Computational kinetic studies of pyruvate metabolism in Carboxydothermus hydrogenoformans Z-2901 for improved hydrogen production

Hydrogen is considered as a renewable energy source and it is also regarded as future fuel. Currently, hydrogen production through a biotechnological approach is a research priority. Hydrogenogens, a microbial species, are of significant interest to researchers because of their ability to produce biological hydrogen. Carboxydothermus hydrogenoformans Z-2901 is one among the hydrogenogens that can grow anaerobically by utilizing pyruvate as a carbon source, and can produce molecular hydrogen. In the present study, we performed an in silico kinetic simulation using the available Kyoto Encyclopedia of Genes and Genomes (KEGG) model and reconstructed pyruvate metabolism in C. hydrogenoformans Z-2901. During this metabolism, dissimilation of pyruvate leads to the formation of energy co-factors, such as ATP and NAD⁺/ NADH, and the level of these co-factors influences the specific growth rate of organism and hydrogen production. Our strategy for improving hydrogen production involves maximizing the ATP and NAD⁺ yield by modification of kinetic properties and adding new reactions in pyruvate metabolism through metabolic pathway reconstruction. Moreover, the influence of phosphoenol pyruvate carboxylase and pyruvate dehydrogenase enzyme concentration on cofactor productions was also simulated. The theoretical molar yield of ATP and NAD⁺ were obtained as 2.32 and 1.83 mM, respectively, from 1 mM/mg of phosphoenol pyruvate (PEP) utilization. A higher yield of ATP is achieved when the PEP level reaches 5 mM/mg. This work also suggests that PEP can be considered as an alternative substrate. In conclusion, the simulation results reported in this paper can be applied to design and evaluate strategies of strain construction for optimal hydrogen yield in C. hydrogenoformans.     

Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.     

Functionally diverse rhizobacteria of Saccharum munja (a native wild grass) colonizing abandoned morrum mine in Aravalli hills (Delhi)

Characterization of the rhizobacteria of native grasses naturally colonizing abandoned mine sites may help in identification of microbial inoculants for ecological-restoration programmes. Eighty one strains of Saccharum munja rhizobacteria isolated from an abandoned mine located on Aravalli mountain and 50 from bulk-region were identified using 16S rRNA sequence analyses. Based on chemical- and biological-assays they were categorized into ecologically diverse functional groups (siderophore-, IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eight genera, 25 species from rhizosphere and 2 genera, 5 species from bulk-region were dominated by Bacillus spp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B. foraminis, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. thuringiensis) and Paenibacillus spp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P. polymyxa, P. popillae). Siderophore-producers were common in rhizosphere and bulk soil, whereas IAA-producers, N₂-fixers and FePO₄-solubilizers dominated rhizosphere samples. During the reproductive phase (winter) of S. munja, siderophore-, ACC-deaminase- and polyP-producers were predominant; however dominance of HCN-producers in summer might be associated with termite-infestation. In vivo ability of selected rhizobacteria (B. megaterium BOSm201, B. subtilis BGSm253, B. pumilus BGSm157, P. alvei BGSm255, P. putida BOSm217, P. aeruginosa BGSm 306) to enhance seed-germination and seedling-growth of S. munja in mine-spoil suggest their significance in natural colonization and potential for ecological-restoration of Bhatti mine.     

Morphological and molecular screening of rice germplasm lines for low soil P tolerance

Phosphorus (P) is an essential macronutrient to all crops including rice and it plays a key role in various plant activities and development. Low availability of P in the soils negatively, influences rice crop growth and causes significant yield loss. In the present study, we characterized a set of 56 germplasm lines for their tolerance to low soil P by screening them at low soil P and optimum soil P levels along with low soil P tolerant and sensitive check varieties. These lines were genotyped for the presence/absence of tolerant allele with respect to the major low soil P tolerance QTL, Pup1, using a set of locus specific PCR-based markers, viz., K46-1, K46-2, K52 and K46CG-1. High genetic variability was observed for various traits associated with low soil P tolerance. The yield parameters from normal and low soil P conditions were used to calculate stress tolerance indices and classify the genotypes according to their tolerance level. Out of the total germplasm lines screened, 15 lines were found to be tolerant to low soil P condition based on the yield reduction in comparison to the tolerant check, but most of them harbored the complete or partial Pup1 locus. Interestingly, two tolerant germplasm lines, IC216831 and IC216903 were observed to be completely devoid of Pup1 and hence they can be explored for new loci underlying low soil P tolerance.

     

"Recruitment signals" from apoptotic cells: invitation to a quiet meal

An evolutionarily conserved machinery exists for engulfment of apoptotic cells from worm to mammals. New observations suggest that corpse clearance is tightly linked to apoptosis and that dying cells use both recruitment and eat-me signals for phagocyte attraction and recognition.     

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.     

A conserved role for SNX9-family members in the regulation of phagosome maturation during engulfment of apoptotic cells

Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution.