Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM.

Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production.

Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man.

Neutralization by insulin of the hypertensive effect of dermcidin isoform 2: an environmentally induced diabetogenic and hypertensive protein.

The effect of dermcidin isoform 2 (dermcidin), an environmentally induced stress protein, was investigated on the genesis of diabetes mellitus and hypertension, the two major atherosclerotic risk factors. The role of dermcidin as an atherosclerotic risk factor related to the impaired systemic insulin level was investigated. Dermcidin was prepared by electrophoresis using plasma from the subjects with acute ischemic heart disease. Injection of 0.2 µM dermcidin in mice increased the blood glucose level from 98 ± 2.45 mg/dL to 350 ± 10.2 mg/dL which was normalized by the oral administration of acetyl salicylic acid (aspirin) after 24 h. Hypertensive subjects with systolic and diastolic blood pressure of 165 mm and 95 mm of Hg, respectively, had plasma dermcidin level of 95 nM. Ingestion of acetyl salicylic acid (aspirin) (150 mg/70 kg body weight) decreased the systolic and diastolic pressures to 125 mm and 80 mm of Hg, respectively, with decrease of dermcidin level to 15 nM. Incubation of kidney cortex cells with 0.2 µM dermcidin-inhibited synthesis of (r)-cortexin, an antihypertensive protein, and the basal (r)-cortexin level was reduced from 33 nM to 15 nM. Addition of 25 µunits of insulin/mL was found to reverse the inhibition of cortexin synthesis. The effect of dermcidin as a diabetogenic and a hypertensive agent could be controlled either by aspirin or by insulin.

Reduction of death rate due to acute myocardial infarction in subjects with cancers through systemic restoration of impaired nitric oxide.

INTRODUCTION: Excessive aggregation of platelets at the site of plaque rupture on the coronary artery led to the formation of thrombus which is reported to precipitate acute myocardial infarction (AMI). Nitric oxide (NO) has been reported to inhibit platelet aggregation and induce thrombolysis through the in situ formation of plasmin. As the plasma NO level in AMI patients from two different ethnic groups was reduced to 0 µM (median) compared to 4.0 µM (median) in normal controls, the effect of restoration of the NO level to normal ranges on the rate of death due to AMI was determined. METHODS AND RESULTS: The restoration of plasma NO level was achieved by a sticking small cotton pad (10×25 mm) containing 0.28 mmol sodium nitroprusside (SNP) in 0.9% NaCl to the abdominal skin of the participants using non-toxic adhesive tape which was reported to normalize the plasma NO level. The participants (8,283) were volunteers in an independent study who had different kinds of cancers and did not wish to use any conventional therapy for their condition but opted to receive SNP "pad" for their condition for 3 years. The use of SNP "pad" which normalized (≈4.0 µM) the plasma NO level that in consequence reduced the death rate due to AMI, among the participants, was found to be significantly reduced compared to the death due to AMI in normal population. CONCLUSION: Our data suggested that the use of SNP "pad" significantly reduced the death due to AMI. TRIAL REGISTRATION: www.ctri.nic.in CTRI/2013/12/004236.

Early clinical outcomes of primary percutaneous coronary intervention in bharatpur, Nepal.

BACKGROUND: Primary percutaneous coronary intervention represents one of the cornerstone management modalities for patients with acute ST-elevation myocardial infarction and has undergone tremendous growth over the past two decades. This study was aimed to determine the early clinical outcomes of primary percutaneous coronary interventions in a tertiary-level teaching hospital without onsite cardiac surgery backup. METHODS: This was a prospective descriptive study which included all consecutive patients who were admitted for primary percutaneous coronary interventions between March 2011 and January 2013 at the College of Medical Sciences and Teaching Hospital, Bharatpur, Nepal. Total 68 patients underwent primary percutaneous coronary interventions as a mode of revascularization. The primary end point of the study was to identify in-hospital as well as 30-day clinical outcomes of primary percutaneous coronary interventions. RESULTS: The mean age was 56.31 ± 11.47 years, with age range of 32 years to 91 years. Of the 68 primary percutaneous coronary interventions performed, 15 (22.05%) were carried out in women and 10 (14.70%) in patients over 75 years of age. Primary percutaneous coronary intervention for anterior wall myocardial infarction was more common than for non-anterior wall myocardial infarction (55.88% vs. 44.12%). Proximal artery stenting was performed in 38.50% and the non proximal artery stenting in 61.50%. The outcomes were mortality (5.88%), cardiogenic shock (5.88%), contrast-induced nephropathy requiring dialysis (2.94%), arrhythmias requiring treatment (4.41%), early stent thrombosis (2.94%) and minor complications (14.70%). CONCLUSION: Primary percutaneous coronary intervention improves the early clinical outcomes in patient with acute ST-elevation myocardial infarction. Despite having no onsite cardiac surgery backup, primary percutaneous coronary intervention was feasible with acceptable complications in a tertiary-care teaching hospital.

Diagnosis of essential hypertension in humans by the determination of plasma renal cortexin using enzyme-linked immunosorbent assay.

BACKGROUND: More than 90% of all hypertensive persons are reported to have essential hypertension (EH), a particular form of elevated blood pressure, for which no diagnostic test is currently available. The level of plasma renal (R) cortexin (PRC), a hypotensive protein produced in the kidney cortex cells, was reported to be reduced from 218 nM in the plasma of normotensive persons (NP) to 0 nM in the plasma of patients with EH. The feasibility of using the determination of PRC by enzyme-linked immunosorbent assay (ELISA) as a diagnostic test for EH was investigated. METHODS: The PRC was determined by ELISA using electrophoretically pure cortexin as the antigen. A total of 344 persons (male and female) with EH, with or without diabetes mellitus (DM), and receiving or not receiving any anti-hypertensive and/or anti-diabetic medication at presentation, as well as an equal number of age- and gender-matched NP participated in the study. RESULTS: All persons with EH, with or without co-existing DM, were found to have 0 nM PRC, regardless of whether they were receiving anti-hypertensive and anti-diabetic drugs, including those who had been taking these medications over an extended period of time (3 months). CONCLUSIONS: The determination of PRC as a marker protein by ELISA, a rapid method that can be carried out in any diagnostic laboratory, was shown to be suitable for the diagnosis of EH, even in those subjects who had co-existing DM and were receiving both anti-hypertensive and anti-diabetic medication.

Percutaneous coronary intervention without onsite cardiac surgery backup.

INTRODUCTION: Mechanical revascularization by percutaneous coronary interventions has now become an established and preferable method of revascularization in patients with acute coronary syndromes. The aim of the study was to identify the clinical indications for percutaneous coronary interventions and in-hospital outcomes of percutaneous coronary interventions in a tertiary-level hospital without onsite cardiac surgery backup. METHODS: This was a prospective descriptive study. All consecutive patients who were admitted for percutaneous coronary interventions, including both primary as well as elective percutaneous coronary interventions, between March 2011 and December 2012 were included in the study. RESULTS: Total 101 percutaneous coronary interventions were performed. The mean age was 58.9 ± 12.3 years. The most frequent indication was ST-elevation myocardial infarction 72 (71.3%). Proximal artery stenting were performed in 39 (38.5%) and the non proximal artery stenting in 62 (61.5%). The outcomes were mortality 5 (4.9%), periprocedural myocardial infarction 2 (1.9%), cardiogenic shock 6 (5.9%), contrast induced nephropathy requiring dialysis in 3 (2.9%), minor complications which were managed conservatively in 13 (12.9%). CONCLUSIONS: Percutaneous coronary intervention was feasible with acceptable complications in a tertiary-level hospital without onsite cardiac surgery backup. ST-elevation myocardial infarction was the major indication and cardiogenic shock was the major complication observed, and non proximal artery stenting was more common than the proximal artery stenting.

The role of dermcidin isoform 2: a two-faceted atherosclerotic risk factor for coronary artery disease and the effect of acetyl salicylic Acid on it.

Hypertension and diabetes mellitus are considered to be two major atherosclerotic risk factors for coronary artery disease (CAD). A stress-induced protein identified to be dermcidin isoform 2 of Mr. 11 kDa from blood plasma of hypertensive persons when injected (0.1 µM) in rabbits increased the systolic pressure by 77% and diastolic pressure by 45% over the controls within 2 h. Ingestion of acetyl salicylic acid (150 mg/70 kg) by these subjects reduced systolic (130 mm Hg) and diastolic pressures (80 mm Hg) with reduction of plasma dermcidin level to normal ranges (9 nM). The protein was found to be a potent activator of platelet cyclooxygenase and inhibited insulin synthesis. Aspirin was found to reduce hypertension by reduction of plasma dermcidin level, neutralized the effect of cyclooxygenase, and restored the pancreatic insulin synthesis through NO synthesis. These results indicated that dermcidin could be a novel atherosclerotic risk factor for its hypertensive and diabetogenic effects.

The Control of Stress Induced Type I Diabetes Mellitus in Humans through the Hepatic Synthesis of Insulin by the Stimulation of Nitric Oxide Production.

The role of stress induced development of Type-1 diabetes mellitus (T1DM) as opposed to autoimmunity remains obscure. It has been reported that a stress induced protein, identified to be dermcidin isoform 2 (dermcidin) inhibited insulin synthesis in both the pancreatic ß cells and the hepatic cells. As dermcidin effect could be neutralized by the increased production of systemic nitric oxide (NO), investigations were carried out to determine the feasibility of controlling stress induced T1DM through the neutralization of dermcidin by systemic increase of NO. To determine the role of plasma dermcidin level in T1DM subjects (n=45), if any, when the plasma dermcidin level were determined, it was found that the protein level was increased in 65% of the participating volunteers. Efforts were made to normalize the plasma glucose level (median=175 mg/dL) in these T1DM subjects by systemic increase of NO by applying a cotton pad containing 0.28mmol sodium nitroprusside on the abdominal skin. It was found that the systemic increase of NO level decreased the blood glucose level of 275 mg/dL (median) to 115 mg/dL (median) in these volunteers within 24 h with concomitant increase of plasma insulin level from 7.5 µunits/dL to 101 µunits/dL at the same time. The increase of plasma insulin level was accompanied by the decrease of dermcidin level of 124.5 nM to 18 nM with increase of NO from 0.43 ± 0.19 nM to 4.1 ± 1.56 nM. The results suggested that the stress induced T1DM by dermcidin could be controlled by the systemic increase of NO which in consequence led to increased synthesis of insulin.

The appearance of dermcidin isoform 2, a novel platelet aggregating agent in the circulation in acute myocardial infarction that inhibits insulin synthesis and the restoration by acetyl salicylic acid of its effects.

Hyperglycemia with severe reduction of plasma insulin level is frequently associated with acute ischemic heart disease. Since insulin is reported to be an anti thrombotic humoral factor, the mechanism of the impaired insulin synthesis was investigated. The plasma from the patients with acute myocardial infarction (AMI) was analyzed by SDS-polyacrylamide gel electrophoresis. Dermcidin isoform 2 (dermcidin) was determined by enzyme linked immunosorbent assay. Insulin synthesis was determined by in vitro translation of glucose induced insulin mRNA synthesis in the pancreatic ß cells. Nitric oxide (NO) was determined by methemoglobin method. SDS-polyacrylamide gel electrophoresis of AMI plasma demonstrated the presence of a novel protein band of Mr 11 kDa that was determined to be dermcidin. Addition of 0.1 µM dermcidin inhibited insulin synthesis by >65 fold compared to control through the inhibition of NO synthesis in the pancreatic cells. The oral administration of 150 mg acetyl salicylic acid (aspirin) to the AMI patients increased the plasma insulin level from 13 (median) to 143 µunits/dl (median) with concomitant decrease of plasma dermcidin level from 112 to 9 nM in these patients within 12 h. It was also found that while the injection of 3.0 ± 0.05 (n = 10) nmol dermcidin with 0.25 ± 0.03 µmol ADP/g body weight caused coronary thrombus in mice, ADP itself at this concentration failed to produce thrombus. These results indicated that dermcidin was a novel platelet aggregating agent, and potentiated the ADP induced thrombosis in the animal model as well as acutely inhibited glucose induced insulin synthesis.

Prevalence of overweight and hypertension among rural and urban school going children and association of hypertension with overweight.

Overweight is a burning problem of school going children especially in affluent society. So urban children are affected more. Hypertension is a major co-morbidity of overweight. The number of overweight and hypertensive children is increasing and more among urban children. In this study sample was taken from rural and urban school going children of various age groups. The prevalence of overweight and hypertension among rural and urban school going children was obtained and relation between overweight and hypertension was established.

Echocardiographic profile of ART naïve human immunodeficiency virus (HIV) infected patients in a tertiary care hospital in Kolkata.

AIMS AND OBJECTIVE: Cardiac affection in human iummunodeficiency virus (HIV) infection is a recognized entity. Some form of heart disease is demonstrable at autopsy in approximately 40 percent of cases and by echocardiography in approximately 25 percent of patients with HIV. the studies indicate that cardiac involvements associated with HIV are mainly characterized by cardiomyopathy and pericardial disease. HIV infection is a global pandemic which is also rapidly spreading in india. We conducted the study to have some insight into the profile oflndian patients. MATERIAL & METHODS: In this cross sectional hospital based study, we evaluated immunological (CD4 count) and echocardiographic status of 45 asymptomatic HIV seropositive patients who did not receive anti-retroviral therapy. The results were compared with age and sex matched controls. Statistical analysis was done using appropriate statistical methods. RESULTS: Most common cardiovascular abnormalities were diastolic dysfunction (18%) followed by pericardial effusion (13%) and systolic dysfunction (7%). When compared with controls the study population had statistically higher number of diastolic dysfunction (p value = 0.035) but not systolic dysfunction (p value = 0.61); none of the control population was having pericardial effusion. Low CD4 count was significantly associated with pericardial effusion (p value 0.048) but the association with diastolic dysfunction (p value = 0.46) or systolic dysfunction (p value = 0.84) was not statistically significant. CONCLUSION: Cardiovascular complications are common among HIV infected patients in india, most common being diastolic dysfunction and pericardial effusion. Low CD4 counts are associated significantly with pericardial effusion. These abnormalities are likely to be found with greater frequency in clinical practice as management of opportunistic infections continues to improve.

Deep vein thrombosis.

Occlusive clot formation in the veins causes venous thrombosis, the site most common in the deep veins of leg, called deep vein thrombosis. The clot can block blood flow and when it breaks off, called an embolism which in turn can damage the vital organs. Venous thrombosis occurs via three mechanisms ie, Virchow's triad. The mechanisms are decreased flow rate of blood, damage to the blood vessel wall and an increased tendency of the blood to clot. There are several factors which can increase a person's risk for deep vein thrombosis. The symptoms of deep vein thrombosis in the legs are pain, swelling and redness of the part. One variety of venous thrombosis is phlegmasia alba dolens where the leg becomes pale and cool. Investigations include Doppler ultrasound examination of the limb, D-dimer blood test, plethysmography of the legs, x-rays to show vein in the affected area (venography). Hospitalisation is necessary in some cases with some risk factors. The mainstream of treatment is with anticoagulants, mostly low molecular weight heparin for 6 months. Deep venous thrombosis is a rising problem. Early diagnosis and treatment is associated with a good prognosis.

The role of leucocytes in the acetyl salicylic acid (aspirin) induced nitric oxide synthesis in the production of interferon-alpha, a potent inhibitor of platelet aggregation and a thrombolytic agent.

The role of aspirin-induced NO synthesis in the production of interferon-alpha (IFN-alpha) in leucocytes and the effect of IFN-alpha on platelet aggregation was studied. Treatment of Platelet Rich Plasma (PRP) with the dialyzed supernatant from the leucocyte suspension incubated with 80 microM aspirin resulted in parallel syntheses of NO and IFN-alpha as determined by methemoglobin assay and enzyme linked immunosorbent assay respectively. Incubation of PRP with 10 nM purified IFN-alpha for 40 min resulted in the maximal inhibition of platelet aggregation through the synthesis of NO due to the activation of nitric oxide synthase in platelets by IFN-alpha. The treatment of clotted PRP with IFN-alpha resulted in the lysis of the clot due to the fibrinolysis. Injection of IFN-alpha was found to protect mice from death due to the lysis of ADP-induced coronary thrombus. Interferon-alpha was found to be a potent inhibitor of platelet aggregation and a thromboprotective agent.

Purification and mechanism of action of "cortexin," a novel antihypertensive protein hormone from kidney and its role in essential hypertension in men.

Because kidney tissue damage is associated with both hypertension and impaired nitric oxide (NO) production, we investigated the possibility whether the kidney tissue contains any activator of endothelial NO synthase (eNOS) that could be important in essential hypertension. An activator protein of M(r) 43000 Da for eNOS from the goat kidney cortex homogenate was purified to homogeneity by chromatographic techniques. This activator trivially, called "cortexin," was determined by enzyme-linked immunosorbent assay using anticortexin antibody. NO was determined by the formation of methemoglobin. Injection of 0.5 nmol cortexin/kg body weight to rabbit pretreated with l-epinephrine that increased the systolic and diastolic pressures to 195 +/- 3.40 mm Hg and 98.14 +/- 6.64 mm Hg, respectively, reduced and kept the elevated pressures at normal ranges of 133.57 +/- 12.14 (systolic) and 51.03 +/- 3.21 (diastolic) for 45 hours with simultaneous increase of plasma NO level. The inhibition of cortexin-induced NO synthesis nullified the antihypertensive effect of cortexin. The plasma cortexin level in newly diagnosed persons with essential hypertension was 0 pmol/mL (median), which contrasted with 218.94 pmol cortexin/mL (median), in normotensive persons (P < .0005; n = 25). We concluded that the impaired production of cortexin in the cortex of kidney might lead to essential hypertension.

Molecular signatures of cell cycle transcripts in the pathogenesis of Glial tumors.

BACKGROUND: Astrocytic brain tumors are among the most lethal and morbid tumors of adults, often occurring during the prime of life. These tumors form an interesting group of cancer to understand the molecular mechanism of pathogenesis. Histological grading of Astrocytoma based on WHO classification does not provide complete information on the proliferation potential and biological behavior of the tumors. It is known that cancer results from the disruption of the orderly regulated cycle of replication and division. In the present study, we made an attempt to identify the cell cycle signatures and their involvement in the clinical aggressiveness of gliomas. METHODS: The variation in expression of various cell cycle genes was studied in different stages of glial tumor progression (low and high grades), and the results were compared with their corresponding expression levels in the normal brain tissue. Macroarray analysis was used for the purpose. RESULTS: Macroarray analysis of 114 cell cycle genes in different grades of glioma indicated differential expression pattern in 34% of the gene transcripts, when compared to the normal tissue. Majority of the transcripts belong to the intracellular kinase networks, cell cycle regulating kinases, transcription factors and transcription activators. CONCLUSION: Based on the observation in the expression pattern in low grade and high grade gliomas, it can be suggested that the upregulation of cell cycle activators are seen as an early event in glioma; however, in malignancy it is not the cell cycle activators alone, which are involved in tumorigenesis. Understanding the molecular details of cell cycle regulation and checkpoint abnormalities in cancer could offer an insight into potential therapeutic strategies.