Characterization and biological activities of melanin pigment from root endophytic fungus, Phoma sp. RDSE17.

Melanins are high molecular weight hydrophobic pigments which have gained popularity for their role in virulence against different pathogens. In the present study, we isolated and characterized the melanin pigment produced by a dark septate endophyte fungus Phoma sp. RDSE17, which was associated with the roots of an indigenous Oryza sativa cv. 'Chakhao amubi' in Manipur, Northeast India. The biological properties of purified melanin from the fungus were evaluated for their antioxidant, antimicrobial and anticancerous activities. The pigment was extracted from Phoma sp. by alkaline-acid hydrolysis method and confirmed as melanin through physico-chemical tests and spectral (UV, FTIR, and EPR) analysis. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen (N) contents, and therefore, would not fall under DOPA class of melanin. Exposure of the fungus to melanin pathway inhibitors revealed a positive melanin inhibition by tricyclazole, but not by kojic acid. Thus, the melanin from Phoma sp. may be a member of the DHN family. Moreover, the purified melanin showed high DPPH (1, 1-Diphenyl-2-picrylhydrazyl) free radical-scavenging activity with an EC50 of 69 µg/mL and inhibited human lung cancer cell (A549 cells) proliferation at 80 µg/mL. The present study demonstrates that melanin from Phoma sp. RDSE17 could be employed as a potential biological (antioxidant) and antimicrobial agent for inhibiting the growth of humans and phytopathogens.

Nanocarriers-based immobilization of enzymes for industrial application.

Nanocarriers-based immobilization strategies are a novel concept in the enhancement of enzyme stability, shelf life and efficiency. A wide range of natural and artificial supports have been assessed for their efficacy in enzyme immobilization. Nanomaterials epitomize unique and fascinating matrices for enzyme immobilization. These structures include carbon nanotubes, superparamagnetic nanoparticles and nanofibers. These nano-based supports offer stable attachment of enzymes, thus ensuring their reusability in diverse industrial applications. This review attempts to encompass recent developments in the critical role played by nanotechnology towards the improvement of the practical applicability of microbial enzymes. Nanoparticles are increasingly being used in combination with various polymers to facilitate enzyme immobilization. These endeavors are proving to be conducive for enzyme-catalyzed industrial operations. In recent years the diversity of nanomaterials has grown tremendously, thus offering endless opportunities in the form of novel combinations for various biotransformation experimentations. These nanocarriers are advantageous for both free enzymes and whole-cell immobilization, thus demonstrating to be relatively effective in several fermentation procedures.

Exposure to drugs of abuse induce effects that persist across generations.

Substance use disorders are highly prevalent and continue to be one of the leading causes of disability in the world. Notably, not all people who use addictive drugs develop a substance use disorder. Although substance use disorders are highly heritable, patterns of inheritance cannot be explained purely by Mendelian genetic mechanisms. Vulnerability to developing drug addiction depends on the interplay between genetics and environment. Additionally, evidence from the past decade has pointed to the role of epigenetic inheritance in drug addiction. This emerging field focuses on how environmental perturbations, including exposure to addictive drugs, induce epigenetic modifications that are transmitted to the embryo at fertilization and modify developmental gene expression programs to ultimately impact subsequent generations. This chapter highlights intergenerational and transgenerational phenotypes in offspring following a history of parental drug exposure. Special attention is paid to parental preconception exposure studies of five drugs of abuse (alcohol, cocaine, nicotine, cannabinoids, and opiates) and associated behavioral and physiological outcomes in offspring. The highlighted studies demonstrate that parental exposure to drugs of abuse has enduring effects that persist into subsequent generations. Understanding the contribution of epigenetic inheritance in drug addiction may provide clues for better treatments and therapies for substance use disorders.

Effects of Paternal Preconception Vapor Alcohol Exposure Paradigms on Behavioral Responses in Offspring.

We and others previously reported that paternal preconception chronic ethanol exposure leads to molecular, physiological, and behavioral changes in offspring including reduced ethanol consumption and preference relative to controls. The goal of the present study was to further explore the impact of paternal ethanol exposure on a wide variety of basal and drug-induced behavioral responses in first generation offspring. Adult male mice were exposed to chronic intermittent vapor ethanol or control conditions for 5-6 weeks before being mated with ethanol-naïve females to produce ethanol (E)- and control (C)-sired offspring. E-sired male offspring showed stress hyporesponsivity in a stress-induced hyperthermia assay and E-sired female offspring had reduced binge-like ethanol consumption in a drinking in the dark assay compared to C-sired offspring. E-sired offspring also showed altered sensitivity to a sedative/hypnotic dose of the GABAergic drug midazolam, but not ketamine or ethanol, in a loss of the righting response assay. E-sired offspring did not differ from controls in marble burying, novel object location, novel object recognition, social interaction, bottle-brush, novelty suppressed feeding, prepulse inhibition, every-other-day ethanol drinking, or home cage activity assays. This study adds to a growing body of literature suggesting that like in utero alcohol exposure, paternal preconception alcohol exposure can also have effects that persist and impact behavior of offspring.

Nitrile hydratase mediated green synthesis of lactamide by immobilizing Rhodococcus pyridinivorans NIT-36 cells on N, N'-Methylene bis-acrylamide activated chitosan.

In this paper green synthesis of an important commodity chemical lactamide has been undertaken using chitosan immobilized Rhodococcus pyridinivorans NIT-36 harbouring nitrile hydratase (NHase) enzyme. The cells immobilization (300 mg/g) is based on the partial entrapment of cells by suspension cross-linking technique facilitated by N, N'-Methylene bis-acrylamide. In the repeated-use experiments, the immobilized cells retained 80% of its initial activity when stored at 4 °C for 30 days. NHase activity of free and immobilized cells was studied over temperature ranging from 25 °C to 60 °C. The activity for free cells showed a sharp decline of 70% when the reaction temperature was elevated from 45 °C to 50 °C whereas chitosan immobilized cells retained their activity in the same temperature range. A fed-batch reaction was designed and the immobilized cells showed 100% similar enzymatic pattern for five consecutive rounds which gradually decreased in following cycles. A volumetric productivity of 20 g/L and catalytic productivity of 8.33 g/g dcw/h for lactamide were achieved.

CRISPR Turbo Accelerated KnockOut (CRISPy TAKO) for Rapid in vivo Screening of Gene Function.

The development of CRISPR/Cas9 technology has vastly sped up the process of mammalian genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous functional knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes an adaptation of the CRISPR/Cas9-mediated method to develop a cohort of homozygous gene-targeted KO animals in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of KO mice as proof-of-concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9 protein. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. Behavioral testing confirmed the hypothesis that successful one-generation KO of MyD88 would reproduce the decreased ethanol-induced sedative/hypnotic effects and increased ethanol consumption in males that were observed in previous studies. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

Random Mutagenesis of Thermophilic Xylanase for Enhanced Stability and Efficiency Validated through Molecular Docking.

BACKGROUND: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability. OBJECTIVE: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability. METHODS: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). RESULTS: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket. CONCLUSION: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.

Purification and characterization of thermostable superoxide dismutase from Anoxybacillus gonensis KA 55 MTCC 12684.

A thermostable superoxide dismutase from thermophilic bacterium Anoxybacillus gonensis KA 55 MTCC 12684 which was isolated from Manikaran hotspring of Himachal Pradesh was purified to apparent homogeneity by fractional ammonium sulphate precipitation and anion exchange chromatography. A purification factor of 33.1-fold was achieved, with the purified enzyme exhibiting specific activity of 5758.4 U/mg protein. The purified superoxide dismutase was optimally active at pH 9.0 and displayed stability over a broad pH range of 7.0-10.0 and was stable up to 70 °C. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide ion. The molecular weight of superoxide dismutase was calculated as 31 kDa by SDS-PAGE. The Km and Vmax values of purified enzyme were found to be 1.002 mM and 14,285.71 U/mg of protein respectively. Tests of inhibitors indicated that the enzyme activity was inhibited by hydrogen peroxide and potassium cyanide but not by sodium azide showing that purified SOD was Cu/ZnSod.

Improving stability and reusability of Rhodococcus pyridinivorans NIT-36 nitrilase by whole cell immobilization using chitosan.

Immobilized enzymes have great significance for industrial processes. In this study whole cell immobilization of Rhodococcus pyridinivorans NIT-36 has been undertaken using chitosan microspheres as an immobilized matrix. R. pyridinivorans NIT-36 harbors a significant intracellular enzyme nitrilase. Chitosan microspheres were generated by supplementing chitosan with glutaraldehyde and results, supported the porous microsphere structure via SEM. The resultant microspheres exhibited cell immobilization capacity of 450mg/g. The immobilized cells exhibited a considerable increase in temperature tolerance at 60°C as compared to free cells. The immobilized microspheres also demonstrated higher substrate tolerance. The immobilized nitrilase retained 80% activity when stored at 4°C for 10 days and retained 50% activity after 7 reuse cycles. It may be concluded that chitosan microspheres are a novel immobilization agent for whole cell immobilization which enhances the stability and reusability of nitrilase enzyme.

Purification, characterization and in-silico analysis of nitrilase from Gordonia terrae.

An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s⁻¹ x 10⁻¹, Km 1.4 mM and Vmax 95 Umg⁻¹ protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzyme's active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.

Monitoring HIV-infected Patients with Diabetes: Hemoglobin A1c, Fructosamine, or Glucose?

BACKGROUND: Published studies report inappropriately low hemoglobin A1C (HbA1c) values that underestimate glycemia in HIV patients. METHODS: We reviewed the charts of all HIV patients with diabetes mellitus (DM) at our clinic. Fifty-nine patients had HbA1c data, of whom 26 patients also had fructosamine data. We compared the most recent HbA1c to finger-stick (FS) glucose averaged over three months, and fructosamine to FS averaged over six weeks. Predicted average glucose (pAG) was calculated as reported by Nathan et al: pAG (mg/dL) = 28.7 × A1C% - 46.7. Data were analyzed using the Statistical Analysis System (SAS) and Kruskal-Wallis test. RESULTS: HbA1c values underestimated (UE) actual average glucose (aAG) in 19% of these patients and overestimated (OE) aAG in 27%. HbA1c estimated aAG within the established range in only 54% of the patients. There were no statistical differences in the types of HIV medication used in patients with UE, OE, or accurately estimated (AE) glycemia. A Spearman correlation coefficient between HbA1c and aAG was r = 0.53 (P < 0.0001). Correlation between fructosamine and aAG was r = 0.47 (P = 0.016). CONCLUSIONS: The correlations between HbA1c and aAG and between fructosamine and aAG were weaker than expected, and fructosamine was not more accurate than HbA1c.

Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34.

Nitrile hydratase (NHase; E.C. 4.2.1.84) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and ß-subunits with a molecular mass of 25 and 30 kDa, respectively. The Km and Vmax values were 102 mM and 350.8 µmol/min/mg using 3-cyanopyridine as substrate. The purified NHase was stable in higher concentration of potassium ions and in acidic pH 5.5 as compared to NHase of the wild R. rhodochrous PA-34. The analysis of the N-terminal amino acid sequence of this enzyme revealed that this enzyme has 90 % homology with the high molecular weight nitrile hydratase of R. rhodochrous J1.

Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli.

The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria-Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

Generation of mutant of Rhodococcus rhodochrous PA-34 through chemical mutagenesis for hyperproduction of nitrile hydratase.

Rhodococcus rhodochrous PA-34 has been reported to produce nitrile hydratase enzyme that converts 3-cyanopyridine to nicotinamide. A mutant of R. rhodochrous PA-34 was generated through chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine (MNNG) that exhibited 2 times higher nitrile hydratase activity as compared to wild strain. The reaction conditions using resting cells of this mutant strain for the conversion of nicotinamide were optimized. Under the optimized reaction conditions the mutant strain exhibited maximum nitrile hydratase activity [7.8 U/mgdcm (milligram dry cell mass)] at 55 degrees C in 0.3 M potassium phosphate buffer (pH 5.5).