Production and antimicrobial activity of silver nanoparticles synthesized from indigenously isolated Pseudomonas aeruginosa from Rhizosphere.

Nanoparticles hold profound biological, pharmaceutical and industrial applications. Green synthesis of nanoparticles is considered amongst the environmentally safe and cost effective method of nanoparticle synthesis. Briefly, Pseudomonas strains were isolated from rhizosphere soil samples. Cell free supernatants of 36% of the isolates showed production of silver nanoparticles. Of these, three isolates, SMS13, SMS100 and SMS124 were selected as the potentially best nanoparticle producers. Amplification and DNA sequencing of 16srRNA gene identified all three strains as Pseudomonas aeruginosa. Scanning electron microscopy showed existence of particles between the ranges of 60nm to 70nm in the diameter with elemental composition of silver varies from 0.48% to 1.61%. Consistently, surface plasmon resonance showed maximum absorbance of nanoparticles between 352nm to 406nm. Finally, antibacterial activity of the synthesized nanoparticles was assessed by spot assay whereas Cell Free Supernatants (CFS) of respective isolates were taken as control against clinical isolates of Salmonella typhi, Shigella dysentriae, Klebsiella pneumoniae, P. aeruginosa, Proteus mirabilis and Streptococcus epidermidis. Nanoparticles suspension from all isolates exhibited antibacterial activity against all the screened isolates, whereas no biological activity was observed in the CFS of corresponding strains.

Pharmacogenetics of sulfonylurea: Presence of CYP2C9*2, CYP2C9*3 and a novel allele, CYP2C9*61, in Type 2 diabetes patients under sulfonylurea therapy.

CYP2C9 is an important member of the cytochrome P450 gene family involved in the metabolism of 15% of the drugs including an oral antidiabetic agent sulfonylurea. This study aims to investigate the frequency of CYP2C9*2 and CYP2C9*3 alleles of the gene in the sulfonylurea treated diabetic subjects in Pakistan. Briefly, total 105 patients were included in the study and segregated as control (24) and test (81) based on the clinical manifestations after taking sulfonylurea. Genomic DNA was extracted from blood of the subjects and amplified using CYP2C9 specific primers for exon 3 and exon 7 and then subjected to DNA sequencing. Alignment of the sequences with the reference sequence shows presence of CYP2C9*3/*3, CYP2C9*1/*3 and CYP2C9*1/*2 genotypes in the test cases but only the latter two were found in the control cases. In addition a novel allele, CYP2C9*61 in the heterozygous state, was also identified frequently in the test cases. Molecular structure comparison also showed variations in the structural features of protein encoded by the allelic variants. To the best of our knowledge, the present data is the first report for CYP2C9 allelic variations in the indigenous diabetic subjects and also report the existence of novel allelic variant of CYP2C9, CYP2C9*61.

High Genetic Diversity in the Himalayan Common Bean (Phaseolus vulgaris) Germplasm with Divergence from Its Center of Origin in the Mesoamerica and Andes.

The common bean is found in the Himalayan region of Pakistan with substantial morphological variability. Genetic diversity within any crop species is a precursor for genetic improvement; however, little is known about common bean genetic diversity in this region. We explored the genetic diversity in the common bean from the Himalayan region (Khyber Pakhtunkhwa, Gilgit-Baltistan, Kashmir) of Pakistan. Microsatellite genotyping was carried out for 147 samples with 40 simple sequence repeat (SSR) markers. The results revealed a clear divergence of the Pakistani population from the primary gene pool (with FST values of 0.2 with Andes and 0.27 with Mesoamerica). However, within the Himalayan germplasm, no clear evidence of spatial structure was observed (with the maximum FST values of only 0.025), probably due to the dispersal of seeds by human activity within the region. This was further elucidated by the discriminant analyses of principal components. Considering the diversity parameters, high genotypic diversity was observed for the indigenous lines (0.990), comparable to the primary gene pool (0.976 for Mesoamerica and 0.976 for Andes populations). A high genotypic diversity was observed within the Himalayan population (ranging from 0.500 for Upper Dir to 0.952 for Mansehra). Gene diversity across loci varied between 0.28 for Chitral to 0.38 for Kurram. Our results suggested a divergent and independent evolution of the Himalayan population, which might have led to the diversification of the common bean germplasm in the region postintroduction into the region. The diversity observed could also be exploited in future breeding programs for the development and introduction of climate-resilient varieties.

Trypanosoma brucei and Trypanosoma cruzi DNA Mismatch Repair Proteins Act Differently in the Response to DNA Damage Caused by Oxidative Stress.

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites' MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei.

Distinct Phenotypes Caused by Mutation of MSH2 in Trypanosome Insect and Mammalian Life Cycle Forms Are Associated with Parasite Adaptation to Oxidative Stress.

BACKGROUND: DNA repair mechanisms are crucial for maintenance of the genome in all organisms, including parasites where successful infection is dependent both on genomic stability and sequence variation. MSH2 is an early acting, central component of the Mismatch Repair (MMR) pathway, which is responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. In addition, recent evidence suggests that MSH2 might also play an important, but poorly understood, role in responding to oxidative damage in both African and American trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the involvement of MMR in the oxidative stress response, null mutants of MSH2 were generated in Trypanosoma brucei procyclic forms and in Trypanosoma cruzi epimastigote forms. Unexpectedly, the MSH2 null mutants showed increased resistance to H2O2 exposure when compared with wild type cells, a phenotype distinct from the previously observed increased sensitivity of T. brucei bloodstream forms MSH2 mutants. Complementation studies indicated that the increased oxidative resistance of procyclic T. brucei was due to adaptation to MSH2 loss. In both parasites, loss of MSH2 was shown to result in increased tolerance to alkylation by MNNG and increased accumulation of 8-oxo-guanine in the nuclear and mitochondrial genomes, indicating impaired MMR. In T. cruzi, loss of MSH2 also increases the parasite capacity to survive within host macrophages. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate MSH2 displays conserved, dual roles in MMR and in the response to oxidative stress. Loss of the latter function results in life cycle dependent differences in phenotypic outcomes in T. brucei MSH2 mutants, most likely because of the greater burden of oxidative stress in the insect stage of the parasite.