Protein engineering and de novo designing of a biocatalyst

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter‐relationships. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluating the Impact of Breastfeeding on Rotavirus Antigenemia and Disease Severity in Indian Children

Objectives

To evaluate the contribution of breastfeeding to Rotavirus (RV)-induced antigenemia and/or RNAemia and disease severity in Indian children (<2 yrs age).

Methods

Paired stool and serum samples were collected from (a) hospitalized infants with diarrhea (n = 145) and (b) healthy control infants without diarrhea (n = 28). Stool RV-antigen was screened in both groups by commercial rapid-test and enzyme immunoassay. The disease severity was scored and real-time-PCR was used for viral-load estimation. Serum was evaluated for RV-antigenemia by EIA and RV-RNAemia by RT-PCR. Data was stratified by age-group and breastfeeding status and compared.

Results

Presence of RV-antigenemia and RV-RNAemia was positively related with presence of RV in stool. Disease severity and stool viral-load was significantly associated with RV-antigenemia[(r = 0.74; CI:0.66 to 0.84; P<0.0001,R² = 0.59) and (r = -0.55; CI:-0.68 to -0.39; P<0.0001,R² = 0.31) respectively], but not with RV-RNAemia. There was significant reduction in RV-antigenemiarate in the breast-fed group compared to non-breastfed infants, especially in 0–6 month age group (P<0.001). Non-breastfed infants were at risk for RV-antigenemia with severe disease manifestations in form of high Vesikari scores correlating with high fever, more vomiting episodes and dehydration.

Conclusion

RV-antigenemia was common in nonbreastfed children with severe RV-diarrhea and correlated with stool RV-load and disease severity.     

The Development and Validation of the Indian Family Violence and Control Scale

The high prevalence of domestic violence (DV) among married women in India and associated negative health repercussions highlight the need for effective prevention strategies and tools to measure the efficacy of such interventions. Literature supporting differing manifestations of DV by culture underscores the need for a culturally-tailored scale to more effectively measure DV in the Indian context. We therefore aimed to develop and validate such a tool, the Indian Family Violence and Control Scale (IFVCS), through a mixed-methods study. The psychometric development of IFVCS is herein discussed. After field pre-testing and expert review, a 63-item questionnaire was administered to a random sample of 630 married women from May-July 2013 in Pune, India. The item response theory approach for binary data to explore the IFVCS structure suggested that IFVCS is reliable, with the majority of items having high (>0.5) and significant factor loadings. Concurrent validity, assessed by comparing responses to IFVCS with the validated, abridged Conflict Tactics Scale-2, was high (r = 0.899, p<0.001) as was the construct validity, demonstrated by its significant association with several established DV correlates. Therefore, initial assessment of the IFVCS psychometric properties suggests that it is an effective tool for measuring DV among married women in India and speaks to its capacity for enhancing understanding of DV epidemiology and for evaluating the effectiveness of future DV interventions.     

The effect of some pesticides in conjunction with nitrogen sources on growth and carbohydrate and nitrogen constituents of sugarcane during formative phase

Summary The effect of gamma isomer of benzene hexachloride and Telodrin both in absence and presence of ammonium and nitrate nitrogen carriers on shoot and root growth and carboydrate and nitrogen constituents of sugarcane was studied. The pesticides induced shoot emergence and development of tillers and roots. Shoot growth, nitrogen intake and insoluble nitrogen content invariably increased by pesticide application while soluble carbohydrate and soluble nitrogen content of the shoot decreased. Pesticides also increased the water-soluble nitrate content of the soil.The effect of pesticides was not confined to ammonical source of nitrogen rather the pesticides proved equally effective under nitrate source of nitrogen and also in absence of additional nitrogen. Beneficial effect of pesticides on growth and development of sugarcane appears largely on account of more efficient utilisation of soil and fertilizer nitrogen. The results raise an interesting possibility of using these pesticides as growth stimulators to increase the efficiency of sugarcane plants growing under sub-optimal conditions of nitrogen     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Effect of piracetam, a nootropic agent, on rat brain monoamines and prostaglandins

Piracetam is the prototype of a new class of psychotropic drugs, the nootropic agents, which are claimed to selectively improve the higher telencephalic integrative activities. The effect of piracetam on rat brain monoamines and prostaglandins (PGs) was assessed so as to garner information on its mode of action. Two doses of the drug were used, a lower dose (20 mg/kg ip) and a higher dose (100 mg/kg, ip), the latter being known to exert a facilitatory effect on learning and memory. Piracetam produced a dose-related effect on rat brain serotonin (5HT) and noradrenaline (NA), with the lower dose inducing a decrease in 5HT levels and an increase in NA concentrations. The higher dose of piracetam produced the opposite effect. Dopamine (DA) levels were not significantly affected. The lower dose of the drug attenuated 5HT turnover and augmented that of NA, whereas the higher dose of piracetam produced the reverse effects, in clorgyline treated rats. The lower dose of piracetam produced a slight and statistically insignificant increase in rat brain PGE2 and PGF2 alpha. However, the higher dose of the drug produced marked increase in the levels of both the PGs. The observed biochemical effects may provide a basis for the nootropic effect of piracetam. However, they may also be due to the GA-BA-mimetic action of the drug, particularly those observed with the lower dose of piracetam.     

The Antimicrobial Efficiency of Root Oil Against Human Pathogenic Bacteria and Phytopathogenic Fungi

The in vitro antimierobial activity of root oil of C. orchioides were studied against human pathogenic bacteria and phytopathogenic fungi by comparing the results with standard microbial susceptibility testing biodises. The oil of C. orchioides in case of bacteria shows significant activity against Bacillus anthracis, B. subtilis, Salmonella pullorum, S. newport, and Staph. aureus. While in case of fungi root oil had excellent activity against Fusarium monili forme, F. solani, Asprgullus flavus and Cladosporium and shows significant activity against all other tested fungi.     

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3)

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the β₂-loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of ¹⁵N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx⁷²⁹DPEEIIVLSDSD⁷⁴⁰), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by β₂-loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of β₂-loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.