Development of Prey Capture in the Indian False Vampire Bat Megaderma Lyra

We studied development of prey capture, under captive conditions, on five newly weaned Indian false vampire bats Megaderma lyra. We tested the hypothesis that the young bats are able to improve the ability of hunting by trial and error. An additional hypothesis was that their rate of prey consumption and method of handling prey improve with age. We separated the juveniles from their mothers and observed them individually at different ages. At 58 d of age, M. lyra roosted 2 m above floor level, flew down only towards moving frogs, landed >1 m away from them and returned to roosts without showing attempt to capture. At day 60, bats roosted at 50‐cm heights, landed approx. 1 m away from frogs and moved towards them. Although bats intercepted frogs, their attempts to capture were still unsuccessful. At day 62, bats landed <1 m from frogs, made similar attempts and most of them were successful. Bats carried frogs, roosted at heights of 100 cm and started consuming. Numbers of jumps made by frogs and attempts made by bats during successful captures decreased with increase in age of bats. Furthermore, the distance between landing sites of bats and places where frogs stayed during landings decreased with advancement of age. Until 75 d of age, M. lyra devoured frogs with legs‐first on significantly more occasions. After 75 d, most of the consumptions started with heads of frogs, similar to adults. Duration of feeding on single frogs was significantly longer compared with that of mothers until young were 75‐d old. The study revealed that juveniles were inefficient on localization, rate of consumption, and handling prey at earlier ages (e.g. 60 d). They became efficient on these behavioural components at later age, i.e. after 75 d. Thus, the results were in accordance with both the hypotheses. Bats did not respond to stationary frogs.     

Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice.⁷ Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, β-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.Key words: sheath blight, Zizyphus jujuba, Ipomoea carnea, Rhizoctonia solani, induced systemic resistance, antimicrobial compoundsSheath blight disease of rice, caused by Rhizoctonia solani, has become a major production constraint in intensive rice cropping systems where semi-dwarf, nitrogen-responsive and high-yielding rice cultivars are grown. The disease causes an annual yield loss of upto 50%.¹ R. solani is both soil- and water-borne, and can infect more than 27 families of both monocot and dicot species.² Natural host genetic resistance to R. solani has not been recorded in cultivars or wild relatives of rice.³ Several broad spectrum fungicides have been recommended for control of sheath blight, however, chemical method of disease management is neither practical due to high cost of fungicides nor sustainable as it can affect the balance of ecosystem by destroying beneficial microbial population. In addition, the environmental pollution problems associated with indiscriminate use of synthetic pesticides have prompted investigations on exploiting bio-pesticides of plant and microbial origin.Plants accumulate an enormous variety of over 100,000 secondary metabolites,⁴ which can act as pre-existing chemical inhibitors to invading pathogens and/or help strengthen defense response of host plant. The pre-formed infectional barriers in plants are generally referred to as “phytoanticipins;” whereas, the antimicrobial compounds that are synthesized de novo in response to pathogen attack are referred to as “phytoalexins.”⁵ Because of years of selective breeding leading to removal of natural products, the endogenous levels of phytoanticipins in commonly cultivated crop species are generally low and often not sufficient to fight pathogen attack, effectively.⁴ Various weed species and wild relatives of crop plants that are not subjected to selective breeding are believed to contain higher levels of antimicrobial compounds, consistent with their ability to fight invading pathogens more effectively than cultivated crop species. Identification of such weed/plant species that are enriched with antimicrobial principles, isolation of bio-active compounds from them, and application in the form of concentrated formulations to crop plants can augment their disease resistance capability by directly inhibiting the growth of pathogen and inducing defense responses. Indeed, the antimicrobial properties of tissue extracts of several weed/plant species have been reported by a number of research groups world-wide, especially in Asia and Latin America.^6–13Earlier, we had evaluated the antimicrobial activity of leaf extracts of 16 different plant species belonging to 16 different families and demonstrated that leaf extracts of most of these plant species exhibit growth-inhibitory activities against R. solani and Xanathomoas oryzae pv. oryzae (Xoo).⁷ Among these, the leaf extracts of Datura metel were found to be the most effective in inhibiting the mycelial growth and sclerotia formation of R. solani, and the growth of Xoo, as well as in reducing the incidence of sheath blight and bacterial blight diseases caused by these pathogens, respectively, under greenhouse condition.⁷ We further demonstrated that rice seedlings treated with leaf extracts of D. metel accumulated significantly higher levels of pathogenesis-related (PR) proteins and other defense related compounds following challenge inoculation with R. solani or Xoo.⁷ Our attempts to identify biologically active compounds from D. metel revealed the presence of a withanolide compound “daturilin” that exhibited remarkable antibacterial activity against Xoo.⁷Apart from D. metel, two other plants species, Zizyphus jujuba and Ipomoea carnea, were found to possess remarkable antifungal activity against R. solani.⁷ Z. jujuba is a thorny rhamnaceous plant that is widely distributed in Europe and South-eastern Asia. I. carnea of convolvulaceae family, commonly known as morning glory, is a toxic weed found in abundance in India, Brazil, the United States and other countries.¹⁴ Both of these plant species have allelopathic effect and are commonly used in folklore medicine for curing multiple diseases.^15–18 The aqueous and methanol leaf extracts of Z. jujuba and I. carnea have been found to be highly effective in reducing in vitro mycelial growth, and therefore, sclerotia production of R. solani.⁷ In the greenhouse experiments, rice seedlings sprayed with leaf extracts of Z. jujuba and I. carnea exhibited 44 and 34% reduction in severity of sheath blight disease over the control, respectively.⁷ While these findings are encouraging, the mechanisms by which the leaf extracts of Z. jujuba and I. carnea modulate defense responses in rice have not yet been explored.Plants are endowed with defense genes which remain quiescent or are expressed at basal levels in healthy plants. Activation of defense genes results in induction of systemic resistance in host plant; this defense response, designated as induced systemic resistance (ISR), plays an important role in development of disease resistance.¹⁹ The onset of ISR in plants correlates with accumulation of phytoalexins and increased activity of PR proteins such as chitinases, β-1,3-glucanases and peroxidases;^20–23 consequently, PR proteins are generally used as ISR markers.¹⁹ The classical inducers of ISR include both biotic and abiotic factors, including disease causing microorganisms themselves,^24,25 plant growth promoting rhizobacteria,^22,26 chemicals^27,28 and natural plant products.^{7,10,12,13,29,30} Plant products have been considered as one of the major groups of compounds that induce ISR. To date, extracts of at least a few plant species have been reported to contain allelopathic substances which can act as elicitors and induce systemic resistance in host plants resulting in reduction or inhibition of disease development.^7,10,12,13In the present study, with the objective of understanding the mechanisms of disease suppression by leaf extracts of Z. jujuba and I. carnea, we investigated their ability to induce ISR in rice by analyzing the activities of ISR markers including PR-proteins and other defense enzymes involved in phenylpropanoid metabolism. The changes in activities of chitinase, β-1,3-glucanase, peroxidase, phenylalanine ammonia-lyase (PAL) and phenolic compounds induced in rice seedlings that were elicited with leaf extracts (at 1:10 dilution; w/v) of Z. jujuba or I. carnea and infected with R. solani were analyzed, and compared to changes in non-elicited and uninfected seedlings. Rice seedlings that were both elicited with leaf extracts of Z. jujuba or I. carnea and infected with R. solani accumulated significantly higher levels (2–5-fold) of ISR markers as compared to non-elicited and/or uninfected seedlings (Fig. 1). About two-fold increase in activities of ISR markers was also observed in seedlings that were either infected but not elicited or elicited but not infected; however, this increase was significantly lower than the changes in seedlings that were both elicited and infected (Fig. 1). Although the activity of all ISR markers began to increase around or after 24 h post-infection, at least two distinct induction patterns were observed. For instance, the activities of chitinase and phenolic substances gradually increased to reach maximum levels at 164 h post-infection (Fig. 1A and E); whereas, the activities of β-1,3-glucanase, peroxidase and PAL reached maximum levels at 72 to 96 h post-infection and decreased thereafter (Fig. 1B–D). The leaf extracts of Z. jujuba were found slightly more effective in inducing ISR markers than the leaf extracts of I. carnea. There was no significant change in the activity of ISR markers in control seedlings sprayed with sterile distilled water (Fig. 1). Collectively, these results suggested that the leaf extracts of Z. jujuba and I. carnea have the ability to induce systemic resistance in rice seedlings infected with R. solani. The fungitoxicity of the leaf extracts of Z. jujuba and I. carnea ⁷ combined with their ability to elicit ISR is possibly responsible for low sheath blight disease incidence observed in rice seedlings treated with these leaf extracts.⁷Open in a separate windowFigure 1Activity of ISR markers and defense-related compounds in rice seedlings elicited with the leaf extracts of Zizyphus jujuba or Ipomoea carnea and challenge inoculated with Rhizoctonia solani. Total activity of chitinase (A), β-1,3-glucanase (B), peroxidase (C) phenylalanine ammonia-lyase (PAL; D) and phenolic substances (E) was analyzed in rice seedlings. The inoculation of rice seedlings with R. solani was performed 45 days after planting. Spraying of leaf extracts (1:10 dilution; w/v) of Z. jujuba or I. carnea was performed two days prior to inoculation. Tissue samples (sheath) from elicited and/or infected seedlings were collected for analysis at various time intervals.The in vitro antimicrobial and in vivo disease inhibitory effects of natural plant products are generally attributed to the allelopathic substances present in them. However, very few attempts have been made to purify and characterize active principles from bio-active natural plant products. We have previously identified a withanolide compound from leaf extracts of D. metel which exhibited antibacterial activity against Xoo.⁷ Both Z. jujuba and I. carnea are rich source of secondary metabolites including alkaloids, terpenoids, flavonoids and phenolic compounds.^31–35 To determine the composition of bio-active ingredients within the leaf extracts of Z. jujuba and I. carnea, we performed thin layer chromatographic separation of alkaloid, terpenoid and phenolic compounds. The partially purified compounds, as reported in

Characterization of cellobiohydrolase from a newly isolated strain of Agaricus arvencis

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency (kcat/Km) of 31.8 mM?1 s?1 for p-nitrophenyl-beta-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.     

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.     

Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181

An isolated gene from Neosartorya fischeri NRRL181 encoding a β-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V _max of 886 μmol min⁻¹ mg-protein⁻¹ and a K _m of 68 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.     

Computational selection of flavonoid compounds as inhibitors against SARS-CoV-2 main protease,RNA-dependent RNA polymerase and spike proteins: A molecular docking study

An outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been recognized as a global health concern. Since, no specific antiviral drug is proven effective for treatment against COVID-19, identification of new therapeutics is an urgent need. In this study, flavonoid compounds were analyzed for its inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. Virtual docking was performed for screening of flavonoid compounds retrieved from PubChem against the main protease of SARS-CoV-2 using COVID-19 docking server. The cut off of dock score was set to >?9 kcal/mol and screened compounds were individually docked against main protease, RNA-dependent RNA polymerase, and spike proteins using AutoDock 4.1 software. Finally, lead flavonoid compounds were subjected to ADMET analysis. A total of 458 flavonoid compounds were virtually screened against main protease target and 36 compounds were selected based on the interaction energy value >?9 kcal/mol. Furthermore, these compounds were individually docked against protein targets and top 10 lead compounds were identified. Among the lead compounds, agathisflavone showed highest binding energy value of ?8.4 kcal/mol against main protease, Albireodelphin showed highest dock score of ?9.8 kcal/mol and ?11.2 kcal/mol against RdRp, and spike proteins, respectively. Based on the high dock score and ADMET properties, top 5 lead molecules such as Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate were identified as potent inhibitors against main protease, RdRp, and spike protein targets of SARS-CoV-2. These all compounds are having non-carcinogenic and non-mutagenic properties. This study finding suggests that the screened compounds include Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate could be the potent inhibitors of SARS-CoV-2 targets.     

Zebavidin - An Avidin-Like Protein from Zebrafish

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.     

Characterization of an L-arabinose isomerase from Bacillus subtilis

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k _cat of 14,504 min⁻¹ and a k _cat/K _m of 121 min⁻¹ mM⁻¹ for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.