Purshia plicata Triggers and Regulates Proteins Related to Apoptosis in HeLa Cancer Cells

Cervical cancer represents a public health problem, develops resistance to traditional therapies and cost-of-treatment is high. These disadvantages have led to the search for alternative bioactive-compound-based therapies. Said bioactive compounds include phenolic compounds, flavonoids, and tannins. The present study aimed to evaluate the therapeutic effect of a P. plicata extract on the HeLa cell line. Viability and apoptosis assays were run on the two cell lines treated with the extract. The peptides, up- and down-expressed in both cell lines, were identified by PDQuest analysis software and high-performance liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS). Our results show that a 500 mg/L treatment deregulated cell viability, with different apoptotic morphologies observed which are associated with the presence of bio-compounds, which up- and down-regulated the peptides. In conclusion, P. plicata regulates proteins associated with apoptosis in HeLa cancer cells.

Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

Conopeptides from the marine cone snails are a mixture of cysteine-rich active peptides, representing a unique and fertile resource for neuroscience research and drug discovery. The ConoServer database includes 8,134 conopeptides from 122 Conus species, yet many more natural conopeptides remain to be discovered. Here, we identified 517 distinct conopeptide precursors in Conus quercinus using de novo deep transcriptome sequencing. Ten of these precursors were verified at the protein level using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The combined gene and protein analyses revealed two novel gene superfamilies (Que-MNCLQ and Que-MAMNV), and three other gene superfamilies (N, P, and I1) were reported for the first time in C. quercinus. From the Que-MAMNV superfamily, a novel conotoxin, Que-0.1, was obtained via cloning and prokaryotic expression. We also documented a new purification process that can be used to induce the expression of conopeptides containing multiple pairs of disulfide bonds. The animal experiments showed that Que-0.1 strongly inhibited neuroconduction; the effects of Que-1.0 were 6.25 times stronger than those of pethidine hydrochloride. In addition, a new cysteine framework (CC-C-C-C-C-C-CC-C-C-C-C-C) was found in C. quercinus. These discoveries accelerate our understanding of conopeptide diversity in the genus, Conus and supply promising materials for medical research.

A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.

Estimation and Evaluation of Mexiletine for Bio-availability and Bio-equivalence studies by Liquid phase extraction using LC-MS/MS

The work aims to develop an appropriate method for mexiletine with 35-65% recovery by the LPE method with efficient and selective efficacy of the IS and analyte for the analysis under Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry. This method also reveals the bio-availability and bio-equivalence report for Internal Standard & working standard. At first, the selection of proper IS. The Internal standard should be structurally more similar to mexiletine. The selection of method plays a major role in which extraction procedure is done either by LPE or SPE. The selection of separation procedure should be either isocratic or gradient. Selection of column on bases separation principle of the compound. Since separation is the major principle for chromatography. Argon and Nitrogen Gas is used as carries with a flow-rate of 2L min. Temperature at 20°C, the pressure at 20psi. If the instrument doesn't show any peak or response in after loading sample, check the columns is an aqueous or reverse-phase and then submit the sample. Check all the solution and column and temperature and system stability before loading the sample. After loading the sample, must form calibration curve it must form linearity. The method found should possess the following parameters Specific & Selectivity, Precision & Accuracy. The work aims to develop a simple, elegant way for quantification of a molecule and the method determined will have recovery of 35-65% worldwide. This quantification will be further utilized in Full-Method Validation.

In Vitro Scolicidal Effects of Sideritis perfoliata Aerial Part Extract against the Protoscoleces of Echinococcus granulosus

Background: Cystic echinococcosis (CE) is commonly located in the liver and lungs of affected hosts. Surgical management is one of the best choices for the treatment of hydatidosis and using effective scolicidal agents during hydatid surgery is essential to prevent secondary infection. The present study was designed to investigate the in vitro scolicidal activity of methanol extract of Sideritis perfoliata against the protoscoleces of hydatid cysts. Methods: The protoscoleces were collected from slaughtered livestock in Adiyaman and the effect of three concentrations of the aerial part extract of S. perfoliata (0.1mg/ml, 0.2mg/ml, and 0.4mg/ml) was assessed over three different exposure periods. All tests were carried in dublicate. Finally, the mortality of protoscoleces was assessed by the eosin exclusion test (0.1% eosin staining). Methanol extract of S. perfoliata was assessed by Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS). Results: The results showed that the scolicidal effect of this extract at exposure periods of 10, 20, and 30 min was 29.6, 32.5, and 43.6% at concentrations of 0.1mg/ml, 37.8, 50, and 58.1% at concentration of 0.2mg/ml and finally 57.9, 71.8, and 79.1% at concentration of 0.4mg/ml, respectively; indicating that the extract requiring a further time to display a potent protoscolicidal effects. Some phenolic acids such as fumaric acid (260,13mg/L), syringic acid (27,92mg/L) and caffeic acid (26,84mg/L) and a flavonoid, luteolin (11,23 mg/L) were detected in high concentrations. Conclusions: The present study has demonstrated that the methanol extract of S. perfoliata has high scolicidal power in vitro, although the low concentration of plant extract may provide a base for future treatment of hydatid cysts. However, more research on the in vivo efficacy of S. perfoliata extract and its potential side effects is recommended.

Relationship Between Nicotine Dependence Scores and Nicotine, Cotinine, 3′-Hydroxycotinine and Nicotine Metabolite Ratio in Chinese Male Smokers

Summary Smoking is mainly sustained by nicotine dependence (ND), which varies across ethnic groups principally due to genetic as well as environmental factors. The Fagerström Test for Nicotine Dependence (FTND) and biomarkers of tobacco exposure are two important approaches to assess ND. However, the relationship between ND and FTND of Chinese smokers has not been studied. The aim of this study was to assess the relationship between FTND scores and nicotine, cotinine, 3′-hydroxycotinine (3HC) and nicotine metabolite ratio (NMR, the concentration ratio of 3HC to cotinine) in Chinese smokers. FTND was carried out and general characteristics were collected using a self-administered smoking questionnaire with 289 smokers. Nicotine, cotinine and 3HC in urine were simultaneously determined by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS). The concentrations of nicotine, cotinine and 3HC in the urine of smokers with a high FTND score were higher than in the urine of those with a low FTND score. There were significant correlations between urinary biomarker and FTND scores. Except for FTND item 2 (difficulty to refrain), the other items showed significant associations with the urinary biomarkers. No relationship was found between the nicotine metabolite ratio (NMR, 3′-hydroxycotinine/cotinine) and FTND scores or general characteristics of the participants. In conclusion, biomarkers of tobacco exposure levels are significantly associated with FTND scores. However, FTND Item 2 and NMR were not found to be associated with nicotine dependence in Chinese smokers.