LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress.

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l-carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

Nummularic acid, a triterpenoid, from the medicinal plant Fraxinus xanthoxyloides, induces energy crisis to suppress growth of prostate cancer cells.

We recently identified and characterized nummularic acid (NA) as a major chemical constituent of Fraxinus xanthoxyloides, a medicinal plant used for over hundred years in traditional medicine. In this study, we describe its potential anti-cancer activity using prostate cancer (PCa) cells as a model. We found that NA treatment (5-60 µM) significantly reduced the proliferation and colony formation capabilities of PCa DU145 and C4-2 cells in a time and dose dependent manner, reduced the migratory and invasive properties and increased apoptotic cell population. Mechanistically, we found that NA treatment to PCa cells resulted in a sustained activation of adenosine monophosphate-activated protein kinase (AMPK). NA simultaneously increased acetyl CoA carboxylase phosphorylation and decreased pS6 phosphorylation, the two major substrates of AMPK. Further, NA treatment significantly elevated the cellular ADP/ATP ratio and altered glycolytic rate. We further observed a reversible decrease in oxygen consumption rate in NA treated cells when compared to the control. Finally, we performed global untargeted metabolomics which showed that NA treatment alters PCa cell metabolism at multiple sites including glycolysis, tricarboxylic acid, and glutamine metabolism which supported our observation of a possible AMPK activation. In summary, we report NA as a novel small molecule activator of AMPK that alters cellular metabolism to induce energy crisis and ultimately cancer cell death. Because of its unique mechanism NA could be potentially applicable against other cancer types.

Sex-Specific Differences in an ApoE(-/-):Ins2(+/Akita) Mouse Model of Accelerated Atherosclerosis.

Diabetic patients have a twofold to fourfold increased risk of cardiovascular disease. Despite a vast amount of research, the underlying mechanisms that predispose individuals with diabetes to the development of cardiovascular disease are unclear. To further our understanding of how diabetes promotes atherosclerosis, we have established, characterized, and manipulated a new model of hyperglycemia-induced atherosclerosis: the apolipoprotein E-deficient (ApoE(-/-)):Ins2(+/Akita) mouse. All mice were fed a standard chow diet. Male ApoE(-/-):Ins2(+/Akita) mice developed chronic hyperglycemia, which significantly accelerated atherosclerosis. Female ApoE(-/-):Ins2(+/Akita) mice presented hyperglycemia that normalized by 15 weeks of age. Despite the transient hyperglycemia, advanced atherosclerosis was observed at 15 weeks of age compared with ApoE(-/-) females. To better understand these differences, subsets of mice were castrated or ovariectomized at 5 weeks of age. Castrated ApoE(-/-):Ins2(+/Akita) mice showed a reduction in blood glucose levels that correlated with the amelioration of atherosclerosis. Interestingly, castrated normoglycemic ApoE(-/-) mice developed larger atherosclerotic lesions than sham-operated on controls. Ovariectomized ApoE(-/-):Ins2(+/Akita) mice presented chronic hyperglycemia, and atherosclerosis appeared to be advanced. We have characterized the distinctive sex-specific phenotypes exhibited by the ApoE(-/-):Ins2(+/Akita) mouse model and present evidence for the action of sex hormones on pancreatic ß-cell function and the vasculature that affect the regulation of blood glucose levels and the development of atherosclerosis. This model will provide a test bed to further delineate these effects.

Glycogen synthase kinase 3α deficiency attenuates atherosclerosis and hepatic steatosis in high fat diet-fed low density lipoprotein receptor-deficient mice.

Studies have implicated signaling through glycogen synthase kinase (GSK) 3α/ß in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr(-/-);Gsk3a(+/+), Ldlr(-/-);Gsk3a(+/-), and Ldlr(-/-);Gsk3a(-/-) mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat-diet fed Ldlr(-/-) mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr(-/-);Gsk3a(+/-) mice, 80% smaller in Ldlr(-/-);Gsk3a(-/-) mice; P < 0.05), compared with Ldlr(-/-);Gsk3a(+/+) controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1-derived macrophages that were pretreated with the GSK3α/ß inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways.

Frequency of lipoprotein(a) testing and its levels in Pakistani population.

BACKGROUND: Lipoprotein(a) [Lp(a)] is a highly atherogenic particle identified as an independent risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). This study aimed to investigate the frequency of Lp(a) testing and the incidence of elevated Lp(a) levels in the Pakistani population. METHODS: For this observational study, Lp(a) and lipid profile data from five years (June 2015 to October 2020) were acquired from the electronic patient records of a diagnostic laboratory with a countrywide network. The association of age and total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), non-HDL, and triglyceride (TG) levels with two thresholds for Lp(a), that is, <30 mg/dL and ≥30 mg/dL, was calculated using the Kruskal-Wallis test, while the association between Lp(a) levels and lipid variables was calculated using Spearman correlation. RESULTS: For five years, 1060 tests were conducted, averaging 212 tests per year. Of these, 37.2% showed Lp(a) levels above 30 mg/dL. No significant differences were observed in the results between males and females. However, younger individuals displayed significantly higher Lp(a) levels. Additionally, there was only a weak correlation between the Lp(a) levels and other lipid variables. CONCLUSION: Despite being recognized as a risk factor for ASCVD in the Pakistani population, only a small proportion of the large population underwent Lp(a) testing. Moreover, a significant proportion of the population exceeded this threshold.

Prognostic value of exercise tolerance and exercise-induced diastolic dysfunction in elderly patients referred for exercise stress echocardiography.

BACKGROUND: We studied the prognostic value of exercise tolerance and exercise-associated diastolic dysfunction (Ex-DD) in elderly patients referred for exercise stress echocardiography (ExE). METHODS: A total of 674 patients ≥60 years who underwent ExE using the Bruce protocol between January 2017 and December 2020 were included in this study. The prognostic factors examined were exercise tolerance, quantified in metabolic equivalents of tasks (METs), and Ex-DD. Patients were followed for a composite endpoint of all-cause death and cardiovascular hospitalization. RESULTS: 122 (18 %) patients had Ex-DD and had a higher incidence of the combined outcome (23 [19 %] vs. 47 [9 %], p = 0.001) compared to patients without Ex-DD. Patients were classified into the following groups: group A (no Ex-DD and METs≥8.5), group B (either Ex-DD or METs<8.5), and group C (both Ex-DD and METs<8.5). Group B did not show higher mortality (HR: 1.6, p = 0.475) or cardiovascular hospitalization (HR: 1.8, p = 0.08) compared to group A, but showed an increased risk for the combined outcome (HR: 1.9, p = 0.038). Group C was associated with higher mortality (HR: 4.8, p = 0.032), cardiovascular hospitalization (HR: 3.9, p < 0.001), and the combined outcome (HR: 4.0, p < 0.001) compared to group A. CONCLUSIONS: Decreased exercise capacity and Ex-DD were associated with poor outcomes, especially when both findings were present.

Endoplasmic reticulum stress and glycogen synthase kinase-3ß activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.

OBJECTIVE: The goal of this study was to examine the role of endoplasmic reticulum (ER) stress signaling and the contribution of glycogen synthase kinase (GSK)-3ß activation in hyperglycemic, hyperhomocysteinemic, and high-fat-fed apolipoprotein E-deficient (apoE(-/-)) mouse models of accelerated atherosclerosis. METHODS AND RESULTS: Female apoE(-/-) mice received multiple low-dose injections of streptozotocin (40 µg/kg) to induce hyperglycemia, methionine-supplemented drinking water (0.5% wt/vol) to induce hyperhomocysteinemia, or a high-fat (21% milk fat+0.2% cholesterol) diet to induce relative dyslipidemia. A subset of mice from each group was supplemented with sodium valproate (625 mg/kg), a compound with GSK3 inhibitory activity. At 15 and 24 weeks of age, markers of ER stress, lipid accumulation, GSK3ß phosphorylation, and GSK3ß activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3ß activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3ß activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3ß activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216. CONCLUSIONS: These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3ß. Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3ß phosphorylation.

Improved accessibility of emergency obstetrics and newborn care (EmONC) services for maternal and newborn health: a community based project.

BACKGROUND: Every year an estimated three million neonates die globally and two hundred thousand of these deaths occur in Pakistan. Majority of these neonates die in rural areas of underdeveloped countries from preventable causes (infections, complications related to low birth weight and prematurity). Similarly about three hundred thousand mother died in 2010 and Pakistan is among ten countries where sixty percent burden of these deaths is concentrated. Maternal and neonatal mortality remain to be unacceptably high in Pakistan especially in rural areas where more than half of births occur. METHOD/DESIGN: This community based cluster randomized controlled trial will evaluate the impact of an Emergency Obstetric and Newborn Care (EmONC) package in the intervention arm compared to standard of care in control arm. Perinatal and neonatal mortality are primary outcome measure for this trial. The trial will be implemented in 20 clusters (Union councils) of District Rahimyar Khan, Pakistan. The EmONC package consists of provision of maternal and neonatal health pack (clean delivery kit, emollient, chlorhexidine) for safe motherhood and newborn wellbeing and training of community level and facility based health care providers with emphasis on referral of complicated cases to nearest public health facilities and community mobilization. DISCUSSION: Even though there is substantial evidence in support of effectiveness of various health interventions for improving maternal, neonatal and child health. Reduction in perinatal and neonatal mortality remains a big challenge in resource constrained and diverse countries like Pakistan and achieving MDG 4 and 5 appears to be a distant reality. A comprehensive package of community based low cost interventions along the continuum of care tailored according to the socio cultural environment coupled with existing health force capacity building may result in improving the maternal and neonatal outcomes. The findings of this proposed community based trial will provide sufficient evidence on feasibility, acceptability and effectiveness to the policy makers for replicating and scaling up the interventions within the health system.

Ameloblastic fibroma: A case report.

Ameloblastic fibroma is a rare mixed odontogenic benign tumor that can occur in either mandible or maxilla but mostly it is found in posterior region of mandible. It can present either peripherally or centrally with a majority of the cases predominantly occurring in first two decades of life and mostly affects male patients. It is characterized by epithelial islands and cords submerged in ectomesenchyme that bear resemblance to the dental papilla and enamel organ but without actual hard tissue formation. Ameloblastic fibroma is a rare odontogenic tumor consisting of neoplastic epithelial and mesenchymal tissues. Recent reports have suggested that this lesion has the potential for high recurrence (18%) and greater chances of recurrent Ameloblastic fibroma transforming into Ameloblastic fibrosarcoma (45%). A 34-year-old male patient presented with pain and swelling in right mandibular posterior region. Intraorally expansion of buccal cortical plate with tenderness over swelling was present. Extraoral examination revealed facial asymmetry on right side. In view of imaging and clinical findings, provisional diagnosis of Odontogenic Keratocyst or Recurrent Ameloblastoma was considered. After obtaining informed consent and general systemic evaluation, the lesion was enucleated under general anesthesia and biopsied which confirmed the diagnosis of Ameloblastic fibroma. Ameloblastic fibroma is a mixed odontogenic tumor composed of odontogenic ectomesenchyme resembling dental papilla with epithelial strands and nests similar to the dental lamina and enamel organ, but with no dental hard tissue formation. Odontogenic tumors, Ameloblasts, Ameloblastoma, Jaw neoplasm.

Angiofibroma of the mandible: Rare sight for juvenile tumor.

Angiofibroma also called juvenile nasopharyngeal angiofibroma are tumors of adolescence and the commonest site is the nasopharynx. Extra nasopharyngeal sites include upper respiratory and digestive tracts, oral cavity, tonsils, larynx, trachea, and esophagus. Intraosseous angiofibroma is the rarest of a rare entity.

SAR Probing of KX2-391 Provided Analogues With Juxtaposed Activity Profile Against Major Oncogenic Kinases.

Tirbanibulin (KX2-391, KX-01), a dual non-ATP (substrate site) Src kinase and tubulin-polymerization inhibitor, demonstrated a universal anti-cancer activity for variety of cancer types. The notion that KX2-391 is a highly selective Src kinase inhibitor have been challenged by recent reports on the activities of this drug against FLT3-ITD mutations in some leukemic cell lines. Therefore, we hypothesized that analogues of KX2-391 may inhibit oncogenic kinases other than Src. A set of 4-aroylaminophenyl-N-benzylacetamides were synthesized and found to be more active against leukemia cell lines compared to solid tumor cell lines. N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-4-chlorobenzamide (4e) exhibited activities at IC50 0.96 µM, 1.62 µM, 1.90 µM and 4.23 µM against NB4, HL60, MV4-11 and K562 leukemia cell lines, respectively. We found that underlying mechanisms of 4e did not include tubulin polymerization or Src inhibition. Such results interestingly suggested that scaffold-hopping of KX2-391 may change the two main underlying cytotoxic mechanisms (Src and tubulin). Kinase profiling using two methods revealed that 4e significantly reduces the activities of some other potent oncogenic kinases like the MAPK member ERK1/2 (>99%) and it also greatly upregulates the pro-apoptotic c-Jun kinase (84%). This research also underscores the importance of thorough investigation of total kinase activities as part of the structure-activity relationship studies.

Performance of the Sulcoflex piggyback intraocular lens in pseudophakic patients.

PURPOSE: To present our experience with a pseudophakic piggyback intraocular lens (IOL) (Sulcoflex; Rayner Intraocular Lenses Ltd) in five eyes of four patients. One patient desired increased spectacle independence after bilateral LASIK and refractive lens exchange with an accommodating IOL. The remaining three patients with residual refractive error desired increased spectacle independence following cataract surgery with a monofocal IOL. METHODS: Four eyes received a multifocal Sulcoflex IOL and one eye received a toric Sulcoflex IOL. RESULTS: All patients achieved uncorrected distance visual acuity (UDVA) of 0.1 logMAR (Snellen 20/25) or better and those who received the multifocal Sulcoflex achieved uncorrected near visual acuity (UNVA) of N6 (Jaeger 4) or better. CONCLUSIONS: The Sulcoflex IOL may be a safe and effective method for enhancing the refractive outcome in pseudophakic eyes, providing good UDVA and UNVA when using the multifocal platform.

Studies on chemical and sensory parameters of coconut oil and its olein blends with sesame oil and palmolein during wheat flour-based product frying.

Blends of coconut oil-coconut oil with sesame oil (blend 1); coconut olein with sesame oil (blend 2); coconut olein with palmolein (blend 3) in 1:1 (v/v) ratio-were used in this study for frying Poori, a traditional Indian fast food prepared from wheat flour. Changes in oil quality were determined by chemical and sensory methods. Free fatty acid content did not change whereas peroxide value increased. Anisidine value increased from 5.5, 0.9 and 4.2 to 34.3, 42.8 and 23.6 for blends 1, 2 and 3, respectively. Iodine value showed marginal decrease in blends 1 and 2. Diene value showed no change in all three blends. Sesamol content in blends 1 and 2, total tocopherols in all the three blends, and ß-carotene content in blend 3 decreased after frying. The blends showed a significant decrease (P ≤ 0.05) in the characteristic coconut oil odour after frying. Blend 3 showed comparatively better frying stability and also overall sensory quality of poori fried in this blend was the highest.

Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1H-Imidazol-5-One Variants.

Structural changes of small-molecule drugs may bring interesting biological properties, especially in the field of kinase inhibitors. We sought to study tirbanibulin, a first-in-class dual Src kinase (non-ATP competitive)/tubulin inhibitor because there was not enough reporting about its structure-activity relationships (SARs). In particular, the present research is based on the replacement of the outer ring of the biphenyl system of 2-[(1,1'-biphenyl)-4-yl]-N-benzylacetamide, the identified pharmacophore of KX chemotype, with a heterocyclic ring. The newly synthesized compounds showed a range of activities in cell-based anticancer assays, agreeing with a clear SAR profile. The most potent compound, (Z)-N-benzyl-4-[4-(4-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl]phenylacetamide (KIM-161), demonstrated cytotoxic IC50 values at 294 and 362 nM against HCT116 colon cancer and HL60 leukemia cell lines, respectively. Profiling of this compound (aqueous solubility, liver microsomal stability, cytochrome P450 inhibition, reactivity with reduced glutathione, and plasma protein binding) confirmed its adequate drug-like properties. Mechanistic studies revealed that this compound does not depend on tubulin or Src kinase inhibition as a factor in forcing HL60 to exit its cell cycle and undergo apoptosis. Instead, KIM-161 downregulated several other kinases such as members of BRK, FLT, and JAK families. It also strongly suppresses signals of ERK1/2, GSK-3α/ß, HSP27, and STAT2, while it downregulated AMPKα1 phosphorylation within the HL60 cells. Collectively, these results suggest that phenylacetamide-1H-imidazol-5-one (KIM-161) could be a promising lead compound for further clinical anticancer drug development.

Hexosamine biosynthesis pathway flux promotes endoplasmic reticulum stress, lipid accumulation, and inflammatory gene expression in hepatic cells.

There is increasing evidence that endoplasmic reticulum (ER) stress contributes to the development of atherosclerosis in diabetes mellitus. The purpose of this study was to determine the effects of increased hexosamine biosynthesis pathway (HBP) flux on ER stress levels and the complications of ER stress associated with diabetes and atherosclerosis in hepatic cells. Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, was overexpressed in HepG2 cells by use of an adenoviral expression system. The ER stress response and downstream effects, including activation of lipid and inflammatory pathways, were determined using real-time PCR, immunoblot analysis, and cell staining techniques. GFAT overexpression resulted in increased expression of ER stress markers, including Grp78, Grp94, calreticulin, and GADD153, relative to cells infected with an empty adenoviral vector. In addition, GFAT overexpression promoted lipid, but not cholesterol, accumulation in hepatic cells as well as inflammatory pathway activation. Treatment with 6-diazo-5-oxo-norleucine, a GFAT antagonist, blocked the effects of GFAT overexpression. Consistent with our in vitro data, hyperglycemic mice presented with elevated markers of hepatic ER stress, glucosamine and lipid accumulation. Together, these data suggest that HBP flux-induced ER stress plays a role in the development of hepatic steatosis and atherosclerosis under conditions of hyperglycemia.

Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.

We have previously shown that glucosamine promotes endoplasmic reticulum (ER) stress in vascular cells leading to both inflammation and lipid accumulation--the hallmark features of atherosclerosis. Pretreatment with glycogen synthase kinase (GSK)-3 inhibitors protects cultured cells from ER stress-induced dysfunction. Here we evaluate the potential role of GSK-3 on the pro-atherogenic effects of hyperglycemia and ER stress. We show that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol under conditions of ER stress. Furthermore, GSK-3 inhibitors, including valproate, attenuate ER stress-induced unesterified cholesterol accumulation in wild-type mouse embryonic fibroblasts. In vivo we show that hyperglycemic apoE-deficient mice have accelerated atherogenesis at the aortic root compared with normoglycemic control mice. Mice fed a diet supplemented with 625 mg/kg valproate have significantly reduced lesion volume relative to nonsupplemented controls. Valproate supplementation has no apparent effect on the plasma levels of either glucose or lipids or on the expression of diagnostic markers of ER stress in the lesion. Significant reductions were observed in total hepatic lipids (>50.4%) and hepatic GSK-3beta activity (>55.8%) in mice fed the valproate diet. In conclusion, dietary supplementation with low levels of valproate significantly attenuates atherogenesis in hyperglycemic apoE-deficient mice. The in vivo anti-atherogenic effects of valproate are consistent with its ability to inhibit GSK-3 and interfere with pro-atherogenic ER stress signaling pathways in vitro.

Sharp injuries and their determinants among health care workers at first-level care facilities in Sindh Province, Pakistan.

SUMMARY OBJECTIVES: To assess the rate and determinants of sharp injuries during the previous 6 months among health care workers at first-level care facilities in two districts of Pakistan. METHODS: Cross-sectional survey at public, general practitioners and non-licensed private practitioners selected through stratified random sampling. At each facility, we interviewed a prescriber and a dispenser/injection provider about knowledge of bloodborne pathogens transmission and preventive practices, risk perception, and use of precautions and sharp injuries received during the previous 6 months. Multivariable Poisson regression was used to assess the factors associated with the number of sharp injuries. RESULTS: Fifty-four percentage of the 233 workers had at least one injury during the previous 6 months. The overall rate of sharp injuries per person per year was 3.7; among non-physician prescribers (9%), it was 4.3; among dispensers (69%), it was 3.7, and among physicians (18%), it was 2.1. In the multivariable model, work experience, risk perception and type of health care worker were significantly associated with receiving sharp injuries during the previous 6 months. In the model including dispensers only, a higher knowledge score was associated with fewer sharp injuries, while perceived severity of disease and lack of professional qualification were associated with more. CONCLUSIONS: Sharp injuries are common in Pakistan. Better knowledge about modes of bloodborne pathogen transmission and professional qualification may reduce their incidence.

Glucosamine-induced endoplasmic reticulum dysfunction is associated with accelerated atherosclerosis in a hyperglycemic mouse model.

Diabetes is a major independent risk factor for cardiovascular disease and stroke; however, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. Our previous studies demonstrated that endoplasmic reticulum (ER) stress-inducing agents, including homocysteine, promote lipid accumulation and activate inflammatory pathways-the hallmark features of atherosclerosis. We hypothesize that the accumulation of intracellular glucosamine observed in diabetes may also promote atherogenesis via a mechanism that involves ER stress. In support of this theory, we demonstrate that glucosamine can induce ER stress in cell types relevant to the development of atherosclerosis, including human aortic smooth muscle cells, monocytes, and hepatocytes. Furthermore, we show that glucosamine-induced ER stress dysregulates lipid metabolism, leading to the accumulation of cholesterol in cultured cells. To examine the relevance of the ER stress pathway in vivo, we used a streptozotocin-induced hyperglycemic apolipoprotein E-deficient mouse model of atherosclerosis. Using molecular biological and histological techniques, we show that hyperglycemia is associated with tissue-specific ER stress, hepatic steatosis, and accelerated atherosclerosis. This novel mechanism may not only explain how diabetes and hyperglycemia promote atherosclerosis, but also provide a potential new target for therapeutic intervention.

Drugs - Do we need them? Applications of non-pharmaceutical therapy in anterior eye disease: A review.

Natural products have been in use long before the introduction of modern drug therapies and are still used in various communities worldwide for the treatment of anterior eye disease. The aim of this review is to look at the current non-pharmaceutical modalities that have been tried and assess the body of existing evidence behind them. This includes alternative medicine, existing non-pharmaceutical therapy and more recent low and high tech solutions. A detailed search of all available databases including MEDLINE, Pubmed and Google was made to look for English-language studies for complementary and alternative treatment modalities (CAM), natural therapies and new modalities for anterior eye disease such as blepharitis, dry eye and microbial keratitis. We have included a broad discussion ranging from traditional treatments like honey and aloe vera which have been used for centuries, to the more recent technological advances like Intense Pulsed Light (IPL), LipiFlow and photoactivated chromophore for corneal cross linking in infectious keratitis (PACK-CXL). Alternative management strategies may have a role in anterior eye diseases and have a potential in changing the way we currently approach them. Some of the available CAM could play a role if incorporated in to current management practices of not only chronic diseases like blepharitis and dry eye, but also acute conditions with significant morbidity like microbial keratitis. Further large-scale randomized control trials stratified by disease severity are required to improve our understanding and to evaluate the use of non-pharmaceutical therapy against current practice.

Pattern of health care utilization and determinants of care-seeking from GPs in two districts of Pakistan.

The aims of the study were to describe the pattern of health care utilization and out-of-pocket expenses incurred in seeking health care, and to identify the determinants of care-seeking from private general practitioners (GP) in two districts of Pakistan. During July-September 2001, we conducted a cross-sectional study in two districts in the Sindh Province of Pakistan. We selected 1,150 participants age > or = 3 months through a two-stage cluster sampling technique. Information was collected about contacts with healthcare providers during the past three months, presenting complaints, type of treatment received, and cost of the latest visit. Of 1,150 participants, 967 (84%) had at least one contact with health care providers during past three months. The mean number of contacts was 1.7. Most of the contacts (66.8%) were with private GPs. The average cost per visit was Pak Rs 106 (US dollar 1.7) and Rs 38 (US dollar 0.6) for GPs and public sector providers, respectively. A multiple logistic regression model revealed those living in urban areas, with monthly household income > Rs 2,500 (US dollar 39.7), an education level > 5 years, and who received both injections and oral drugs were more likely to visit private general practitioners.