Proteomics analysis of human coronary atherosclerotic plaque: a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.     

Production of alkaline protease from an alkaliphilic actinomycete

The repression of alkaline protease synthesis from alkaliphilic actinomycete was studied by using glucose, peptone, yeast extract, KH2PO4 and amino acids; tyrosine, tryptophan, lysine, and arginine. There was a critical limit of stimulation of enzyme production by these components. Crude components such as molasses, wheat flour, and wheat bran were found to be effective for growth and enzyme production. The high level of enzyme production using agro-industrial by-products is commercially significant due to cheap nature of these sources. The findings are quite attractive, as only few actinomycetes, particularly alkaliphilic ones, have so far been explored for their enzymatic potential and regulation of enzyme synthesis.     

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.     

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of bisoprolol in human plasma using multiplexing technique (two HPLC units connected to one MS). Bisoprolol was extracted from human plasma using solid-phase extraction technique using metoprolol as internal standard. A Betabasic 8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2-->116.1 for bisoprolol and m/z 268.2-->191.0 for metoprolol. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.5-70.0 ng/mL with correlation coefficient > or =0.9991. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for bisoprolol and metoprolol were 93.89% and 77.65%, respectively. Total MS run time was 0.90 min only. The developed method was applied for the determination of pharmacokinetic parameters of bisoprolol following a single oral administration of a 10mg bisoprolol tablet in 18 healthy male volunteers.     

Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India

BackgroundXpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India.MethodsThis demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates.ResultsIn the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST.ConclusionIntroduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.     

UDP-glucose 4, 6-dehydratase activity plays an important role in maintaining cell wall integrity and virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.     

Development and validation of a selective and sensitive LC-MS/MS method for determination of cycloserine in human plasma: application to bioequivalence study

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20-30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

A single dose of the DENV-1 candidate vaccine rDEN1Δ30 is strongly immunogenic and induces resistance to a second dose in a randomized trial

Dengue is an emerging infectious disease that has become the most important arboviral infection worldwide. There are four serotypes of dengue virus, DENV-1, DENV-2, DENV-3, and DENV-4, each capable of causing the full spectrum of disease. rDEN1Δ30 is a live attenuated investigational vaccine for the prevention of DENV-1 illness and is also a component of an investigational tetravalent DENV vaccine currently in Phase I evaluation. A single subcutaneous dose of rDEN1Δ30 was previously shown to be safe and immunogenic in healthy adults. In the current randomized placebo-controlled trial, 60 healthy flavivirus-naive adults were randomized to receive 2 doses of rDEN1Δ30 (N = 50) or placebo (N = 10), either on study days 0 and 120 (cohort 1) or 0 and 180 (cohort 2). We sought to evaluate the safety and immunogenicity of this candidate vaccine in 50 additional vaccinees and to test whether the humoral immune response could be boosted by a second dose administered 4 or 6 months after the first dose. The first dose of vaccine was well tolerated, infected 47/50 vaccinees and induced seroconversion in 46/50 vaccinees. Irrespective of dosing interval, the second dose of vaccine was also well tolerated but did not induce any detectable viremia or ≥4-fold rise in serum neutralizing antibody titer.Only five subjects had an anamnestic antibody response detectable by ELISA following a second dose of vaccine, demonstrating that the vaccine induced sterilizing humoral immunity in most vaccinees for at least six months following primary vaccination.The promising safety and immunogenicity profile of this vaccine confirms its suitability for inclusion in a tetravalent dengue vaccine.