy and apoptosis in bladder cancer cells. All the molecular events responsible for Gambogic acid induced autophagy and apoptosis are mediated by reactive oxygen species. GENERAL SIGNIFICANCE: Since Gambogic acid targets various cell survival molecules therefore, it may be considered as a potential anticancer agent against bladder cancer.

Physical activity, smoking, and genetic predisposition to obesity in people from Pakistan: the PROMIS study.

BACKGROUND: Multiple genetic variants have been reliably associated with obesity-related traits in Europeans, but little is known about their associations and interactions with lifestyle factors in South Asians. METHODS: In 16,157 Pakistani adults (8232 controls; 7925 diagnosed with myocardial infarction [MI]) enrolled in the PROMIS Study, we tested whether: a) BMI-associated loci, individually or in aggregate (as a genetic risk score--GRS), are associated with BMI; b) physical activity and smoking modify the association of these loci with BMI. Analyses were adjusted for age, age(2), sex, MI (yes/no), and population substructure. RESULTS: Of 95 SNPs studied here, 73 showed directionally consistent effects on BMI as reported in Europeans. Each additional BMI-raising allele of the GRS was associated with 0.04 (SE = 0.01) kg/m(2) higher BMI (P = 4.5 × 10(-14)). We observed nominal evidence of interactions of CLIP1 rs11583200 (P(interaction) = 0.014), CADM2 rs13078960 (P(interaction) = 0.037) and GALNT10 rs7715256 (P(interaction) = 0.048) with physical activity, and PTBP2 rs11165643 (P(interaction) = 0.045), HIP1 rs1167827 (P(interaction) = 0.015), C6orf106 rs205262 (P(interaction) = 0.032) and GRID1 rs7899106 (P(interaction) = 0.043) with smoking on BMI. CONCLUSIONS: Most BMI-associated loci have directionally consistent effects on BMI in Pakistanis and Europeans. There were suggestive interactions of established BMI-related SNPs with smoking or physical activity.

Global blood pressure screening during the COVID-19 pandemic: results from the May Measurement Month 2021 campaign.

BACKGROUND: Raised blood pressure (BP) remains the biggest risk factor contributing to the global burden of disease and mortality, despite the COVID-19 pandemic. May Measurement Month (MMM), an annual global screening campaign aims to highlight the importance of BP measurement by evaluating global awareness, treatment and control rates among adults with hypertension. In 2021, we assessed the global burden of these rates during the COVID-19 pandemic. METHODS: Screening sites were set up in 54 countries between May and November 2021 and screenees were recruited by convenience sampling. Three sitting BPs were measured, and a questionnaire completed including demographic, lifestyle and clinical data. Hypertension was defined as a systolic BP at least 140 mmHg and/or a diastolic BP at least 90 mmHg (using the mean of the second and third readings) or taking antihypertensive medication. Multiple imputation was used to impute the average BP when readings were missing. RESULTS: Of the 642 057 screenees, 225 882 (35.2%) were classified as hypertensive, of whom 56.8% were aware, and 50.3% were on antihypertensive medication. Of those on treatment, 53.9% had controlled BP (<140/90 mmHg). Awareness, treatment and control rates were lower than those reported in MMM campaigns before the COVID-19 pandemic. Minimal changes were apparent among those testing positive for, or being vaccinated against COVID-19. Of those on antihypertensive medication, 94.7% reported no change in their treatment because of the COVID-19 pandemic. CONCLUSION: The high yield of untreated or inadequately treated hypertension in MMM 2021 confirms the need for systematic BP screening where it does not currently exist.

South Asian medical cohorts reveal strong founder effects and high rates of homozygosity.

The benefits of large-scale genetic studies for healthcare of the populations studied are well documented, but these genetic studies have traditionally ignored people from some parts of the world, such as South Asia. Here we describe whole genome sequence (WGS) data from 4806 individuals recruited from the healthcare delivery systems of Pakistan, India and Bangladesh, combined with WGS from 927 individuals from isolated South Asian populations. We characterize population structure in South Asia and describe a genotyping array (SARGAM) and imputation reference panel that are optimized for South Asian genomes. We find evidence for high rates of reproductive isolation, endogamy and consanguinity that vary across the subcontinent and that lead to levels of rare homozygotes that reach 100 times that seen in outbred populations. Founder effects increase the power to associate functional variants with disease processes and make South Asia a uniquely powerful place for population-scale genetic studies.

LIM kinase 1 - dependent cofilin 1 pathway and actin dynamics mediate nuclear retinoid receptor function in T lymphocytes.

BACKGROUND: It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation. RESULTS: Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1. CONCLUSION: We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.

RNA-activated protein kinase differentially modulates innate immune response mediated by supraphysiological concentrations of thyroid hormone.

Nuclear hormone receptor ligands are known to modulate innate immunity by dampening the immune response induced by pathogens. Here, we report that unlike other ligands, 3,3',5-triiodo-l-thyronine (T3) induced the type 1 IFN response and expression of IFN-stimulated genes (ISGs). T3 action was found to be significantly amplified at supraphysiological concentrations (SPC) and in combination with double-stranded RNA mimic polyinosinic-polycytidylic acid. Induction by T3 was due to non-genomic mechanisms involving integrin binding, calcium mobilization, and phosphatidyl-inositol 3-kinase-AKT pathways, but was independent of TLR3, RIG-I, and IFN-ß1 pathways. Whereas siRNA-induced knockdown of RNA-activated protein kinase (PKR) was found to abrogate the T3-induced expression of select ISGs, expression of other T3-induced ISGs was strongly induced by PKR knockdown, indicating the differential role of PKR in modulating T3 action. Together, we describe a novel role of T3 in modulating the innate immune response and identify the importance of PKR in regulating T3-induced immune activation. These findings have important implications in the basic understanding of the mechanisms of T3 function at SPCs and crosstalk involved in the thyroid hormone function and the innate immune response.

GADD34 attenuates HIV-1 replication by viral 5'-UTR TAR RNA-mediated translational inhibition.

Role of GADD34, a protein that is induced following cellular stress, in HIV-1 replication was investigated. GADD34 was induced during the late phase of HIV-1 infection. siRNA-knockdown of GADD34 stimulated whereas overexpression of GADD34 inhibited HIV-1 replication. GADD34 N-terminal ER-binding-helix amino acid region 1-192 alone was found to be sufficient for the inhibition of HIV-1 replication whereas protein-phosphatase -1-binding domain and eIF-2α-phosphatase activity of GADD34 were not crucial for anti-HIV-1 activity. GADD34 did not alter the HIV-1 RNA levels but reduced the viral protein expression suggesting that GADD34 interferes in HIV protein synthesis. Studies on the effect of HIV-1-5'-UTR and its mutants on a human promoter-driven luciferase expression indicated that GADD34-inhibition was mediated by 5'-UTR/TAR RNA, probably by modulating TAR RNA structure. In summary, our data support a novel function of GADD34 as a putative anti-HIV-1 restriction factor.

Awareness of the risk factors, presenting features and complications of hypertension amongst hypertensives and normotensives.

OBJECTIVES: To assess the level of awareness with regards to risk factors, presenting features and complications of hypertension. Moreover, to Compare the level of awareness amongst patients (hypertensives) and nonpatients (normotensives) in Karachi, Pakistan. METHODS: This cross-sectional study was carried out on a random sample of 440 people (220-normotensives/220-hypertensives) using an interview based questionnaire. Data entry was performed on Epi-info v 6 and managed and analyzed on SPSS v 14. Proportions were calculated for categorical data, means and standard deviations were calculated for continuous data. RESULTS: Hypertensives had a higher mean awareness score than the normotensives (p<0.001). It was observed that people below 30 years of age were significantly more aware than people above this age (p<0.001). Patients with higher awareness scores were more compliant to therapy (p<0.001). CONCLUSION: Poor levels of awareness regarding the risk factors, presenting features and complications of hypertension were observed in both the groups. The hypertensive population was relatively more aware than the normotensive population.

Radiative MHD thin film flow of Williamson fluid over an unsteady permeable stretching sheet.

In this research work we have examined the flow of Williamson liquid film fluid with heat transmission and having the impact of thermal radiation embedded in a permeable medium over a time dependent stretching surface. The fluid flow of liquid films is assumed in two dimensions. By using suitable similarity transformation the governing non-linear partial differential equations have been transformed into non-linear differential equations. An optimal approach has been used to acquire the solution of the modelled problem. The convergence of the technique has been shown numerically. The impact of the Skin friction and Nusslet number and their influence on thin film flow are shown numerically. Thermal radiation, unsteadiness effect and porosity have mainly focused in this paper. Furthermore, for conception and physical demonstration the entrenched parameters, like porosity parameter k , Prandtl number Pr , unsteadiness parameter S , Radiation parameter R d , Magnetic parameter M , and Williamson fluid parameter have been discussed graphically in detail with their effect on liquid film flow.

Physician assessment of stroke risk in hypertensive patients in the Middle East and Africa: results of the action survey.

OBJECTIVES: In the absence of reliable, contemporary national data, the ACTION survey was designed to: a) provide preliminary data on stroke risk in the MEA (Middle East and Africa); b) describe the contribution of specific cardiovascular risk factors; 3) assess blood pressure (BP) control. DESIGN AND PATIENTS: This was a multi-center observational study in nine countries in the MEA region. From 2003 to 2005, 562 physicians from a variety of specialties recorded observations of cardiovascular risk factors in 4,747 hypertensive patients, aged 54-80 years. The 10-year absolute stroke risk was calculated using a scoring system based on the Framingham Heart Study observations, and comparisons made with an age-matched cohort. RESULTS: The mean 10-year stroke risk was estimated at 22.7% and was significantly higher for men (25.4%) than for women (19.5%) (P < .001) and for diabetics (28.2%) than for non-diabetics (19.4%) (P < .001). Compared with an age-matched Framingham cohort, the estimated stroke risk in our population was almost double, and was significantly higher for females (212%) than for males (192%) (P < .001). Hypertension, diabetes, left ventricular hypertrophy, and smoking were major contributing risk factors, as were physical inactivity and elevated cholesterol. Blood pressure was controlled in only 18% of the population and in 12% of diabetics. CONCLUSION: Physicians of all specialties were willing to participate in stroke risk assessment. The risk of stroke in hypertensive patients in the MEA region is high, and is higher than would be predicted using Framingham data, particularly for females. Hypertension appears to be poorly controlled in more than 80% of hypertensive patients in the MEA region.

Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a mendelian randomisation analysis.

BACKGROUND: The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease. METHODS: In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease. FINDINGS: The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90-0·97; p<0·0001) per 1-SD increment in LPA KIV2 repeats after adjustment for lipoprotein(a) concentration and conventional lipid concentrations. The OR for myocardial infarction was 1·10 (1·05-1·14; p<0·0001) per 1-SD increment in lipoprotein(a) concentration, after adjustment for LPA KIV2 repeats and conventional lipids. Genome-wide analysis identified rs2457564 as a variant associated with smaller apolipoprotein(a) isoform size, but not lipoprotein(a) concentration, and rs3777392 as a variant associated with lipoprotein(a) concentration, but not apolipoprotein(a) isoform size. In 60 801 patients with coronary heart disease and 123 504 controls, OR for myocardial infarction was 0·96 (0·94-0·98; p<0·0001) per 1-SD increment in apolipoprotein(a) protein isoform size in serum due to rs2457564, which was directionally concordant with the OR observed in PROMIS for a similar change. The OR for myocardial infarction was 1·27 (1·07-1·50; p=0·007) per 1-SD increment in lipoprotein(a) concentration due to rs3777392, which was directionally concordant with the OR observed for a similar change in PROMIS. INTERPRETATION: Human genetic data suggest that both smaller apolipoprotein(a) isoform size and increased lipoprotein(a) concentration are independent and causal risk factors for coronary heart disease. Lipoprotein(a)-lowering interventions could be preferentially effective in reducing the risk of coronary heart disease in individuals with smaller apolipoprotein(a) isoforms. FUNDING: British Heart Foundation, US National Institutes of Health, Fogarty International Center, Wellcome Trust, UK Medical Research Council, UK National Institute for Health Research, and Pfizer.

General practitioners' approach to hypertension in urban Pakistan: disturbing trends in practice.

BACKGROUND: Control of blood pressure (BP) remains suboptimal worldwide. The objective of this study was to determine whether (primary) general practitioners' (GPs) approach to high BP is in accordance with international guidelines. METHODS AND RESULTS: We conducted a cross-sectional survey of 1000 randomly selected GPs from urban areas in Pakistan during 2002. A rigorously developed questionnaire on (1) type of practice and (2) detection, (3) evaluation, (4) treatment, and (5) source of information about high BP was administered by trained medical personnel. A total of 1051 GPs were approached, and 1000 (95%) consented to enroll; 766 were male and 655 had been in practice > or =10 years. The average number of patients (SD) seen per day was 48.2 (42.7). Overall, 30.6% (29.0% to 32.3%) and 79.7% (78.3% to 81.0%) of GPs used incorrect BP cutoffs to diagnose hypertension in patients <60 and > or =60 years, respectively. Appropriate therapy for hypertension in the elderly was initiated by only 34.7% (33.0% to 36.3%) of GPs. The use of sedatives either alone (23.8%) or in combination with antihypertensive agents as first-line medication for lowering BP was reported by 45.0% (43.2% to 46.7%). Thiazide diuretics were rarely prescribed (4.2%). Sublingual antihypertensive agents were prescribed by 68.7% (67.1% to 70.3%) of GPs for treating very high levels of BP. The practices of recent graduates from medical school were not better than those of older graduates. CONCLUSIONS: GPs in Pakistan underdiagnose and undertreat high BP, especially in the elderly. Our findings underscore the need for urgent revision of teaching curricula in medical schools with regard to the risks, complications, and management of hypertension, as well as the initiation of widespread and intensive continuing medical education for all physicians involved in the management of patients with hypertension. Particular efforts are needed to encourage the use of low-cost thiazide diuretics as antihypertensive agents in developing countries.

Pentoxifylline triggers autophagy via ER stress response that interferes with Pentoxifylline induced apoptosis in human melanoma cells.

Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor is known to inhibit the growth of various cancer cells including melanoma. Here in this study, we have found that PTX induces autophagy in human melanoma cell lines (A375 and MeWo). Induction of autophagy is associated with the increase in Atg5 expression as knockdown of Atg5 effectively inhibited PTX mediated autophagy. A decrease in mTOR activation was also observed after PTX treatment. We observed that autophagy was activated as a downstream effector mechanism of ER stress induced by PTX. ER stress response was confirmed by upregulation of IRE-1α, GRP78 and CHOP expression. PTX treatment also resulted in an increase in intracellular calcium (Ca(2+)) level. Ca(2+) is the central player as blocking Ca(2+) by intracellular calcium chelator (BAPTA-AM) effectively inhibited the PTX induced ER stress response as well as autophagy. Moreover, silencing of CHOP also resulted in autophagy inhibition with a decrease in Atg5 expression. Collectively, PTX triggers ER stress response followed by induction of autophagy via involvement of Ca(2+)→CHOP→Atg5 signalling cascade. Interestingly, inhibition of intracellular calcium level by BAPTA-AM significantly increased PTX mediated cell death by augmenting intrinsic apoptotic pathway. Inhibition of autophagy by the ATG5 siRNA and pharmacological inhibitor, chloroquine also enhances PTX induced cell death. Taken together, our results clearly indicate that activation of ER stress response and autophagy provides resistance to PTX mediated apoptosis, and thus, interferes with the anticancer activity of PTX in human melanoma cells.