02108nas a2200241 4500008004100000022001400041245007600055210006900131260008100200300001200281490000700293520126000300653002801560653002601588653002401614653001401638653001401652100001701666700002201683700002401705700002501729856011201754 2015 eng d a0959-665800aInsect trehalase: physiological significance and potential applications0 aInsect trehalase physiological significance and potential applic aJOURNALS DEPT, 2001 EVANS RD, CARY, NC 27513 USAbOXFORD UNIV PRESS INCcAPR a357-3670 v253 a

Trehalose, a non-reducing disaccharide, is widespread throughout the biological world. It is the major blood sugar in insects playing a crucial role as an instant source of energy and in dealing with abiotic stresses. The hydrolysis of trehalose is under the enzymatic control of trehalase. The enzyme trehalase is gaining interest in insect physiology as it regulates energy metabolism and glucose generation via trehalose catabolism. The two forms of insect trehalase namely, Tre-1 and Tre-2, are important in energy supply, growth, metamorphosis, stress recovery, chitin synthesis and insect flight. Insect trehalase has not been reviewed in depth and the information available is quite scattered. The present mini review discusses our recent understanding of the regulation, mechanism and biochemical characterization of insect trehalase with respect to its physiological role in vital life functions. We also highlight the molecular and biochemical properties of insect trehalase that makes it amenable to competitive inhibition by most glycosidase inhibitors. Due to its crucial role in carbon metabolism in insects, application of inhibitors against trehalose can form a promising area towards formulating strategies for insect pest control.

10acarbohydrate metabolism10aglycosidase inhibitor10aglycoside hydrolase10atrehalase10atrehalose1 aShukla, Ekta1 aThorat, Leena, J.1 aNath, Bimalendu, B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/insect-trehalase-physiological-significance-and-potential-applications-002224nas a2200205 4500008004100000245011400041210006900155260000800224300001200232490000700244520150600251100001701757700001801774700002101792700002201813700001601835700001701851700002501868856012501893 2016 eng d00aMolecular cloning and in silico studies of physiologically significant trehalase from drosophila melanogaster0 aMolecular cloning and in silico studies of physiologically signi cNOV a282-2920 v923 aTrehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275 bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55 degrees C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure. (C) 2016 Elsevier B.V. All rights reserved.1 aShukla, Ekta1 aThorat, Leena1 aBhavnani, Varsha1 aBendre, Ameya, D.1 aPal, J., K.1 aNath, B., B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/molecular-cloning-and-silico-studies-physiologically-significant-trehalase-drosophila02008nas a2200157 4500008004100000245013300041210006900174260000800243300001200251490000700263520138000270100001701650700002801667700002501695856013001720 2017 eng d00aConformational and functional transitions and in silico analysis of a serine protease from conidiobolus brefeldianus (MTCC 5185)0 aConformational and functional transitions and in silico analysis cMAY a387-3970 v983 aThis work describes functional and structural transitions of a novel protease isolated from Conidiobolus brefeldianus MTCC 5185 (Cprot), in detail using biophysical and bioinformatics tools. The commercial importance of Cprot in silk and leather industries made it an interesting candidate for structural investigations. Cprot possesses 8.2%alpha-helix, 31.1% beta-sheet and 23.8% turns. The enzyme was found to be active over a wide pH range and up to 55 degrees C. The protease was also stable in organic solvents up to 50% (v/v) concentration of alcohols and DMSO for >24 h and in 2 M guanidine hydrochloride for >12 h. Cprot was also resistant to trypsin, chymotrypsin, proteinase K and fluorinated alcohols (5-10%). The melting temperatures observed for the native Cprot and for the enzyme treated under various stress conditions correlated well with the corresponding structural and functional transitions obtained. The structural information was supported by the homology model of its closest homologue from C. coronatus; revealing its similarity to PA clan of proteases (Proteases of mixed nucleophile, superfamily A), with His-64, Asp-113 and Ser-208 as putative catalytic triad. Three tryptophan residues in Cprot are surrounded by positively charged residues, as evident from solute quenching studies and homology model. (C) 2017 Elsevier B.V. All rights reserved.1 aShukla, Ekta1 aAgrawal, Sanskruthi, B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/conformational-and-functional-transitions-and-silico-analysis-serine-protease-conidiobolus02644nas a2200277 4500008004100000022001400041245011100055210006900166260000800235300000800243490000600251520174300257653002102000653002302021653001102044653002402055653001402079100001702093700001802110700002202128700002002150700002102170700002402191700002502215856012602240 2018 eng d a2190-572X00aCloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus0 aCloning and characterization of trehalase a conserved glycosidas cAUG a3520 v83 a

Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57-92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71-81). The modelled 3D tertiary structure of CrTre depicts a (alpha/alpha)(6) barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.

10aConserved motifs10aHomology modelling10aMidges10aSecondary structure10atrehalose1 aShukla, Ekta1 aThorat, Leena1 aBendre, Ameya, D.1 aJadhav, Santosh1 aPal, Jayanta, K.1 aNath, Bimalendu, B.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/cloning-and-characterization-trehalase-conserved-glycosidase-oriental-midge-chironomus02188nas a2200217 4500008004100000022001500041245008900056210006900145260000800214300001200222490000800234520147700242100002001719700001701739700001901756700001901775700002501794700001501819700002101834856011501855 2018 eng d a 0141-813000aThe Leishmania donovani IMPACT-like protein possesses non-specific nuclease activity0 aLeishmania donovani IMPACTlike protein possesses nonspecific nuc cNOV a962-9730 v1193 aIMPACT (Imprinted and Ancient)-like proteins are known to be regulators of GCN2 (General control non-derepressible 2) kinases involved in translation regulation. Here, we report on cloning and characterization of an IMPACT-like protein, LdIMPACT from Leishmania donovani which harbours two domains. 'RWD domain' at the N-terminal end that mediates GCN2 regulation, while a conserved 'ancient domain' lies at the C-terminal end whose function remains elusive. Interestingly, our observations indicated that LdIMPACT has a novel nonspecific nuclease activity. In silico analysis further revealed the resemblance of ancient domain of LdIMPACT to RNase PH domain (known to bind to nucleic acids). The recombinant LdIMPACT exhibited a Mg2+-dependent nuclease activity. Moreover, thermostability and pH stability assays of the protein suggest it to be a stress responsive protein. Circular dichroism studies elucidated the conformational transitions of the enzyme in response to various temperature and pH conditions which correlated well with the activity profiles. Thus, the current study highlights the structural and functional characteristics of LdIMPACT which interestingly also possesses a novel nuclease activity. With its physiological relevance unresolved, the multifaceted LdIMPACT might therefore lie in a hitherto unknown network, whose perturbation could be an attractive therapeutic approach for treating leishmaniasis. (C) 2018 Elsevier B.V. All rights reserved.1 aRao, Shilpa, J.1 aShukla, Ekta1 aBhatia, Varsha1 aLohiya, Bharat1 aGaikwad, Sushama, M.1 aKar, Anita1 aPal, Jayanta, K. uhttps://library.ncl.res.in/content/leishmania-donovani-impact-protein-possesses-non-specific-nuclease-activity02329nas a2200229 4500008004100000022001400041245010400055210006900159260000800228300001200236490000700248520151100255653001301766653003601779653003801815653002501853653002901878100002201907700001701929700002601946856012701972 2020 eng d a1572-388700aFunctional stability and structural transitions of a kunitz trypsin inhibitor from chickpea (CaTI2)0 aFunctional stability and structural transitions of a kunitz tryp cAUG a350-3570 v393 a

Enzymes are important tools for various applications. We have studied structural transitions and functional stability of a Kunitz trypsin inhibitor from Chickpea (CaTI2), a potent insect gut-protease inhibitor, under different stress conditions like non-neutral pH, elevated temperature and co-solvent concentrations. CaTI2 was cloned and expressed in an eukaryotic systemP. pastorisand was investigated for conformational transitions using circular dichroism spectroscopy, differential scanning fluorimetry and activity assay. Native CaTI2 has a sheet dominant structure with 40% beta sheets and possess a single tryptophan residue situated in the hydrophobic core of the enzyme. The recombinant inhibitor maintained its maximum activity under alkaline pH with its secondary structure intact between pH 6-10. CaTI2 was observed to be thermally stable up to 55 degrees C with aT(m) of 61.3 degrees C above which the protein unfolds. On treating with chemical denaturant (urea), the CaTI2 lost its inhibitory potential and native conformation beyond 2 M urea concentration. Moreover, the protein unfolded at lower temperatures as the concentration of denaturant increased, suggesting more complex structural changes. Further, the stability of the inhibitor was found to be directly proportional to the solvent polarity. The data, herein offers significant information of inhibitor stability and activity which could be exploited for its further development into an effective pesticide. [GRAPHICS] .

10aChickpea10aCircular dichroism spectroscopy10aDifferential scanning fluorimetry10aFunctional stability10aKunitz trypsin inhibitor1 aBendre, Ameya, D.1 aShukla, Ekta1 aRamasamy, Sureshkumar uhttps://library.ncl.res.in/content/functional-stability-and-structural-transitions-kunitz-trypsin-inhibitor-chickpea-cati202434nas a2200277 4500008004100000022001400041245011600055210006900171260000900240300001200249490000800261520151600269653001501785653001501800653002801815653002701843653001501870653002001885100001801905700002101923700001701944700001701961700002201978700002402000856013202024 2020 eng d a0141-813000aStructural and physical analysis of underwater silk from housing nest composites of a tropical chironomid midge0 aStructural and physical analysis of underwater silk from housing cNOV  a934-9420 v1633 a

Chironomids are an abundant group of aquatic silk spinning insects. They offer a unique opportunity of silk harvestation without killing them; however, they remained underappreciated models in silk research. Here, we investigate the structural and biomechanical characteristics of silk from the midge, Chironomus ramosus. A combination of microscopic (SEM), spectroscopic (CD and IR), structural (XRD), thermal (DSC and TGA) and mechanical measurement tools and techniques were employed to gain critical insights on midge silk. Maximum yield of silk was obtained from Chironomus in similar to 2.5 h, the shortest time reported among insects. The network of water-insoluble silk fibres possessed the smallest diameter of 110 +/- 35 nm, known for any insect silk, qualifying its superiority in fibre fineness. We demonstrate a cruelty-free silk extraction method in contrast to the conventional violent techniques. Structural characterization indicated coexistence of various secondary conformations, beta sheets being predominant. We compare and contrast these features to well-characterized caddisfly and silk-worm silks and highlight the uniqueness in midge silk that render mechanical stability and potentially contribute to its multi-functionalization. We thus propose Chironomus as an emerging candidate of water-borne silk, especially in the context of the `Peace silk' industry, aiming to develop non-violent methods for silk harvestation from animals. (C) 2020 Elsevier B.V. All rights reserved.

10abiopolymer10aChironomus10aCommercial applications10aHousing nest composite10aPeace silk10aUnderwater silk1 aThorat, Leena1 aJoseph, Emmanuel1 aNisal, Anuya1 aShukla, Ekta1 aRaviKumar, Ameeta1 aNath, Bimalendu, B. uhttps://library.ncl.res.in/content/structural-and-physical-analysis-underwater-silk-housing-nest-composites-tropical-chironomid01267nas a2200181 4500008004100000022001400041245013200055210006900187260000800256300001600264490000700280520060300287100001700890700002200907700001700929700001500946856012400961 2025 eng d a0022-326300aIpso-cyclization via a dienone-phenol rearrangement of alkynyloxy cyclohexadienones for the synthesis of oxaspiro[4.5]trienones0 aIpsocyclization via a dienonephenol rearrangement of alkynyloxy  cSEP a13399-134060 v903 a

Herein, we present a convenient method to access oxaspiro[4.5]trienone compounds from alkynyloxy cyclohexadienones. The reactions are catalyzed by Lewis/Bronsted acids, yielding the desired products in moderate to good yields under very mild reaction conditions. The process, which involves a dienone-phenol rearrangement, is characterized by its simplicity and the absence of any additional activators. The synthesis of the oxaspiro[4.5]trienone compounds is amenable to gram-scale reactions along with subsequent product transformations, making this approach practical and attractive.

1 aRai, Archana1 aKundu, Ananya, I.1 aShukla, Ekta1 aDas, Utpal uhttps://library.ncl.res.in/content/ipso-cyclization-dienone-phenol-rearrangement-alkynyloxy-cyclohexadienones-synthesis