Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

Effect of mode of operation of bioreactors on the biosynthesis of penicillin amidase in Escherichia coli

Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of penicillin amidase in Escherichia coli. Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.     

Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by Trichoderma harzianum

Statistical design was used to determine the optimal levels of medium components, the optimal initial pH of the enzyme production medium, the temperature of fermentation, age of the organism in the slant growth and the age of the inoculum for the production of chitinase in shake flask fermentations. The use of high concentrations of chitin and ammonium sulphate and exclusion of peptone and urea from the medium resulted in the production of higher level of the enzyme. The optimal concentrations of the medium components were 12.5 kg/m³ and 4.2 kg/m³ for the chitin and ammonium sulphate respectively. The effect of the addition of peptone and urea to the optimized medium was studied. The optimal values of initial pH and temperature were 5.6 and 28 °C respectively. The optimal age of the slant and the inoculum were found to be 105 h and 43 h respectively. The highest level of chitinase before optimization of the above variables was 0.054 U which was maximized to the level of 0.197 U.     

Parameters optimization of chitin hydrolysis by Trichoderma harzianum chitinase under assay conditions

A kinetic analysis and optimization of reaction conditions for the enzymatic hydrolysis of chitin using chitinase produced by Trichoderma harzianum NCIM 1185 was carried out. Swollen chitin was used as the substrate for chitinase. The central composite design was followed for this optimization. The required volume ratio of the major reactants for maximum hydrolysis was determined. The pH and temperature optima were found to be 4.75 and 47 °C respectively. K _m and V _max for this enzyme were 4.643 kg/m³ and 0.1542 U respectively.     

A comparative account of the microbiological characteristics of soils under natural forest,grassland and cropfield from Eastern India

Microbiological and physico-chemical characteristics of tropical forest, grassland and cropfield soils from India were investigated. The study revealed that the conversion of natural forest led to a reduction of soil organic C (26–36%), total N (26–35%), total P (33–44%), microfungal biomass (44–66%) and total microbial biomass C, N and P (25–60%) over a period of 30–50 years. Comparative analysis of microbial activity in terms of basal soil respiration revealed maximum activity in the forest and minimum in the cropfield soil. Analysis of microbial metabolic respiratory activity (qCO₂) indicated relatively greater respiratory loss of CO₂-C per unit microbial biomass in cropfield and grassland than in forest soil. Considering the importance of the microbial component in soil, we conclude that the conversion of the tropical forest to different land uses leads to the loss of biological stability of the soil.     

Efficacy of foliar extracts against pre- and post-harvest diseases of sponge-gourd fruits

S.K. AHMAD AND J.S. PRASAD. 1995. The inhibitory potential of aqueous foliar extracts of Adhatoda vasica, Azadirachta indica, Catharanthes roseus, Datura fistulosa, Lantana camara, Muraya exotica, Ocimum sanctum, Ricinus communis and Strychnos nux-vomica were evaluated against soft rot diseases of sponge-gourd fruits caused by Helminthosporium spiciferum and Fusarium scirpi. Conidial germination of F. scirpi and H. spiciferum were reduced to about 75% when their spores were treated with A. indica, C. roseus, D. fistulosa, L. camara, M. exotica and O. sanctum. Considerable reductions (50%) in mycelial dry weights and colony diameters were recorded in liquid media containing (20%) aqueous extracts of C. roseus, D. fistulosa and M. exotica . Other extracts reduced the mycelial growth but to a lesser extent. Post-infection treatment of sponge-gourd fruits with the extracts of A. indica, L. camara, M. exotica, O. sanctum, D. fistulosa and C. roseus almost fully inhibited the spread of disease. Treatment of fruits with A. indica and C. roseus before fungal infection reduced the spread of disease caused by both the pathogens, whereas M. exotica, O. sanctum and D. fistulosa reduced the spread of soft-rot caused by F. scirpi but not by H. spiciferum .     

Free and total thyroid hormones in humans at extreme altitude

Alterations in circulatory levels of total T₄ (TT₄), total T₃ (TT₃), free T₄ (FT₄), free T₃ (FT₃), thyrotropin (TSH) and T₃ uptake (T₃U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized lowlanders, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT₃ and FT₄ occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T₃U was found to be higher at high altitudes. A decline in TSH and hormone binding proteins and an increase in the free moiety of the hormones is indicative of a subtle degree of tissue hyperthyroidism which may be playing an important role in combating the extreme cold and hypoxic environment of high altitudes.     

The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.     

Cytomorphology of induced octoploid Chili pepper (Capsicum annuum L.)

Summary Octoploidy was induced in Chili pepper (Capsicum annuum cultivar cerasiformis) through the application of colchicine and the cytomorphological features of two octoploid plants were described. In general, the octoploids did not exhibit gigas characters when compared to the tetraploids; on the contrary they were less vigorous, suggesting that the optimum and desirable ploidy level for Capsicum is probably tetraploid. Chromosome associations such as octovalents and hexavalents, in addition to IVs, IIIs, IIs and Is, were recorded at diakinesis and metaphase I. Meiosis was highly irregular and the pollen and seed fertility was very low. Cytological features of octoploid Chili peppers are compared with octoploids of Physalis and Petunia.