Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 306.1 → 233.0) and IS (m/z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg (E)-entacapone tablet formulation under fasting conditions.     

Mass spectrometry‐compatible silver staining of histones resolved on acetic acid‐urea‐Triton PAGE

Acetic acid‐Urea‐Triton (AUT) PAGE is commonly used method to separate histone variants and their post‐translationally modified forms. Coomassie staining is the preferred method for protein visualization; however, its sensitivity is less than that of silver staining. Though silver staining of histones in AUT‐PAGE has been reported, the method is time‐consuming, dependent on prior staining by Amido black and has not been reported suitable for mass spectrometry. Here, we propose ‘SDS‐Silver’ method for rapid, sensitive and mass spectrometry‐compatible staining of histones resolved on AUT‐PAGE.     

Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni

Background  

Background: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.     

Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents

A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC(50) = 5.67 microm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC(50) = 6.1 microm) and oral (IC(50) = 4.17 microm) cancers. These compounds could be of use in designing new anti-cancer agents.     

Vibrio cholerae Cytolysin Causes an Inflammatory Response in Human Intestinal Epithelial Cells That Is Modulated by the PrtV Protease

Background

Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV.

Methodology/Principal Findings

Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-α (TNF-α) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis.

Conclusions/Significance

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.     

Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.     

Informed Consent in Health Research: Challenges and Barriers in Low‐and Middle‐Income Countries with Specific Reference to Nepal

Obtaining ‘informed consent’ from every individual participant involved in health research is a mandatory ethical practice. Informed consent is a process whereby potential participants are genuinely informed about their role, risk and rights before they are enrolled in the study. Thus, ethics committees in most countries require ‘informed consent form’ as part of an ethics application which is reviewed before granting research ethics approval. Despite a significant increase in health research activity in low‐and middle‐income countries (LMICs) in recent years, only limited work has been done to address ethical concerns. Most ethics committees in LMICs lack the authority and/or the capacity to monitor research in the field. This is important since not all research, particularly in LMICs region, complies with ethical principles, sometimes this is inadvertently or due to a lack of awareness of their importance in assuring proper research governance. With several examples from Nepal, this paper reflects on the steps required to obtain informed consents and highlights some of the major challenges and barriers to seeking informed consent from research participants. At the end of this paper, we also offer some recommendations around how can we can promote and implement optimal informed consent taking process. We believe that paper is useful for researchers and members of ethical review boards in highlighting key issues around informed consent.