Isolation of ß-glucan from Eleusine coracana and its antibiofilm, antidiabetic, antioxidant, and biocompatible activities.

Eleusine coracana (Finger millet) has high nutritional value with numerous health benefits and is of low cost. Isolation of beta-glucan (ßG) from E. coracana (Ec-ßG) has gained increasing research attention. UV-vis spectroscopy used to measure the surface plasmon resonance at 361 nm to confirm the presence of polysaccharides (glucan molecules) in Ec-ßG. X-ray diffraction analysis of Ec-ßG displayed a crystalline nature and confirmed the presence of the ßG molecule. Further, the bioactive compounds of Ec-ßG were screened using gas chromatography-mass spectrometry. The antibacterial activity of Ec-ßG against both Gram-positive (Lysinibacillus fusiformis, Enterococcus faecalis) and Gram-negative (Proteus vulgaris, Shigella sonnei) bacteria were assessed through minimum inhibitory concentrations <70 µg/ml of Ec-ßG. In addition, the antibiofilm activity and bacterial viability of Ec-ßG at 100 µg/ml was confirmed by light and confocal laser scanning microscopy. Furthermore, Ec-ßG inhibits α-amylase and α-glucosidase at an IC50 -value of 1.23 and 1.42 µg/ml, respectively. Superoxide anion scavenging activity at IC50-1.4 µg/ml and DPPH radical scavenging activity at IC50-1.2 µg/ml showed that Ec-ßG had potential antioxidant property. The in vitro hemolysis assay for biocompatibility of Ec-ßG at 200 µg/ml showed 0.06 ± 0.09%. Therefore, Ec-ßG has the potential to act as a suggestive agent for antibacterial, antidiabetic, and antioxidant activity.

Antibiofilm and anticancer potential of ß-glucan-binding protein-encrusted zinc oxide nanoparticles.

ß-Glucan-binding protein (ßGBP) is important for the rational expansion of molecular biology. Here, zinc oxide nanoparticle (ZnONP) was synthesized using ßGBP from the crab Scylla serrata (Ss-ßGBP-ZnONP). Ss-ßGBP-ZnONP was observed as a 100 kDa band on sodium dodecyl sulfate polyacrylamide gel and characterized with UV-vis spectroscopy at 350 nm. X-ray diffraction analysis displayed values consistent with those for zincite. Fourier transform infrared spectroscopy revealed the presence of functional groups, including amide, alcohol, alkane, alkyl halide, and alkene groups. The zeta potential (-5.36 mV) of these particles indicated their stability, and transmission electron microscopy revealed the presence of 50 nm nanocones. Ss-ßGBP-ZnONPs were tested at 100 µg/mL against the gram-positive Enterococcus faecalis and gram-negative Pseudomanas aeruginosa using confocal laser scanning microscopy and the bacterial viability assay was also performed. The growth of MCF7 breast cancer cells was inhibited following treatment with 75 µg/mL Ss-ßGBP-ZnONPs. Thus, Ss-ßGBP-ZnONPs have the ability to control the growth of pathogenic bacteria and inhibit the viability of MCF7 breast cancer cell lines.

Antimicrobial and biochemical characterization of a C-type lectin isolated from pearl spot (Etroplus suratensis).

The present study reveals purification and characterization of a C-type lectin from the serum of pearl spot, Etroplus suratensis (Es-Lec). The Es-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 75 kDa in SDS-PAGE. The surface morphology of purified Es-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 2.958 min was appeared in high performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis expresses a single peak at 31.8372̊ and MALDI-TOF peaks which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Es-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy and haemagglutination inhibition was also done. In addition, purified Es-Lec showed the broad spectrum of antibacterial activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila. Antibiofilm potential of purified Es-Lec against selected Gram-negative bacteria exhibited the disruption of biofilm architecture at the concentration of 50 µg ml-1 and also it exhibited antiviral and anticancer activity.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties.

The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp-Lec). The Pp-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 155 kDa in SDS-PAGE. The surface morphology of purified Pp-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X-ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp-Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp-Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp-Lec showed the broad spectrum of antibacterial activity against Gram-positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp-Lec against selective Gram-negative bacteria showed the disruption of biofilm architecture at the concentration of 50 µg ml-1.

Haemolytic and antibiofilm properties of haemocyanin purified from the haemolymph of Indian white shrimp Fenneropenaeus indicus.

Haemocyanin (Hc) is an important non-specific immune macromolecule present in the haemolymph of both mollusks and crustaceans. In the present study, Hc was purified from the haemolymph of Indian white shrimp Fenneropenaeus indicus by gel filtration chromatography and it exhibits a single band with a molecular weight of 74 kDa on SDS-PAGE. The X-ray diffraction (XRD) and High performance liquid chromatography (HPLC) result of purified Hc express single peak at 31.5° be a sign of crystalline nature and appear as a single peak with a retention time of 5.6 min signify the homogeneity nature of the protein respectively. The purified Hc exhibited haemolytic activity against chicken erythrocytes. The haemolytic activity of purified Hc in optimum conditions observed to be pH 6.0, temperature 40 °C, in the presence of calcium. As well purified Hc exhibited the antibiofilm activity against both Gram positive and Gram negative bacteria. Moreover, the haemolysis can be inhibited to different degrees by osmoprotectants of diverse molecular masses, signifying that it follows a colloid-osmotic mechanism. This study conclude that purified Hc from F. indicus remarkably possess haemolytic and antibiofilm activity.

Identification and analysis of the salt tolerant property of AHL lactonase (AiiATSAWB ) of Bacillus species.

Bacterial biofilms communicate by a process called Quorum Sensing. Gram negative bacterial pathogens specifically talk through the production, detection, and response to the signal or autoinducer called Acyl Homoserine Lactones. Bacterial lactonases are important AHL hydrolysing or quorum quenching enzymes. The present study deals with ten endospore forming gram positive isolates of the saltern soil. Preliminary screening for Quorum Quenching activity with the QS Inhibition indicator strain Chromobacterium violaceum ATCC 12472, showed positive activity in four isolates namely TS2, TS16, TSAWB, and TS53B. AHL lactonase (AiiA) specific primers amplified Acyl Homoserine Lactone lactonase gene in the TSAWB genome alone. Phylogenetic relationship of the identified AiiATSAWB confirmed its evolutionary relationship with bacterial AiiA like AHL lactonase of the metallo-beta-lactamase super family. Our in vitro AHL hydrolysis assay under wide percentage (0-5) of salt solutions with TSAWB isolate and also its intracellular soluble protein fraction showed halotolerant AHL hydrolysis ability of the AiiATSAWB enzyme. In silico determination of putative tertiary structure, the ESBRI derived conserved salt bridges, aminoacid residue characterization with high mole percent of acidic and hydrophobic residues reaffirmed the halotolerant ability of the enzyme. So we propound the future use of purified AiiATSAWB , as hypertonic suspension for inhalation to substitute the action of inactivated host's paraoxonase in treating Pseudomonas aeruginosa infection in cystic fibrosis patients.

Hybrid of Metapenaeus dobsoni lectin and platinum nanoparticles exert antimicrobial and immunostimulatory effects to reduce bacterial bioburden in infected Nile tilapia.

A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential "green" application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics.

OsPRP3, a flower specific proline-rich protein of rice, determines extracellular matrix structure of floral organs and its overexpression confers cold-tolerance.

Proline-rich protein (PRP), a cell wall protein of plant, has been studied in many plant species. Yet, none of the PRPs has been functionally elucidated. Here we report a novel flower-specific PRP designated OsPRP3 from rice. Expression analysis showed that the OsPRP3 transcript was mainly present in rice flower and accumulated abundantly during the late stage of the flower development. To study the function of OsPRP3, we constructed and transformed a binary vector containing a full clone of OsPRP3 in sense orientation and also an RNAi vector to achieve overexpression and knockout of the gene, respectively. Our overexpression plants showed a significant increase in cold tolerance than the WT plants which is conferred by the accumulation of OsPRP3 protein during cold treatment. Further the microscopic analysis revealed that OsPRP3 enhances the cell wall integrity in the cold tolerant plant and confers cold-tolerance in rice. Microscopic analysis of the RNAi mutant flower revealed that blocking OsPRP3 function caused significant defects in floral organogenesis. Taken together, the results suggested that OsPRP3 is a cell wall protein, playing a crucial role in determining extracellular matrix structure of floral organs.

Genome Sequence of Bacillus vallismortis TD3, a Salt-Tolerant Strain Isolated from the Sediments of a Solar Saltern in Tamil Nadu, India.

Various Bacillus spp. capable of producing enzymes with industrially desirable properties have been isolated from adverse environments. Here, we announce the 3.91-Mbp draft genome sequence of a moderately salt-resistant Bacillus vallismortis strain, TD3, capable of producing several industrially relevant enzymes.

Genome Sequence of a Moderately Halophilic Bacillus cereus Strain, TS2, Isolated from Saltern Sediments.

We report the 5.3-Mbp genome sequence of Bacillus cereus strain TS2, which was isolated from the sediments of a solar saltern in southern India. Genome analysis of B. cereus TS2, a salt-resistant strain, will improve our understanding of how B. cereus, a food pathogen, responds to hyperosmotic stress.

Phenoloxidase activation, antimicrobial, and antibiofilm properties of ß-glucan binding protein from Scylla serrata crab hemolymph.

In this study, we purified ß-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata ß-GBP (Ss-ß-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with ß-GBP of Astacus leptodactylus. Ss-ß-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-ß-GBP. The purified Ss-ß-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-ß-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-ß-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60µg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-ß-GBP at 50 and 100µg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-ß-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-ß-GBP might be widely used to suppress the growth of pathogenic bacteria.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity.

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Synthesis and larvicidal activity of low-temperature stable silver nanoparticles from psychrotolerant Pseudomonas mandelii.

Applications based on silver nanoparticles (AgNPs) are limited by low temperatures, which cause aggregation of the nanoparticle fraction, leading to reduced efficacy of their products. We aimed at studying AgNP synthesis by psychrotolerant bacteria, its stability under long-term storage, and larvicidal activity under low-temperature conditions. Electron and atomic force microscopy studies revealed that 6 among 22 psychrotolerant isolates synthesized AgNPs with an average diameter of 1.9-14.1 nm. Pseudomonas mandelii SR1 synthesized the least-sized AgNPs with an average diameter of 1.9-10 nm, at temperatures as low as 12 °C without aggregate formation, and the synthesized nanoparticles were stable for up to 19 months of storage period. On studying their larvicidal activity, LC90 (lethal concentration) values against Anopheles subpictus and Culex tritaeniorhynchus larvae were at 31.7 and 35.6 mg/L, respectively. Stable non-aggregate AgNPs at low-temperature conditions from P. mandelii SR1, coupled with their larvicidal property, can be applied to control larval populations in water bodies located in seasonal or permanently cold environments.

ß-Keto esters from ketones and ethyl chloroformate: a rapid, general, efficient synthesis of pyrazolones and their antimicrobial, in silico and in vitro cytotoxicity studies.

BACKGROUND: Pyrazolones are traditionally synthesized by the reaction of ß-keto esters with hydrazine and its derivatives. There are methods to synthesize ß-keto esters from esters and aldehydes, but these methods have main limitation in varying the substituents. Often, there are a number of methods such as acylation of enolates in which a chelating effect has been employed to lock the enolate anion using lithium and magnesium salts; however, these methods suffer from inconsistent yields in the case of aliphatic acylation. There are methods to synthesize ß-keto esters from ketones like caboxylation of ketone enolates using carbon dioxide and carbon monoxide sources in the presence of palladium or transition metal catalysts. Currently, the most general and simple method to synthesize ß-keto ester is the reaction of dimethyl or ethyl carbonate with ketone in the presence of strong bases which also requires long reaction time, use of excessive amount of reagent and inconsistent yield. These factors lead us to develop a simple method to synthesize ß-keto esters by changing the base and reagent. RESULTS: A series of ß-keto esters were synthesized from ketones and ethyl chloroformate in the presence of base which in turn are converted to pyrazolones and then subjected to cytotoxicity studies towards various cancer cell lines and antimicrobial activity studies towards various bacterial and fungal strains. CONCLUSION: The ß-keto esters from ethyl chloroformate was successfully attempted, and the developed method is simple, fast and applicable to the ketones having the alkyl halogens, protecting groups like Boc and Cbz that were tolerated and proved to be useful in the synthesis of fused bicyclic and tricyclic pyrazolones efficiently using cyclic ketones. Since this method is successful for different ketones, it can be useful for the synthesis of pharmaceutically important pyrazolones also. The synthesized pyrazolones were subjected to antimicrobial, docking and cytotoxicity assay against ACHN (human renal cell carcinoma), Panc-1 (human pancreatic adenocarcinoma) and HCT-116 (human colon cancer) cell line, and lead molecules have been identified. Some of the compounds are found to have promising activity against different bacterial and fungal strains tested.