Sonication-assisted production of biodiesel using soybean oil and supercritical methanol

High temperature and pressure are generally required to produce biodiesel using supercritical methanol. We reduced the harsh reaction conditions by means of sonicating the reaction mixture prior to transesterification using supercritical methanol. Soybean oil was selected as the raw material for transesterification. As soybean oil contains more unsaturated fatty acid triglycerides, the biodiesel degraded more at high temperature. The reactants were sonicated for 60 min at 35 °C prior to transesterification to avoid degradation of the product and to enhance biodiesel yield at temperatures <300 °C. The process parameters were optimized using central composite design. The variables selected for optimization were temperature, time, and the oil to methanol molar ratio. The temperature and oil to methanol molar ratios were varied from 250 to 280 °C and 1:40–1:50, respectively. The reaction time was tested between 4 and 12 min. The biodiesel was analyzed for any possible degradation by gas chromatography–mass spectroscopy and for the wt% of fatty acid methyl esters (FAME) obtained. The maximum FAME yield (84.2 wt%) was obtained at a temperature of 265.7 °C, an oil to alcohol molar ratio of 1:44.7, and a time of 8.8 min. The optimum yield was obtained at a pressure of 1,500 psi. The pressure and optimum temperature used to obtain the maximum yield were the lowest reported so far without the use of a co-solvent. Thus, the severity of the supercritical reactions was reduced by adding sonication prior to the reaction.     

A review on interplay between small RNAs and oxidative stress in cancer progression

Molecular and Cellular Biochemistry - Oxidative stress has been known to be the underlying cause in many instances of cancer development. The new aspect of cancer genesis that has caught the...     

Biochemical and structural insights into bacterial organelle form and biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.     

Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patients

Highly successful bacterial clones have the ability to effectively colonize environmental niches and patients. However, the factors which determine the complex interplay between the colonization of environmental niches and patients are mainly unknown. In this study we show that Pseudomonas aeruginosa clone C strains are distributed worldwide and highly prone to infect cystic fibrosis (CF) patients in Canada, England, France and Germany. In Hanover, Germany and Vancouver, Canada, clone C strains are highly prevalent in the CF patient community, although the mechanisms of acquisition may have been different. All clone C strains showed highly related macrorestriction fragment pattern of the whole genome as visualized by pulsed-field gel electrophoresis and harboured the 102 kbp plasmid pKLC102. Comparison of three prevalent P. aeruginosa clones with different distribution between the environment and patients revealed that neither enhanced biofilm formation nor antibiotic resistance was responsible for the spread of clone C. Clone M, which was highly prevalent in the clinical environment such as sanitary facilities, lacked motility, which could explain its relatively low prevalence in CF patients. Elucidation of the mechanisms which lead to the prevalence of clone C strain in patients and the environment requires the investigation of additional phenotypes.     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.     

Adaptive expression of foreign genes in the clonal variants of bacteria: from proteomics to clinical application

Clonal variants of bacteria are able to colonize environmental niches and patients. The factors, that determine the interplay between the colonization of diverse habitats and adaptation, are acquired through horizontal gene transfer. Elucidation of mechanisms, which lead to the prevalence of dominant bacterial clones in patients and the environment, requires the knowledge of complex phenotypes. It was found in the genomes of most bacteria, that upon a conserved chromosomal backbone there were regions of plasticity achieved by insertions, deletions and rearrangements of genomic islands and islets as well as large chromosomal inversions. However, it had been shown that environmental and clinical isolates are indistinguishable in certain pathogenic and biodegradative properties. For example, clonal variants of Pseudomonas aeruginosa exhibit convergent phenotypes despite the presence of numerous DNA insertions in the genome. Apart from this feature, expression of a few genes from the acquired genetic material is important for niche-based adaptation of this organism. Protein expression patterns at the cellular and sub-cellular levels showed common virulence factors and novel drug targets among clonal variants of bacteria. This review will give a short overview on proteomics of different clonal variants of bacteria with respect to clinical applications.     

Wnt signaling regulates the proliferation potential and lineage commitment of human umbilical cord derived mesenchymal stem cells

Relatively less is known about the interactions that tightly regulate the mesenchymal stem cells (MSCs) to maintain their pluripotency. Recent studies reports that Wnt proteins might play an important role in governing the MSC cell fate. In this study, we tested the hypothesis that Wnt proteins differentially regulate in vitro differentiation of human umbilical cord derived MSCs. Stromal cells from human umbilical cord (hUCMSCs) were isolated and treated with Wnt inhibitor/activator. FACS analysis of hUCMSCs for CD29, CD90, CD73, CD44, CD45 marker expression and gene expression of Wnt target genes and lineage specific genes were performed after Lithium Chloride (LiCl) and Quercetin treatment for 6 days. The cultured primary hUCMSCs demonstrated elevated MSC surface marker expression with clonogenic properties and differentiation potentials towards osteogenic, adipogenic and chondrogenic lineages. Downregulation in the expression of Wnt with Quercetin treatment was noted. LiCl treatment increased cellular proliferation but did not influence differentiation suggesting that the cells retain pluripotency whereas Quercetin treatment downregulated stemness markers, Wnt target gene expression and promoted osteogenesis as demonstrated by FACS analysis, calcium estimation and gene expression studies. Shift of differentiation potential after the inhibition of Wnt signaling by Quercetin was evident from the gene expression data and elevated calcium production, driving MSCs towards probable osteogenic lineage. The findings in particular are likely to open an interesting avenue of biomedical research, summarizing the impact of Wnt signaling on lineage commitment of MSCs.

     

Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs

Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.