Identification and characterization of novel plasma proteins in drug resistant HIV/AIDS patients by SWATH-MS

Background and ObjectivesAcquired immunodeficiency syndrome is one of the most important diseases caused by human immunodeficiency virus. Understanding its molecular pathogenesis is essential to manage the disease at the population level. In this study, a quantitative analysis of plasma proteins was carried out in drug resistant and drug respondent patients using the SWATH-MS.MethodsSequential window acquisition of all theoretical mass spectra (SWATH-MS) is a prime technique to seek the key plasma proteins involved in virus replication and drug metabolism during therapy.ResultsIn total, 204 proteins were identified and quantified, 57 proteins were differentially expressed, 25 proteins were down regulated and 32 proteins were upregulated in drug resistant patients. Six proteins such as complement C4-A, immunoglobulin heavy variable 1-2, carboxylic ester hydrolase, fibulin-1, immunoglobulin lambda constant 7, secreted phosphoprotein 24 were statistically expressed in drug resistant patients compared to the drug respondent patients. Gene ontology study and protein-protein interaction networks were established in six statistically significant differentially expressed proteins of the drug resistant patients.Interpretation and ConclusionOur findings high lights the novel proteins that were differentially expressed in drug resistant patients. A label-free quantitative proteomics method for depleted human plasma samples by SWATH-MS that can be useful in plasma proteomics research in any biological system.

Label-Free Proteomics Reveals the Molecular Mechanism of Subculture Induced Strain Degeneration and Discovery of Indicative Index for Degeneration in Pleurotus ostreatus

Pleurotus ostreatus is one of the widely cultivated edible fungi across the world. Mycelial subculture is an indispensable part in the process of cultivation and production for all kinds of edible fungi. However, successive subcultures usually lead to strain degeneration. The degenerated strains usually have a decrease in stress resistance, yield, and an alteration in fruiting time, which will subsequently result in tremendous economic loss. Through proteomic analysis, we identified the differentially expressed proteins (DEPs) in the mycelium of Pleurotus ostreatus from different subcultured generations. We found that the DNA damage repair system, especially the double-strand breaks (DSBs), repairs via homologous recombination, was impaired in the subcultured mycelium, and gradual accumulation of the DSBs would lead to the strain degeneration after successive subculture. The TUNEL assay further confirmed our finding about the DNA breaks in the subcultured mycelium. Interestingly, the enzyme activity of laccase, carboxylic ester hydrolase, α-galactosidase, and catalase directly related to passage number could be used as the characteristic index for strain degeneration determination. Our results not only reveal for the first time at the molecular level that genomic instability is the cause of degeneration, but also provide an applicable approach for monitoring strain degeneration in process of edible fungi cultivation and production.

Biochemical Characterization and Kinetics of Carboxylesterase Isolated from Rabbit Liver and Lung in order to Application in the Detoxification of Environmental Pollutants

Objective: A carboxylesterase (CbE) or carboxylic-ester hydrolase (EC 3.1.1.1) is an enzyme that catalyzes the hydrolysis of carboxylesters to alcohol and carboxylate. The objective of this study is to obtain this enzyme from liver and lung of rabbit with a comparison between the effects of two specific substrates, α-naphthyl acetate (α-NA) and p-nitrophenyl acetate (p-NPA) on the enzymes activity. The kinetic characteristics of the enzymes have been also investigated in details. Methods: CbE was obtained from rabbit liver and lung with Tris/HCl buffer (pH 7.4) extraction, ammonium sulfate precipitation, dialysis, and lyophilized. Results: The lyophilized enzyme appeared to be homogeneous on native polyacrylamide gel electrophoresis and its molecular mass was estimated to be 30 kDa suggesting a monomeric structure for the obtained enzyme. The enzyme exhibited a broad activity at different pH and temperature. The pH optimum was 7.5 and optimum temperature was 35ºC. The enzyme showed specific activities of 1.36 and 1.04 μmol α-naphthol hydrolyzed/mg protein/min for liver and lung CbEs, respectively using α-naphthyl acetate (α-NA). However, the specific activities of the liver and lung CbE were 0.44 and 0.23 μmol p-nitrophenol hydrolyzed/mg protein/min, respectively using p-nitrophenyl acetate (p- NPA). The relationships between estimates of Km and Vmax calculated from the Michaelis-Menten equation have been explored. The Km values of liver and lung CbEs were 59.28 and 28.10 μM with α-NA, respectively however, the values were 86.39 and 90.05 μM with p-NPA. The Vmax values of liver and lung CbEs were 2.32 and 1.05 with α-NA, respectively however, the values were 0.55 and 0.19 with p- NPA. The enzyme showed residual activity when incubated at wide range of pesticides chlorpyrifos and methomyl however it was totally inhibited by concentrations higher than 200 μM. The extent of the inhibition was different, as estimated by the values of the inhibition constant Ki that were found to be 2.14 and 5.14 for chlorpyrifos and methomyl, respectively. Conclusion: Conclusion: This enzyme may play an important role in the detoxification of organophosphorous and carbamate pesticides at low levels.