Comparative assessment of in situ bioremediation potential of cadmium resistant acidophilic Pseudomonas putida 62BN and alkalophilic Pseudomonas monteilli 97AN strains on soybean

A study was undertaken to examine the effects of the acidophilic strain 62BN (pH 5.5) and alkalophilic strain 97AN (pH 9.0) on remediation of cadmium and their subsequent effects on soybean (Glycine max var. PS-1347) in acidic and alkaline soils, respectively. The effect of cadmium on soybean plants was studied in acidic (pH 6.3 ± 0.2) and alkaline (8.5 ± 0.2) soil amended with 124 μM CdCl₂ concentration, respectively, and the cadmium toxicity was evident from stunted growth, poor rooting, and cadmium accumulation in each case. Furthermore, 16S ribosomal DNA (rDNA) sequencing identified 62BN as a Pseudomonas putida strain and 97AN as a Pseudomonas monteilli strain. In situ studies showed that on seed bacterization, both the P. putida 62BN strain and P. monteilli 97AN strain were able to enhance plant growth in terms of agronomical parameters, in the presence of cadmium in acidic and alkaline soils, respectively. Apart from this, strain 62BN and 97AN reduced cadmium concentration in plant and soil significantly (p < 0.05) in their respective soil types. Further comparative analysis revealed that P. putida 62BN was more effective than P. monteilli 97AN strain in remediation of cadmium. The bacterial strains offer promise as inoculants to improve the growth of plants in the presence of toxic Cd concentrations in the environment with their optimum pH.     

A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.KEY WORDS: GAA repeat, Friedreich’s ataxia, FRDA, YG8sR, Mouse model     

Prediction of protein targets of kinetin using in silico and in vitro methods: a case study on spinach seed germination mechanism

Kinetin, a cytokinin which promotes seed germination by inhibiting the action of abscisic acid, is an important molecule known to trigger various molecular mechanisms by interacting with an array of proteins shown from experimental observations in various model organisms. We report here the prediction of most probable protein targets of kinetin from spinach proteome using in silico approaches. Inverse docking and ligand-based similarity search was performed using kinetin as molecule. The former method prioritized six spinach proteins, whereas the latter method provided a list of protein targets retrieved from several model organisms. The most probable protein targets were selected by comparing the rank list of docking and ligand similarity methods. Both of these methods prioritized chitinase as the most probable protein target (ΔG_pred = 5.064 kcal/mol) supported by the experimental structure of yeast chitinase 1 complex with kinetin (PDB: 2UY5) and Gliocladium roseum chitinase complex with 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (caffeine; 3G6M) which bears a 3D similarity of 0.43 with kinetin. An in vitro study to evaluate the effect of kinetin on spinach seed germination indicated that a very low concentration of kinetin (0.5 mg/l) did not show a significant effect compared to control in inducing seed germination process. Further, higher levels of kinetin (>0.5 mg/l) constituted an antagonist effect on spinach seed germination. It is anticipated that kinetin may have a molecular interaction with prioritized protein targets synthesized during the seed germination process and reduces growth. Thus, it appears that kinetin may not be a suitable hormone for enhancing spinach seed germination in vitro.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-015-0135-3) contains supplementary material, which is available to authorized users.     

Microbial diversity of the Soldhar hot spring,India, assessed by analyzing 16S rRNA and protein-coding genes

Bacterial diversity of the Soldhar hot spring, located in the Chamoli district of Uttarakhand, India, was investigated using a clone library, denaturing gradient gel electrophoresis (DGGE) and functional genes. Physicochemical analysis of sediment samples indicated an oligotrophic environment with very low sulfur content. Based on the 16S rRNA gene studies Proteobacteria was the most predominant group in all the three approaches. Other dominant phyla were Deinococcus-Thermus and Aquificae. Pyrobaculum was the only archaeal genus detected by DGGE. In the functional gene analysis, the nifH library showed a single operational taxonomic unit (OTU) related to the genus Paenibacillus whereas the aoxB library showed three OTUs related to Acidovorax, Aminobacter and Agrobacterium. Our results demonstrate for the first time both the bacterial and archaeal diversity in the Soldhar hot spring by culture-independent techniques, thereby providing important information that will increase our understanding of the microbial ecology of the Soldhar hot spring.     

Bacillus enclensis sp. nov., isolated from sediment sample

A novel bacterial strain, designated SGD-1123^T was isolated from Chorao Island, in Goa Province, India. The strain was found to be able to grow at 15–42 °C, pH 5–12 and 0–12 % (w/v) NaCl. The whole cell hydrolysates were found to contain meso-diaminopimelic acid, galactose and arabinose. The major fatty acids were identified as iso-C_15:0 and anteiso-C_15:0, MK-7 was identified as the predominant menaquinone and the predominant polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The genomic DNA G+C content was determined to be 44.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate within the genus Bacillus and further revealed that strain SGD-1123^T had highest sequence similarity with Bacillus aquimaris, and forms a separate clade with its closest relatives i.e. B. aquimaris, Bacillus vietnamensis and Bacillus marisflavi, with which it shares 94.5, 94.1 and 94.1 % similarity respectively. The phylogenetic, chemotaxonomic and phenotypic analyses indicated that strain SGD-1123^T represents a novel species within the genus Bacillus, for which the name Bacillus enclensis is proposed. The type strain is SGD-1123^T (NCIM 5450^T=CCTCC AB 2011125^T).     

Application of Modeling to Scale-up Dissolution in Pharmaceutical Manufacturing

Liquid mixing scale-up in pharmaceutical industry has often been based on empirical approach in spite of tremendous understanding of liquid mixing scale-up in engineering fields. In this work, we attempt to provide a model-based approach to scale-up dissolution process from a 2 l lab-scale vessel to a 4,000 l scale vessel used in manufacturing. Propylparaben was used as a model compound to verify the model predictions for operating conditions at commercial scale that would result in similar dissolution profile as observed in lab scale. Geometric similarity was maintained between both of the scales to ensure similar mixing characteristics. We utilized computational fluid dynamics (CFD) to ensure that the operating conditions at laboratory and commercial scale will result in similar power per unit volume (P/V). Utilizing this simple scale-up criterion of similar P/V across different scales, results obtained indicate fairly good reproducibility of the dissolution profiles between the two scales. Utilization of concepts of design of experiments enabled summarizing scale-up results in statistically meaningful parameters, for example −90% dissolution in lab scale at a given time under certain operating conditions will result in 75–88% at commercial scale with 95% confidence interval when P/V is maintained constant across the two scales. In this work, we have successfully demonstrated that scale-up of solid dissolution can be done using a systematic process of lab-scale experiments followed by simple CFD modeling to predict commercial-scale experimental conditions.     

Role of 4-hydroxynonenal and its metabolites in signaling

Available evidence from a multitude of studies on the effects of 4-hydroxynonenal (HNE) on cellular processes seem to converge on some common themes: (i) concentration-dependent opposing effects of HNE on key signaling components (e.g. protein kinase C, adenylate cyclase) predict that certain constitutive levels of HNE may be needed for normal cell functions - lowering of this constitutive HNE level in cells promotes proliferative machinery while an increase in this level promotes apoptotic signaling; (ii) HNE is a common denominator in stress-induced apoptosis caused by H(2)O(2), superoxide, UV, heat or oxidant chemicals such as doxorubicin; and (iii) HNE can modulate ligand-independent signaling by membrane receptors such as EGFR or Fas (CD95) and may act as a sensor of external stimuli for eliciting stress-response. Against a backdrop of various reported effects of HNE, in vitro and in vivo, we have critically evaluated the above mentioned hypotheses suggesting a key role of HNE in signaling.     

Possible involvement of queuine in regulation of cell proliferation

An increase in cell number is one of the most prominent characteristics of cancer cells. This may be caused by an increase in cell proliferation or decrease in cell death. Queuine is one of the modified base which is found at first anticodon position of specific tRNAs. It is ubiquitously present throughout the living system except mycoplasma and yeast. The tRNAs of Q-family are completely modified to Q-tRNAs in terminally differentiated somatic cells, however hypomodification of Q-tRNA is closely associated with cell proliferation and malignancy. Queuine participates at various cellular functions such as regulation of cell proliferation, cell signaling and alteration in the expression of growth associated proto-oncogenes. Like other proto-oncogenes bcl2 is known to involve in cell survival by inhibiting apoptosis. Queuine or Q-tRNA is suggested to inhibit cell proliferation but the mechanism of regulation of cell proliferation by queuine or Q-tRNA is not well understood. Therefore, in the present study regulation in cell proliferation by queuine in vivo and in vitro as well as the expression of cell death regulatory protein Bcl2 are investigated. For this DLAT cancerous mouse, U87 cell line and HepG2 cell line are treated with different concentrations of queuine and the effect of queuine on cell proliferation and apoptosis are studied. The results indicate that queuine down regulates cell proliferation and expression of Bcl2 protein, suggesting that queuine promotes cell death and participates in the regulation of cell proliferation.