Current Issue : January - March Volume : 2018 Issue Number : 1 Articles : 5 Articles
Background: A sensitive, rapid and selective UHPLCââ?¬â??MS/MS method has been developed and validated for the\nquantification of Nicotine (NT) and Cotinine (CN) using Continine-d3 as internal standard (IS) as per FDA guidelines.\nSample preparation involved simple protein precipitation of 20 Ã?¼L mouse plasma or brain homogenate using acetonitrile\nat 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode\nusing electro spray ionization technique and the transitions of m/z 163.2 ââ? â?? 132.1, 177.2 ââ? â?? 98.0 and 180.2 ââ? â?? 101.2\nwere used to measure the NT, CN and IS, respectively. The elution of NT, CN and IS are at 1.89, 1.77 and 1.76 min,\nrespectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile\nand methanol (3:1, v/v) at a flow rate of 0.3 mL/min on a Kinetex EVO C18 column. The method was validated with a\nlower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes.\nResults: A linear response function was established for the range of concentrations 3ââ?¬â??200 (r > 0.995) for NT and\n3ââ?¬â??600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are\nstable in the battery of stability studies viz., stock solution, bench-top and auto-sampler.\nConclusion: This method was successfully utilized to validate a newly developed preclinical smoking model in mice....
Background: Current malaria diagnostic methods require blood collection, that may be associated with pain and the\nrisk of transmitting blood-borne pathogens, and often create poor compliance when repeated sampling is needed.\nOn the other hand, the collection of saliva is minimally invasive; but saliva has not been widely used for the diagnosis\nof malaria. The aim of this study was to evaluate the diagnostic performance of saliva collected and stored at room\ntemperature using the OMNIgene�®â�¢ORAL kit for diagnosing Plasmodium falciparum malaria.\nMethods: Paired blood and saliva samples were collected from 222 febrile patients in Cameroon. Saliva samples\nwere collected using the OMNIgene�®â�¢ORAL (OM-501) kit and stored at room temperature for up to 13 months. Thick\nblood film microscopy (TFM) was used to detect P. falciparum blood-stage parasites in blood. Detection of P. falciparum\nDNA in blood and saliva was based on amplification of the multi-copy 18 s rRNA gene using the nested-polymerase\nchain reaction (nPCR).\nResults: Prevalence of malaria detected by TFM, nPCR-saliva and nPCR-blood was 22, 29, and 35%, respectively.\nUsing TFM as the gold standard, the sensitivity of nPCR-saliva and nPCR-blood in detecting P. falciparum was 95 and\n100%, respectively; with corresponding specificities of 93 and 87%. When nPCR-blood was used as gold standard, the\nsensitivity of nPCR-saliva and microscopy was 82 and 68%, respectively; whereas, the specificity was 99 and 100%,\nrespectively. Nested PCR-saliva had a very good agreement with both TFM (kappa value 0.8) and blood PCR (kappa\nvalue 0.8). At parasitaemia > 10,000 parasites/�¼l of blood, the sensitivity of nPCR-saliva was 100%. Nested PCR-saliva\ndetected 16 sub-microscopic malaria infections. One year after sample collection, P. falciparum DNA was detected in\n80% of saliva samples stored at room temperature.\nConclusions: Saliva can potentially be used as an alternative non-invasive sample for the diagnosis of malaria and\nthe OMNIgene�®â�¢ORAL kit is effective at transporting and preserving malaria parasite DNA in saliva at room temperature.\nThe technology described in this study for diagnosis of malaria in resource-limited countries adds on to the\narmamentarium needed for elimination of malaria....
Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected\non ice, processed ice cold and stored frozen at âË?â??80 Ã?°C, in liquid nitrogen (LN2), or freeze dried and stored at\nroom temperature in a desiccator (FDRT) or freeze dried and stored at âË?â??20 Ã?°C for 1 year (FD-20). In a separate experiment,\nEDTA plasma samples were collected onto ice, processed ice cold and maintained on ice Ã?± protease inhibitors\nversus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography\nand tandem mass spectrometry (LCââ?¬â??ESIââ?¬â??MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST\nalgorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at\nroom temperature. Random sampling by LCââ?¬â??ESIââ?¬â??MS/MS showed that peptides from C4B were undetectable on ice,\nbut peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room\ntemperature. The frequency and intensity of precursors within Ã?± 3 m/z of the C4B peptide NGFKSHALQLNNR was\nconfirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target\nsequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature\nchain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the\nmature C4B protein, to reveal the thioester reaction site, consistent with LCââ?¬â??ESIââ?¬â??MS/MS and Western blot. Random\nsampling showed that proteolytic peptides from complement component C4B were rarely observed with long term\nstorage at âË?â?? 80 Ã?°C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at âË?â?? 20 Ã?°C (FD-20 Ã?°C) or freeze\ndrying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at\nleast 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to\nsamples on ice for up to 72 h or stored at âË?â?? 80 Ã?°C, LN2, FDRT or FD-20 Ã?°C for up to a year....
Introduction. Chinesemedicine syndrome diagnosis is the key requisite in the treatment of male infertility with traditional Chinese\nmedicine (TCM). Kidney-Yang deficiency syndrome (KYDS) is the critical Chinese medicine syndrome of male infertility. To\nexplore the modernized mechanisms of KYDS in male infertility, this study aims to investigate the metabolomics of males with\nKYDS. Methods. The gas chromatography-mass spectrometry method was applied to analyze the plasma samples of 67 infertile\nmales with KYDS compared with 55 age-matched healthy controls. The chemometric methods including principal component\nand partial least squares-discriminate analyses were employed to identify the potential biochemical patterns.With the help of the\nvariable importance for the projection and receiver operating characteristic curve analyses, the potential biomarkers were extracted\nto define the clinical utility. Simultaneously the high-quality KEGG metabolic pathways database was used to identify the related\nmetabolic pathways. Results. The metabolomics profiles of infertile males with KYDS including 10 potential biomarkers and six\nmetabolic pathways were identified. They precisely distinguished infertile males with KYDS from healthy controls. Conclusions.\nThese potential biomarkers and pathways suggest the substantial basis of infertile males with KYDS. The metabolomics profiles\nhighlight the modernized mechanisms of infertile males with KYDS....
Background: Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) is the causative pathogen in up to 20% of\nstreptococcal-induced infective endocarditis (IE) cases. However, the underlying mechanisms of pathogenesis in S.\ngallolyticus have not yet been solved. Pathogens causing IE need to employ virulent strategies to initiate and\nestablish infections, such as escape the bloodstream, invade the host-cell, and persist intracellularly. In this study,\nwe examined the induction of inflammation by different S. gallolyticus strains in relation to their survival in whole\nblood and cell culture models as well as their ability to induce platelet aggregation. Phagocytosis of these bacteria\nby macrophages, followed by intracellular survival, was also quantified.\nMethods: In whole blood and THP-1 cell culture assays bacterial growth kinetics was determined by plating, followed\nby colony counting. Induction of interleukin (IL)-6 expression in whole blood of three healthy volunteers, caused by\ndifferent strains, was quantified by ELISA. Gene expression of cytokines (IL1B, IL6 and IL8) was quantified by real-time\nPCR after stimulating THP-1 monocytes with bacteria. Induction of platelet aggregation was analyzed by light\ntransmission aggregometry using the BORN method. A macrophage model was used to analyze phagocytosis of\nstrains and their survival in macrophages within 48 h.\nResults: Strains promoted IL-6 secretion in a time-dependent fashion. For example, DSM16831 induced IL-6 secretion\nin whole blood earlier than other isolates, and was eliminated in the whole blood of one volunteer, whereas UCN34\ncould grow. Platelet aggregation depended on the different isolates used and on the individual platelet donor. Two\nstrains (AC1181 and 010672/01) induced cytokine gene expression in THP-1 monocytes only marginally, compared to\nother strains. The phagocytosis rate of S. gallolyticus isolates differed significantly, and the isolates UCN34 and BAA-2069\ncould persist for a considerable time in the phagocytes.\nConclusion: The strain-dependent differences of S. gallolyticus isolates, observed during interaction with human blood\ncells, support the hypotheses that divergences in individual virulence factors determine a distinct pathogenicity of the\nisolates. These data constitute an additional step towards the elucidation of mechanisms in the complex, multifactorial\npathogenesis of this IE pathogen....
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