Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl2 was assessed and compared with the conventional method using the FeCl3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl3, due to Mn2+ ions present in the supernatant. Addition of Mn2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl3 reagents showed that Mn2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl2 supernatant was treated with FeCl3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.     

Production of a thermophilic,extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl₂. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India     

Is Sustained Virological Response a Marker of Treatment Efficacy in Patients with Chronic Hepatitis C Viral Infection with No Response or Relapse to Previous Antiviral Intervention?

Background

Randomised clinical trials (RCTs) of antiviral interventions in patients with chronic hepatitis C virus (HCV) infection use sustained virological response (SVR) as the main outcome. There is sparse information on long-term mortality from RCTs.

Methods

We created a decision tree model based on a Cochrane systematic review on interferon retreatment for patients who did not respond to initial therapy or who relapsed following SVR. Extrapolating data to 20 years, we modelled the outcome from three scenarios: (1) observed medium-term (5 year) annual mortality rates continue to the long term (20 years); (2) long-term annual mortality in retreatment responders falls to that of the general population while retreatment non-responders continue at the medium-term mortality; (3) long-term annual mortality in retreatment non-responders is the same as control group non-responders (i.e., the increased treatment-related medium mortality “wears off”).

Results

The mean differences in life expectancy over 20 years with interferon versus control in the first, second, and third scenarios were -0.34 years (95% confidence interval (CI) -0.71 to 0.03), -0.23 years (95% CI -0.69 to 0.24), and -0.01 (95% CI -0.3 to 0.27), respectively. The life expectancy was always lower in the interferon group than in the control group in scenario 1. In scenario 3, the interferon group had a longer life expectancy than the control group only when more than 7% in the interferon group achieved SVR.

Conclusions

SVR may be a good prognostic marker but does not seem to be a valid surrogate marker for assessing HCV treatment efficacy of interferon retreatment. The SVR threshold at which retreatment increases life expectancy may be different for different drugs depending upon the adverse event profile and treatment efficacy. This has to be determined for each drug by RCTs and appropriate modelling before SVR can be accepted as a surrogate marker.     

Endothelial nitric oxide synthase gene polymorphisms and the risk of acute myocardial infarction in a South Indian population

Myocardial infarction (MI) is a complex multi-factorial, polygenic disorder which results from an interaction between a person’s genetic makeup and various environmental factors. Nitric oxide (NO), a potent vasodilator produced by endothelial cells, plays an important role in the regulation of blood pressure, regional blood flow and also inhibits platelet aggregation, vascular smooth muscle cell proliferation and leukocyte adhesion to vascular endothelium. Our aim was to analyze the association of NOS3 (endothelial nitric oxide synthase 3) 894G>T and ?786T>C gene polymorphisms and MI risk in the South Indian population. A total of 287 MI patients, 279 risk control patients and 321 healthy controls were recruited for the retrospective study. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was no significant association observed between NOS3 894G>T, ?786T>C polymorphisms and MI. A significant difference was observed in the distribution of GT genotype of the NOS3 894G>T polymorphism between the cases and the risk controls (p = 0.05) but the odds ratio (0.6) did not show risk for MI. The present study showed lack of association between NOS3 gene polymorphisms and MI in South Indian population.     

Medical Therapies for Uterine Fibroids – A Systematic Review and Network Meta-Analysis of Randomised Controlled Trials

Background

Uterine fibroids are common, often symptomatic and a third of women need repeated time off work. Consequently 25% to 50% of women with fibroids receive surgical treatment, namely myomectomy or hysterectomy. Hysterectomy is the definitive treatment as fibroids are hormone dependent and frequently recurrent. Medical treatment aims to control symptoms in order to replace or delay surgery. This may improve the outcome of surgery and prevent recurrence.

Purpose

To determine whether any medical treatment can be recommended in the treatment of women with fibroids about to undergo surgery and in those for whom surgery is not planned based on currently available evidence.

Study Selection

Two authors independently identified randomised controlled trials (RCT) of all pharmacological treatments aimed at the treatment of fibroids from a list of references obtained by formal search of MEDLINE, EMBASE, Cochrane library, Science Citation Index, and ClinicalTrials.gov until December 2013.

Data Extraction

Two authors independently extracted data from identified studies.

Data Synthesis

A Bayesian network meta-analysis was performed following the National Institute for Health and Care Excellence—Decision Support Unit guidelines. Odds ratios, rate ratios, or mean differences with 95% credible intervals (CrI) were calculated.

Results and Limitations

A total of 75 RCT met the inclusion criteria, 47 of which were included in the network meta-analysis. The overall quality of evidence was very low. The network meta-analysis showed differing results for different outcomes.

Conclusions

There is currently insufficient evidence to recommend any medical treatment in the management of fibroids. Certain treatments have future promise however further, well designed RCTs are needed.     

Transforming growth factor-beta: a neuroprotective factor in cerebral ischemia

Transforming growth factor-β (TGF-β) has diverse and multiple roles throughout the body. This review focuses on the evidence supporting its functions in the central nervous system, with a particular emphasis on its purported role in cerebral ischemia. Numerous studies have documented that TGF-β1 levels are enhanced in the brain following cerebral ischemia. As evidence that such an upregulation is beneficial, agonist studies have demonstrated that TGF-β1 reduces neuronal cell death and infarct size following middle cerebral artery occlusion (MCAO), while conversely, antagonist studies have shown increased neuronal cell death and infarct size after MCAO. These studies suggest that TGF-β1 has a neuroprotective role in cerebral ischemia. Recent work with adenoviral-mediated overexpression of TGF-β1 in vivo in mice has further implicated a neuroprotective role for TGF-β1 in cerebral ischemia, as evidenced by a reduction in neuronal cell death, infarct size, and neurological outcome. Additionally, numerous in vitro studies have documented the neuroprotective ability of TGF-β1 in neurons from a variety of species, including rats, mice, chicks, and humans. Of significant interest, TGF-β1 was shown to be protective against a wide variety of death-inducing agents/insults, including hypoxia/ischemia, glutamate excitotoxicity, β-amyloid, oxidative damage, and human immunodeficiency virus. The mechanism of TGF-β1-mediated neuroprotection remains to be resolved, but early evidence suggests that TGF-β1 regulates the expression and ratio of apoptotic (Bad) and antiapoptotic proteins (Bcl-2, Bcl-x₁), creating an environment favorable for cell survival of death-inducing insults. Taken as a whole, these results suggest that TGF-β1 is an important neuroprotective factor that can reduce damage from a wide-array of death-inducing agents/insults in vitro, as well as exert protection of the brain during cerebral ischemia. The authors’ research is supported by research grants (HD-28964 and AG-17186 to DWB) from the National Institutes of Health, NICHD, and NIA.     

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.     

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.