Secretory proteins of Mycobacterium habana induce a protective immune response against experimental tuberculosis

The capacity of proteins of Mycobacterium habana TMC 5135 secreted into culture medium during the mid-exponential growth phase (secretory proteins, SPs) to induce protective immunity against Mycobacterium tuberculosis H37Rv was studied in the mouse model. Mice immunized with SPs followed by a challenge with M. tuberculosis H37Rv showed lesser M. tuberculosis bacilli in their lung and spleen and survived longer than unimmunized controls. The findings suggest that SP antigens of M. habana are protective against tuberculosis infection.     

Bioactivity assessment of natural compounds using machine learning models trained on target similarity between drugs

Natural compounds constitute a rich resource of potential small molecule therapeutics. While experimental access to this resource is limited due to its vast diversity and difficulties in systematic purification, computational assessment of structural similarity with known therapeutic molecules offers a scalable approach. Here, we assessed functional similarity between natural compounds and approved drugs by combining multiple chemical similarity metrics and physicochemical properties using a machine-learning approach. We computed pairwise similarities between 1410 drugs for training classification models and used the drugs shared protein targets as class labels. The best performing models were random forest which gave an average area under the ROC of 0.9, Matthews correlation coefficient of 0.35, and F1 score of 0.33, suggesting that it captured the structure-activity relation well. The models were then used to predict protein targets of circa 11k natural compounds by comparing them with the drugs. This revealed therapeutic potential of several natural compounds, including those with support from previously published sources as well as those hitherto unexplored. We experimentally validated one of the predicted pair’s activities, viz., Cox-1 inhibition by 5-methoxysalicylic acid, a molecule commonly found in tea, herbs and spices. In contrast, another natural compound, 4-isopropylbenzoic acid, with the highest similarity score when considering most weighted similarity metric but not picked by our models, did not inhibit Cox-1. Our results demonstrate the utility of a machine-learning approach combining multiple chemical features for uncovering protein binding potential of natural compounds.     

Origin and Genetic Diversity of Aromatic Rice Varieties, Molecular Breeding and Chemical and Genetic Basis of Rice Aroma

Aroma is an important quality attribute of rice and is a key determinant of its market value. Among the different groups of aromatic rice varieties ‘Basmati’ from the Indian subcontinent and ‘Jasmine’ from Thailand occupy prime position in the international market. In addition, there are a large number of premium short-grain aromatic rice varieties cultivated by farmers in India and South-East Asia that have not been fully commercially utilised as yet. The origin and evolution of aromatic rice varieties is being unravelled by application of genomic tools. The common alleles of aroma gene seem to have their origin in the aromatic group of rice varieties native to the Sub-Himalayan region. Of more than two hundred volatile compounds present in the rice grain, 2-acetyl-l-pyrolline (2-AP) is considered as the key aroma compound present in almost all the aromatic rice varieties. However, there is significant variation in the type and intensity of aroma in the different groups of aromatic rice varieties suggesting involvement of additional chemical compounds in varying proportions. Studies have been undertaken to understand the genetics of rice aroma and to map the genes or quantitative trait loci (QTL) controlling aroma expression. Of the three mapped aroma QTL, qaro8.1 located on rice chromosome S is the most significant and it represents a non-functional allele of BADH2 gene coding for enzyme betaine aldehyde dehydrogenase. Functional allele of the BADH2 gene makes rice non-aromatic. Similarly, specific alleles of BADH1 gene located on rice chromosome 4 within the aroma QTL qaro4.1 show association with the aromatic rice varieties. The gene underlying QTL qaro3.1 on chromosome 3 has not yet been deciphered. Functional molecular markers have been developed for the major aroma QTL on chromosome S and marker-assisted breeding for high yielding aromatic rice varieties is now a reality. To safeguard the reputation of Basmati rice an international code of practice has been developed where DNA markers help check the purity of commercial samples. There is need to use advanced genomic and metabolomic approaches to further study the minor genes controlling rice aroma and understand the variation in type, intensity and stability of rice aroma. It is also required to improve the production and marketing of short grain aromatic rice varieties.     

Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.     

N-Glycoproteome of E14.Tg2a Mouse Embryonic Stem Cells

E14.Tg2a mouse embryonic stem (mES) cells are a widely used host in gene trap and gene targeting techniques. Molecular characterization of host cells will provide background information for a better understanding of functions of the knockout genes. Using a highly selective glycopeptide-capture approach but ordinary liquid chromatography coupled mass spectrometry (LC-MS), we characterized the N-glycoproteins of E14.Tg2a cells and analyzed the close relationship between the obtained N-glycoproteome and cell-surface proteomes. Our results provide a global view of cell surface protein molecular properties, in which receptors seem to be much more diverse but lower in abundance than transporters on average. In addition, our results provide a systematic view of the E14.Tg2a N-glycosylation, from which we discovered some striking patterns, including an evolutionarily preserved and maybe functionally selected complementarity between N-glycosylation and the transmembrane structure in protein sequences. We also observed an environmentally influenced N-glycosylation pattern among glycoenzymes and extracellular matrix proteins. We hope that the acquired information enhances our molecular understanding of mES E14.Tg2a as well as the biological roles played by N-glycosylation in cell biology in general.     

A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of <Emphasis Type="Italic">peste des petits ruminants</Emphasis> virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T_m of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ～0.0001 cell culture infectious dose 50% units (TCID₅₀). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Application of ozone based treatments of secondary effluents

The present work was aimed at studying the efficiency of ozone in oxidation processes, coliform inactivation and Disinfection Byproducts (DBPs) formation, associated with the potential of ozone to increase the Biodegradable Dissolved Organic Carbon (BDOC) in secondary effluent with applied ozone doses of 5.0, 10.0 and 15.0 mg/L for contact times of 2, 5 and 10 min. The wastewater used in this work was collected from the Bhagwanpur Sewage Treatment Plant, Varanasi, India. Results of this experiment showed that 10 mg O₃/L O₃ for 5 min exposure was found most suitable dose for highest degradation of COD, TOC, UV₂₅₄, color, turbidity and total nitrogen parameters. The inactivation range of microbial biomass range was found in between 95% and 98%. Experiment revealed the fact that aldehydes and carboxylic acid formation were significantly related with the ozone dose and exposure time and ozone might enhance the treatment efficiency of secondary effluent treatment.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.