Current Biotechnology - Volume 6, Issue 1, 2017

Background: Ebola virus (EBOV) is a single-stranded RNA filovirus and its genome consists of seven genes. Among these seven genes, the glycoprotein (GP) gene encodes the only transmembrane protein of EBOV which consists of disulfide linked glycoprotein 1 (GP1) and glycoprotein 2 (GP2). The two glycoproteins i.e. GP1 and GP2 are furin-cleavage fragments of GP. Both GP1 and GP2 are heavily glycosylated. The GP1 and GP2 heterodimers forms trimeric spikes and are critical for EBOV attachment to host cells. The GP1 and GP2 are also involved in the catalysis of membrane fusion following viral attachment. Hence both are key targets to develop therapeutics to combat the deadly viral disease. Methods: ZMapp is an antibody mixture consisting of three chimeric monoclonal antibodies against GP. The three antibody components are called c13C6, c2G4, and c4G7 that were isolated from mice immunized with a recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein was replaced with the GP from Ebola virus. The recombinant component antibodies of ZMapp were produced using a plant expression system. Results: During the recent Ebola outbreak, ZMapp was used to treat EBOV infection in a number of patients in the USA and abroad on compassionate use basis under the expanded access program of US FDA (United States Food and Drug Administration). Seven patients were treated with ZMapp and five of the treated patients survived. Hence, ZMapp gained worldwide attention to treat Ebola. Conclusions: ZMapp appears to be safe and effective treatment for Ebola. This article discusses the biochemical characteristics of ZMapp in relation to the development of the antibody mixtures as biotherapeutics and the efforts to produce ZMapp component antibodies in mammalian cells which would increase the production yield and efficiency along with the possible decrease in cost of treatment.

Background: There are some chapters written on microbiology of alcohol-making, where grapes and certain regional and seasonal fruits have been used to produce wines or specialized fruit beverages. This overview deals with the types of microorganisms involved in alcohol (ethanol) fermentation using various types of substrates, such as agricultural, industrial by-products and residual substrates, and seasonal and regional fruits. The type of micro-flora controls the process of alcohol production, which could be pure saccharomyces yeasts, exo- and endogenous non-saccharomyces yeasts, and bacteria depending on the type of substrate used. Review incorporates information on some processes which have been recently developed for the use of immobilized microbial-cells in fermentation, simultaneous alcoholic and malolactic wine fermentation, simultaneous saccharification and fermentation and co-culture system to process different types of substrates, available globally as by-products or residues. Methods: A search of bibliographic references for peer-reviewed research literature was undertaken focussing on two aspects - first on the types of substrate have been used for ethanol production, and sec- ond on specific types of microorganisms employed to ferment these substrates. Results: Forty-seven most relevant papers were included in the review of this topic. Information gathered on both aspects as described in methods, has been structured in suitable sections of review. Conclusion: The findings of this review confirm the importance of selection of a suitable microorganism , Saccharomyces, or non-Saccharomyces is important in ethanol fermentation, particularly depending on the type of substrate being used for the fermentation, a traditional fruit or agro-industrial waste. Certain modifications and adaptations could be incorporated in the fermentation system using these microorganisms as biocatalyst, to improve their performances and the overall productivity and the quality of the product.

Background: Halogenases are involved in the halogenation of secondary metabolites with pharmaceutical relevance, such as the antibiotics chloramphenicol and vancomycin. Hereby flavin- or FADH2-dependent halogenases represents the major class of halogenases. Previous investigations on flavin-dependent halogenases have focused on Actinomycetes and sponge-associated microorganisms. In order to extend our knowledge on the diversity and distribution of halogenases we have applied a PCR-based screening approach to show the wide distribution of flavin-dependent halogenases in metagenomes of microbial communities from different environments, such as soils and sediments. Methods: For the screening of new flavin-dependent halogenases a metagenomic approach was used. Metagenomic libraries were constructed and screened by homology-driven PCR based on degenerate primers designed from known flavin-dependent halogenases. Results: We screened 55 environmental samples that we collected from different habitats by a PCRbased screening for novel flavin-dependent halogenases. The phylogenetic analysis of the putative halogenases together with the 16S Amplicon sequencing results of the environmental samples demonstrates the wide distribution of the halogenases and the large sequence diversity. In addition, metagenomic libraries were constructed to screen for flavin-dependent halogenases. Conclusion: The metagenomic approach described in this paper is a promising method to explore novel putative halogenases from different environments.

Introduction: CD4+ regulatory T-cells that constitutively express CD25 have a potent mechanism of action of immunomodulation and hence hold a great therapeutic potential for the treatment of autoimmune diseases and tolerance induction in the context of solid organ transplantation. Methods: For in vitro generation of Tregs volunteer donor’s adipose tissue was resected and mesenchymal stem cells (MSC) were generated. MSC were co-cultured with post-renal transplantation recipient’s peripheral blood derived peripheral blood mononuclear cells (PBMC) and with irradiated PBMC from same recipient with the addition of exogenous IL-2. Controls were carried out without use of MSC. In vitro generated Tregs were subjected for quantitative and qualitative analysis of Tregs specific markers. Results: Quantitative analysis revealed increased cell counts of Tregs in comparison to PBMC. Tregs specific markers evaluation using flocytometer showed rise in all Treg specific cell population: CD4+25+127Neg with mean 7.6 ± 6.1%, CD4+FoxP3+ mean of 21.7 ± 15.3% and CD4+CTLA-4+ mean of 16.9 ± 13.7%. Conclusion: We conclude that MSC induce in vitro generation of Tregs. In vitro generated Tregs hold a great promise as “magic bullet” for various disease prevention and/or treatment.

Background: Forkhead box protein 3 (Foxp3) is a transcription factor expressed by regulatory T (Treg) cells in the immune system. Detection of Foxp3 gene expression is an indispensable tool for the diagnosis of autoimmune disorders. Here we report the application of a polyacrylamide hydrogel sensor for the detection of Foxp3 gene via DNA hybridization. Methods: Polyacrylamide based hydrogels were generated using acrydite-Foxp3 DNA probes via covalent attachment during gel polymerization. The presence of the target Foxp3 gene allows for the formation of a bridge like structure via DNA hybridization. The bridge like structure is formed via hybridization of the target DNA, fluorescent-labeled Foxp3 DNA probe and the hydrogel anchor DNA. A wash step allows efficient removal of any unbound DNA. Analysis was done via fluorescence intensity of the dye. Results: The proposed sensor was able to detect the presence of the target DNA with a detection limit of 50nM. Image capture using a handphone camera allows for simple data collection and reporting. The images were analyzed using Adobe Photoshop CS2 software. The comparison of the data obtained from standard laboratory fluorescence spectrophotometer showed good correlation with the data obtained using the Adobe Photoshop CS2 software. Conclusion: Our proposed hydrogel for Foxp3 gene detection is inexpensive and can be molded into any desired shape or size for easy handling. The proposed hydrogel based sensor is an interesting alternative for nucleic acid diagnostic applications.

Background: Predicting thermostability of a protein, given its sequence or structure is a challenging job. Predicting which mutations can render mesophilic proteins thermostable is further challenging. A guided approach for the same is still elusive. Prediction can be done on the three hierarchies of protein organization: nucleotide sequence of the gene, primary amino acid sequences and the three dimensional structure of proteins. However it is still unclear that which level leads to a better predictor of protein thermostability and what combination of parameters in each level is responsible for thermostability of proteins. Methods: Present paper addresses this question by testing numerous unsupervised and supervised machine learning methodologies with multitude of prediction parameters to discriminate thermostable and mesostable proteins and to elucidate parameters in each level that lead to protein thermostability. The present method used 33 features of nucleotide sequences, 19 amino acid composition features and 17 structural features. Numerous machine learning approaches: 11 weighting algorithms, 5 unsupervised, 3 supervised learners and 2 lazy modeling algorithms with different parameters were tested and applied on the three datasets. Results: Results showed that amino acid datasets outperforms the nucleotide and structural datasets in generating the best prediction model for protein thermostability. LibSVM with polynomial kernel when applied on amino acid dataset with 10 fold cross validation results in the best prediction model for classifying thermostable proteins with 90.97% accuracy of prediction. Results also show that CG codons are not deterministic of thermostability. Decrease in Gln content, increase in hydrophobic residues and increase in main chain to main chain hydrogen bonds followed by gamma turns and aromatic aromatic interactions are responsible for protein thermostability. Conclusion: Present study shows that studying different datasets can bring about the importance of features in each hierarchy of protein organization which is responsible for protein thermostability. The results support the long drawn conclusion that protein amino acid composition is highly correlated to protein thermostability. Thus further insight into all these features can lead to the development of a better predictor and classifier of thermostable proteins. The results support the long drawn conclusion that protein amino acid composition is correlated to protein thermostability. The study also shows that testing of multitude of criteria is important before reaching at a definitive conclusion.

Purpose: The ability of spermidine to increase the selectivity of anticancer agents has been studied extensively. In this research we report the combination of this polyamine and GM1 ganglioside micelles and characterize their behavior for drug delivery. Methods: Dynamic light scattering and electron microscopy were used to characterize size and morphology of micelles. Zeta potential was determined using a Nano-zeta potential analyzer. Size-exclusion chromatography was used to separate different populations. Cytotoxic effect of micelles was evaluated on Hep2 cell line. Results: Covalent conjugation of spermidine to gangliosides produces a clear reduction of the electronegative z potential of micelle surface. DLS analysis shows no significant differences between both micelles, while TEM image shows a smaller size of Spermidine-GM1. These modified micelles load hydrophilic or hydrophobic antitumor drugs and conjugation does not affect the stability of micelles/drug in solution. Spermidine-GM1/Doxo micelles show faster drug release into cells as compared with GM1/Doxo micelles; however, no evidence can be found for the participation of the polyamine transport system in the uptake of modified micelles. Conclusion: While Spermidine-GM1 and GM1 micelles show similar physical properties, spermidine modified micelles are most efficient to release drugs, making this an interesting alternative to consider for drug delivery.

Background: The antitumor activity of L-asparaginase isolated from natural sourceshas been proven thriving in treatment of acute lymphoblastic leukemia and other lymphoproliferative disorders however production efficiency and associated glutaminase activity has always been a limitation to its use. Asparaginase also induces apoptosis and cytoprotective autophagy in chronic myeloid leukemia cells. Together these findings make L-asparaginase a useful drug for the treatment of Acute Lymphocytic Leukemia and various other tumors. Methods: In the present study, the purification and characterization of a glutaminasefree intra-cellular L-asparaginase was done from Bacillus brevis, originally isolated from a cold desert region of Himachal Pradesh, India. Results: The enzyme was purified to 90-fold by a SulphopropylSephadex columnchromatography and found to be 32 kDa in molecular weight under reduced conditions. The enzyme possessed significant amount of activity at human physiological conditions of temperature (37°C) and pH (7.4) and have a half-life of 70 minutes at 25ºC. The Km and Vmax values of L-asparaginase were found to be 3.5 X 10-2 M and 0.77 IU, respectively. The L-asparaginase purified from B. brevis showed comparable in vitro toxicity on Hep-2C cell lines (91% survival) in comparison to commercial L-asparaginase preparation (90% survival) obtained from E. coli. Conclusion: L-asparaginase with high purity and high affinity for L-asparagine will bemost suitable for the treatment of lymphoblastic leukemia. The glutaminase free L-asparaginase obtained from B. brevis thus provides an additional therapeutic agent and could be used against for the treatment of various malignant diseases.

Background: Saurauia punduana Wallich (Actinidiaceae) is a therapeutically important threatened plant species. Habitat destruction due to anthropogenic activities and over-collection for medicinal purpose has reduced the natural population. Present communication reports successful somatic embryogenesis and plant regeneration from in vitro cotyledonary leaves. Method: Saurauia punduana seeds were extracted from 5-7 weeks old fruits and cultured on MS medium fortified BA (2μM) for germination. The cotyledonary leaves were harvested from in vitro raised seedlings and cultured on MS fortified medium. Embryogenic callus was maintained on MS medium fortified with sucrose (3%), NAA (1.5 μM) and BA (4 μM) for callus proliferation. The callus was subsequently maintained for embryo development and maturation. Results: About 68% of the leaf pieces formed embryogenic callus on MS medium fortified with sucrose (3%, w/v), NAA (1.5 μM) and BA (4 μM). The embryogenic cultures registered ~348% proliferation of inoculum callus after four weeks on subsequent subculture onto proliferation medium. The proembryos converted into cotyledonary embryos and torpedo shaped embryos when transferred onto MS medium fortified with sucrose (4%) and BA (4 μM); ~62% globular embryos converted to cotyledonary embryos. In addition as many as 38 embryos per 200 mg of embryogenic cultures were formed. The somatic embryos germinated on the conversion medium accompanied by secondary somatic embryo formation. The germinated embryos could be easily separated from the clump and transferred to potting mix and maintained in a polyhouse for 5-6 weeks, where ~75% seedlings survived after 6 weeks in the potting mix. Conclusion: This is a reproducible protocol for somatic embryogenesis and secondary embryogenesis of Saurauia punduana and their conversion into embling. The protocol developed will help in production of clonal planting materials and propagation of this species.