Heterotrophic Nitrifying and Oxygen Tolerant Denitrifying Bacteria from Greenwater System of Coastal Aquaculture

In this work, herbivorous fish Mugil cephalus has been cultured to secrete protein rich green slime, which helps nitrifying and oxygen tolerant denitrifying bacteria to grow and colonize. Four strains representing Alcaligenaceae family have been isolated from greenwater system and characterized using biochemical test, fatty acid methyl ester (GC-FAME) analysis, 16S rRNA and functional gene approaches. They were tested for an ability to nitrify ammonia and nitrite aerobically. Two strains showed notable nitrification activity, when grown in a mineral salts medium containing ammonium sulfate and potassium nitrite. Functional gene analysis confirmed the presence of nitrous oxide reductase (nosZ) gene showing that they have an oxygen-tolerant denitrification system. It has been proposed that Alcaligenes faecalis strains heterotrophically nitrify ammonia into nitrite via formation of hydroxyl amine, which is oxidized to nitrous oxide using oxygen or nitrite as electron acceptor. These results provide a possible advantage of having nitrification and denitrification capabilities in the same organism, which plays an important role in biological wastewater system.     

An Efficient Method for Agrobacterium-Mediated Genetic Transformation and Plant Regeneration in Cumin (Cuminum cyminum L.)

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg’s B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD₆₀₀, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T₀ plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.     

Expression, Purification, and Partial In Vitro Characterization of Biologically Active Human Coagulation Factor VIII Light Chain (A3-C1-C2) in Pichia pastoris

Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1–A2) and light chains (A3–C1–C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.     

rmpM Genosensor for Detection of Human Brain Bacterial Meningitis in Cerebrospinal Fluid

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5′-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087?(μA/cm²)/ng with DPV and limit of detection was 3 ng/6 μL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.     

Role of a Repeated Hexapeptide Motif GIHFAP Near C-terminus in Assembly, Stability, and Activity of “HCH Dehydrochlorinase LinA”

Enzyme “hexachlorocyclohexane (HCH) dehydrochlorinase LinA” mediates first step of aerobic microbial degradation of a chlorinated insecticide γ-HCH. The archetypal LinA-type1 consists of 156 amino acids that include a directly repeated hexapeptide motif GIHFAP at positions 141–146 and 148–153. Analysis of a series of LinA mutants, containing none, one, two, or three units of this repeated motif revealed that two units, as present in wild-type LinA, are required for its optimal activity and stability. Moreover, the presence of a bend in its secondary structure due to a proline residue that precedes the distal repeated unit contributes to enhanced LinA activity.     

Advances in arsenic speciation techniques

The present review describes the speciation techniques of arsenic. The principal advanced techniques discussed are gas chromatography, reversed-phase liquid chromatography, ion chromatography, capillary electrophoresis. Some other techniques are also mentioned. The extraction procedures of arsenic species from unknown samples are also discussed. Arsenic speciation is summarized in tabular form and optimizing parameters are also discussed.     

Accurate measurement of uranium isotope ratios in soil samples using thermal ionization mass spectrometry equipped with a warp energy filter

A chemical and mass-spectrometric procedure for uranium isotopic analysis using a thermal ionisation mass spectrometer equipped with a Wide Aperture Retardation Potential energy filter has been developed and applied to uranium isotopic measurements for various soil samples. Soil samples were digested using a microwave digestor. Uranium was isolated from soil samples by the chemical separation procedure based on the use of anion-exchange resin and UTEVA extraction chromatography column. The isotope ratios were measured for two certified reference materials by using a VG Sector 54-30 thermal ionisation mass spectrometer in dynamic mode with Faraday cup and Daly ion counting system. Replicates of standard reference materials showed excellent analytical agreement with established values supporting the reliability and accuracy of the method. Precision of the ²³⁵U/²³⁸U ratio was achieved by a correction factor of 0.22% amu as a function of ion-beam intensity with sample loads of around 250?ng of U. The resulting reproducibility for standards and soil samples was better than 0.2% at two standard deviations (SD). Uranium isotopic compositions have been determined in several reference soil samples such as Buffalo river sediment, NIST 2704, river sediment SRM 4350b and ocean sediment NIST-4357 and a Chernobyl soil sample. There was a significant deviation from the natural uranium in comparison with Chernobyl soil samples.     

Synthesis and structural studies of some copper-benzoate complexes

The reaction of CuCl₂·2H₂O with 3,5-diisopropylpyrazole (Pz^iPr2H) in the presence of sodium parafluorobenzoate (Na-p-FBz) resulted in the formation of an oxo-chloro-bridged tetranuclear complex [Cu₄(Pz^iPr2H)₄(μ-O)(μ-Cl)₆] 1, whereas the reaction of Cu(NO₃)₂·3H₂O with Pz^iPr2H in the presence of different benzoates gave [Cu(Pz^iPr2H)₂(μ-OCH₃)]₂(NO₃)₂ 2, [Cu(Pz^iPr2H)₃(NO₃)(p-ClBz)]·CH₃CN 3, [Cu(p-CH₃Bz)₂(Pz^iPr2H)]₂·2CH₃CN 4, [Cu(p-OCH₃Bz)₂(CH₃CN)]₂·4CH₃CN 5 and [Cu(p-CNBz)(CH₃CN)]₂ 6. Single-crystal X-ray diffraction studies confirmed these formulations. DNA binding studies for these complexes were performed by means of UV-visible absorption titration and viscosity measurements. Gel electrophoresis studies showed that hydroxyl radicals are involved in DNA cleavage in the presence of the complexes.     

Thermoluminescence studies of UV-irradiated Y2O3:Eu3+ doped phosphor

The present paper reports thermoluminescence (TL) glow curves of Eu³⁺-doped Y₂O₃ phosphor with different ultraviolet (UV) exposure times. The glow peak shows second-order kinetics of Eu³⁺-doped Y₂O₃, and corresponding kinetic parameters were evaluated using the peak shape method. Calculations of trap depth were carried out using different methods. The kinetics order, activation energy, and frequency factor were calculated. The recorded glow curve shifts towards higher intensity with longer UV exposure. The heating rate used for recording TL was 3.0 °C s^?1. Particle size and structure were verified by X-ray diffraction (XRD) pattern and morphology by scanning electron microscopy (SEM) imaging.     

An efficient and facile synthesis of Zn(II) complexes with 2-substituted benzothiazoles and glycine- and alanine-based ligands having antifungal and antibacterial activities

We carried out an efficient and facile synthesis of Zn(II) complexes with heterocyclic ligands based on 2-substituted benzothiazole moieties, 2-(2′-hydroxynaphthyl)benzothiazole, 2-(2′-hydroxyphenyl)benzothiazole, and 2-(2′-merceptophenyl)benzothiazole, and amino acids, glycine and alanine, are reported and also examined for antifungal and antibacterial activities. Ligands and complexes were characterized by FTIR, ¹H NMR, and elemental analysis.