Green Synthesis of Selenium Nanoparticles Mediated by Nilgirianthus ciliates Leaf Extracts for Antimicrobial Activity on Foodborne Pathogenic Microbes and Pesticidal Activity Against Aedes aegypti with Molecular Docking.

The present study deals with the synthesis of selenium nanoparticles (SeNPs) using Nilgirianthus ciliatus leaf extracts, characterized by UV-Vis spectrophotometer, XRD, FTIR, FE-SEM, HR-TEM, DLS, and zeta potential analysis. The antimicrobial activity against Staphylococcus aureus (MTCC96), Escherichia coli (MTCC443), and Salmonella typhi (MTCC98) showed the remarkable inhibitory effect at 25 µl/mL concentration level. Furthermore, the characterized SeNPs showed a great insecticidal activity against Aedes aegypti in the early larval stages with the median Lethal Concentration (LC50) of 0.92 mg/L. Histopathological observations of the SeNPs treated midgut and caeca regions of Ae. aegypti 4th instar larvae showed damaged epithelial layer and fragmented peritrophic membrane. In order to provide a mechanistic approach for further studies, molecular docking studies using Auto Dock Vina were performed with compounds of N. ciliatus within the active site of AeSCP2. Overall, the N. ciliates leaf-mediated biogenic SeNPs was promisingly evidenced to have potential larvicidal and food pathogenic bactericidal activity in an eco-friendly approach.

Cloning, expression, homology modelling and molecular dynamics simulation of four domain-containing α-amylase from Streptomyces griseus.

In the present study, α-amylase from Streptomyces griseus TBG19NRA1 was amplified, cloned and successfully expressed in E. coli BL21/DE3. Sequence analysis of S. griseus α-amylase (SGAmy) revealed the presence of four domains (A, B, C and E). Alpha-amylases with E domain (also known as carbohydrate binding module 20 (CBM20)) are capable of degrading raw starch and this property holds great potential for application in starch processing industries. Though α-amylase is a well-studied and characterized enzyme, there is no experimental structure available for this four domain-containing α-amylases. To gain more insight about SGAmy structure and function, homology modelling was performed using a multi-template method. The template α-amylase from Pseudoalteromonas haloplanktis (PDB ID 1AQH) and E domain of Cyclodextrin glucanotransferase from Bacillus circulans (PDB ID 1CGY) was found to have significant similarity with the complete target sequence of SGAmy. Therefore, homology model for SGAmy was generated from the crystal structure of 1AQH and 1CGY and the resulting structure was subjected to 10 ns molecular dynamics (MD) simulation. Remarkably, CBM20 domain of SGAmy showed greater flexibility in MD simulation than other three domains. This observation is highly rational as this part of SGAmy is strongly implicated in substrate (raw starch) binding. Thus, conformational plasticity at CBM20 is functionally beneficial.Communicated by Ramaswamy H. Sarma.

Antiquorum sensing and antibiofilm potential of biosynthesized silver nanoparticles of Myristica fragrans seed extract against MDR Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers and typhoid patients.

Globally, Salmonella infection poses a major public health problem. Here, we report antibiofilm activity and quorum sensing inhibition of aqueous seeds extract of Myristica fragrans (nutmeg) and biosynthesized silver nanoparticles (AgNPs) against multidrug resistant (MDR) Salmonella enterica serovar Typhi (S. Typhi) isolated from typhoid patients and asymptomatic carriers. S. Typhi isolates revealed higher percentage (46%) of biofilm production identified by tissue culture plate (TCP) than Congo red agar (CRA) and tube adherence (TA) methods. The inhibition of biofilm-producing MDR S. Typhi isolates and pigment production of Chromobacterium violaceum (indicator bacteria) demonstrated the quorum sensing potential of nutmeg. The aqueous seed extract of nutmeg exhibited 87% of antibiofilm activity, while the biosynthesized AgNPs showed 99.1% of antibiofilm activity. Molecular docking studies of bioactive compounds of nutmeg against transcriptional regulatory protein RcsB and sensor kinase protein RcsC revealed interaction with the target proteins. It is proposed that biosynthesized AgNPs could be used as one of the effective candidates in treating asymptomatic typhoid carriers or typhoid patients and to control the subsistence of biofilm-producing S. Typhi strains or other pathogenic bacteria in the environment or industrial settings.

Isolation, characterization, and therapeutic activity of bergenin from marlberry (Ardisia colorata Roxb.) leaf on diabetic testicular complications in Wistar albino rats.

Bergenin is one of the phytochemical constituents in marlberry (Ardisia colorata Roxb.) having antioxidant, anti-diabetic, and anti-inflammatory properties. A. colorata has been used as an herbal medicine in Southeast Asia particularly in Northeast India to treat diabetes. Bergenin was isolated from methanol extract of A. colorata leaf (MEACL) by column chromatography and TLC profiling. Characterization and structural validation of bergenin were performed by spectroscopic analyses. A LC-ESI-MS/MS method was developed for the quantitation of bergenin and validated as per the guidelines of FDA and EMA. The validated method was successfully utilized to quantify bergenin concentration in MEACL samples. Therapeutic efficacy of bergenin was investigated on streptozotocin-induced diabetic rats by following standard protocols. Bergenin supplementation significantly improved the physiological and metabolic processes and in turn reverses diabetic testicular dysfunction via increasing serum testosterone concentrations and expression pattern of PCNA, improving histopathological and histomorphometric manifestations, modulating spermatogenic events and germ cell proliferation, restoring sperm quality, reducing sperm DNA damage, and balancing the antioxidant enzymes levels. Hence, A. colorata leaf is one of the alternate rich resources of bergenin and could be used as a therapeutic agent for diabetic testicular complications.

Testicular toxicity and sperm quality following copper exposure in Wistar albino rats: ameliorative potentials of L-carnitine.

Copper is a persistent toxic and bio-accumulative heavy metal of global concern. Continuous exposure of copper compounds of different origin is the most common form of copper poisoning and in turn adversely altering testis morphology and function and affecting sperm quality. L-carnitine has a vital role in the spermatogenesis, physiology of sperm, sperm production and quality. This study was designed to examine whether the detrimental effects of long-term copper consumption on sperm quality and testis function of Wistar albino rat could be prevented by L-carnitine therapy. The parameters included were sperm quality (concentration, viability, motility, and morphology), histopathology, serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), serum urea, serum creatinine, serum testosterone and testis antioxidant enzyme levels (superoxide dismutase and glutathione-S-transferase), and biomarkers of oxidative stress (lipid peroxidation and expression of heat shock protein 70 in testis). Three-month-old male Wistar rats (n = 30) were divided into six groups as group 1 (G1, 0.9% saline control), group 2 (G2, CuSO4 200 mg/kg dissolved in 0.9% saline water), groups 3 and 4 (G3 and G4, L-carnitine 50 and 100 mg/kg dissolved in 0.9% saline water, respectively), and groups 5 and 6 (G5 and G6, CuSO4 200 mg/kg plus L-carnitine, 50 and 100 mg/kg dissolved in 0.9% saline water, respectively). Doses of copper (200 mg/kg) and L-carnitine (50 and 100 mg/kg) alone and in combinations along with untreated control were administered orally for 30 days. The following morphological, physiological, and biochemical alterations were observed due to chronic exposure of copper (200 mg/kg) to rats in comparison with the untreated control: (1) generation of oxidative stress through rise in testis lipid peroxidation (12.21 vs 3.5 nmol MDA equivalents/mg protein) and upregulation of heat shock protein (overexpression of HSP70 in testis), (2) liver and kidney dysfunction [elevation in serum ALT (81.65 vs 48.08 IU/L), AST (156.82 vs 88.25 IU/L), ALP (230.54 vs 148.16 IU/L), urea (12.65 vs 7.45 mmol/L), and creatinine (80.61 vs 48.25 µmol/L) levels], (3) significant decrease in body (99.64 vs 106.09 g) and organ weights (liver-3.48 vs 4.99 g; kidney-429.29 vs 474.78 mg; testes-0.58 vs 0.96 g), (4) imbalance in hormonal and antioxidant enzyme concentrations [significant decline in serum testosterone (0.778 vs 3.226 ng/mL), superoxide dismutase (3.07 vs 8.55 µmol/mg protein), and glutathione-S-transferase (59.28 vs 115.58 nmol/mg protein) levels], (5) severe alterations in the testis histomorphology [sloughed cells (90.65%, score 4 vs 15.65%, score 1), vacuolization (85.95%, score 4 vs 11.45%, score 1), cellular debris along with degenerative characteristics, accentuated germ cell depletion in the seminiferous epithelium, severe damage of spermatogonia and Sertoli cells (73.56%, score 3 vs 0%, score 1)], (6) suppression of spermatogenic process [hypospermatogenesis (low Jhonsen testicular biopsy score 4 vs 9.5), decrease in tubules size (283.75 vs 321.25 µm in diameter), and no. of germ cells (81.8 vs 148.7/100 tubules), Leydig cells (5.2 vs 36.65/100 tubules), and Sertoli cells (8.1 vs 13.5/100 tubules)], (7) sperm transit time was shorter in caput and cauda and ensued in incomplete spermatogenic process and formation of immature sperm leading to infertility, (8) sperm quality was affected significantly [decreased daily sperm production (13.21 vs 26.9 × 106 sperms/mL), sperm count (96.12 vs 154.25 × 106/g), sperm viability (26.88 vs 91.65%), and sperm motility (38.48 vs 64.36%)], and (9) increase of head (32.82 vs 2.01%) and tail (14.85 vs 0.14%) morphologic abnormalities and DNA fragmentation index (88.37 vs 11.11%). Oxidative stress and their related events appear to be a potential mechanism involved in copper testicular toxicity and L-carnitine supplementation significantly modulated the possible adverse effects of copper on seminiferous tubules damage, testes function, spermatogenesis, and sperm quality. It was validated that the use of L-carnitine at doses of 50 and 100 mg/kg protects against copper-induced testicular tissue damage and acts as a therapeutic agent for copper heavy metal toxicity.

Biosterilant effects of Bacillus thuringiensis kurstaki HD-73 extract on male Wistar albino rats.

Chemosterilants have long been used in sterilization programs for managing pet, stray, and wild animals but adverse effects such as trauma, incomplete responses to treatment, and complete abolition of gonadal sources of testosterone often occurs. This study describes the biosterilant effects of administering three doses of Bacillus thuringiensis kurstaki HD-73 (Bt 5, 50, and 250 mg/kg; 20 rats/dose) extract in adult male Wistar albino rats on testicular parameters, function, histology, and a number of biochemical markers of overall health, free radical production, and cell proliferation. Intratesticular administration of Bt extract to rats induces testicular oxidative stress and damages and consequently, perturb spermatogenesis, degeneration of testis, reduction in testes size, and depletion of testosterone and antioxidant enzyme concentrations in a dose-dependent manner because of free radical-mediated lipid peroxidation. No morbidity or mortality adverse effects were observed in both the saline control and Bt extract-treated rats. Significant variation was noted in clinical manifestations, weight and volume of testes, and hormonal and biochemical profiles between Bt doses in comparison with the saline control. Aspermia/azoospermia (100%) resulted in Bt-treated rats without any adverse effects. Histopathological analysis showed degeneration, necrosis, vacuolation, fewer germ cells, formation of multinucleated giant cells, and a lack of elongated spermatids in atrophic seminiferous tubules in Bt extract-treated groups in the presence of low concentrations of testosterone, antioxidant enzymes, and suppression of germ cell proliferation. Dose-dependent effects were evident in most parameters that were measured. The vast array of tests that were undertaken also provides some important indicators of the physiological effects associated with the treatments that were applied. Intratesticular injection of Bt extract impairs spermatogenesis and induces permanent sterility in rats.

Influence of shifting cultivation practices on soil-plant-beetle interactions.

Shifting cultivation (jhum) is a major land use practice in Mizoram. It was considered as an eco-friendly and efficient method when the cycle duration was long (15-30 years), but it poses the problem of land degradation and threat to ecology when shortened (4-5 years) due to increased intensification of farming systems. Studying beetle community structure is very helpful in understanding how shifting cultivation affects the biodiversity features compared to natural forest system. The present study examines the beetle species diversity and estimates the effects of shifting cultivation practices on the beetle assemblages in relation to change in tree species composition and soil nutrients. Scarabaeidae and Carabidae were observed to be the dominant families in the land use systems studied. Shifting cultivation practice significantly (P < 0.05) affected the beetle and tree species diversity as well as the soil nutrients as shown by univariate (one-way analysis of variance (ANOVA), correlation and regression, diversity indices) and multivariate (cluster analysis, principal component analysis (PCA), detrended correspondence analysis (DCA), canonical variate analysis (CVA), permutational multivariate analysis of variance (PERMANOVA), permutational multivariate analysis of dispersion (PERMDISP)) statistical analyses. Besides changing the tree species composition and affecting the soil fertility, shifting cultivation provides less suitable habitat conditions for the beetle species. Bioindicator analysis categorized the beetle species into forest specialists, anthropogenic specialists (shifting cultivation habitat specialist), and habitat generalists. Molecular analysis of bioindicator beetle species was done using mitochondrial cytochrome oxidase subunit I (COI) marker to validate the beetle species and describe genetic variation among them in relation to heterogeneity, transition/transversion bias, codon usage bias, evolutionary distance, and substitution pattern. The present study revealed the fact that shifting cultivation practice significantly affects the beetle species in terms of biodiversity pattern as well as evolutionary features. Spatiotemporal assessment of soil-plant-beetle interactions in shifting cultivation system and their influence in land degradation and ecology will be helpful in making biodiversity conservation decisions in the near future.

Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress.

Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560 ± 48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350 nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

AP-APSE dpol intein: A novel family A DNA polymerase intein domain.

Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in organisms from all three kingdoms of life - eucarya, bacteria and archaea, but their distribution is sporadic. Here we report the identification and bioinformatics characterization of intein in DNA polymerase A gene of bacteriophage APSE (Acyrthosiphon pisum Secondary Endosymbiont bacteriophage) infecting the Aphid secondary endosymbionts of eukaryotic insects such as Acyrthosiphon pisum, Uroleucon rudbeckiae. The insertion site of intein within APSE family A DNA polymerase extein was identified to be dpola. Hence we propose this as a unique intein of family A DNA polymerase (dpola insertion site) and only reported intein in podoviridae family.

Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine ß-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55.

The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.