Seasonal Prevalence of Enteropathogenic Vibrio and Their Phages in the Riverine Estuarine Ecosystem of South Bengal

Diarrheal disease remains an unsolved problem in developing countries. The emergence of new etiological agents (non-cholera vibrios) is a major cause of concern for health planners. We attempted to unveil the seasonal dynamics of entero-pathogenic Vibrios in Gangetic riverine-estuarine ecosystem. 120 surface water samples were collected for a period of one year from 3 sampling sites on the Hooghly river. Five enteropathogenic Vibrio species, V. cholerae (35%), V. parahaemolyticus (22.5%), V. mimicus (19.1%), V. alginolyticus (15.8%) and V. vulnificus (11.6%), were present in the water samples. The vibriophages, V. vulnificus ɸ (17.5%), V. alginolyticus ɸ (17.5%), V. parahaemolyticus ɸ (10%), V. cholerae non-O1/O139 ɸ (26.6%) and V. mimicus ɸ (9.1%), were also detected in these samples. The highest number of Vibrios were noted in the monsoon (20–34°C), and to a lesser extent, in the summer (24–36°C) seasons. Samples positive for phages for any of the identified Vibrio species were mostly devoid of that particular bacterial organism and vice versa. The detection of toxin genes and resistance to β-lactam antibiotics in some environmental enteropathogenic Vibrio species in the aquatic niches is a significant outcome. This finding is instrumental in the south Bengal diarrhoeal incidence.     

Determining Maximum Glycolytic Capacity Using Extracellular Flux Measurements

Measurements of glycolytic rate and maximum glycolytic capacity using extracellular flux analysis can give crucial information about cell status and phenotype during normal operation, development of pathology, differentiation, and malignant transformation. They are also of great use when assessing the effects of chemical or drug treatments. Here, we experimentally define maximum glycolytic capacity, demonstrate how it differs from glycolytic rate, and provide a protocol for determining the basal glycolytic rate and maximum glycolytic capacity in cells using extracellular flux measurements. The results illustrate the power of extracellular flux analysis to describe the energetics of adherent cells in culture in a fully quantitative way.     

Circulating microRNAs Reveal Time Course of Organ Injury in a Porcine Model of Acetaminophen-Induced Acute Liver Failure

ConclusionsMicroRNAs were released passively into the circulation in response to acetaminophen-induced cellular damage. A significant increase in global microRNA was detectable prior to significant increases in miR122, miR192 and miR124-1, which were associated with clinical evidence of liver, kidney and brain injury respectively.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Evidence That Msh1p Plays Multiple Roles in Mitochondrial Base Excision Repair

Mitochondrial DNA is thought to be especially prone to oxidative damage by reactive oxygen species generated through electron transport during cellular respiration. This damage is mitigated primarily by the base excision repair (BER) pathway, one of the few DNA repair pathways with confirmed activity on mitochondrial DNA. Through genetic epistasis analysis of the yeast Saccharomyces cerevisiae, we examined the genetic interaction between each of the BER proteins previously shown to localize to the mitochondria. In addition, we describe a series of genetic interactions between BER components and the MutS homolog MSH1, a respiration-essential gene. We show that, in addition to their variable effects on mitochondrial function, mutant msh1 alleles conferring partial function interact genetically at different points in mitochondrial BER. In addition to this separation of function, we also found that the role of Msh1p in BER is unlikely to be involved in the avoidance of large-scale deletions and rearrangements.DEPLETION of mitochondrial function has been implicated in the human aging process as well as in several inherited and aging-related disorders (Wallace 2005; Weissman et al. 2007). Much of this dysfunction may be attributed to mitochondrial genome instability, as the respiratory capacity of the mitochondria is dependent on an intact genome. Since respiration is essential for the survival of eukaryotic obligate aerobes, the facultative anaerobe Saccharomyces cerevisiae is an ideal model system for mitochondrial studies. Despite the difference in size between the mitochondrial genomes of yeast and humans, the encoded components are required for the same process, cellular energy production (Foury et al. 1998). Therefore, studying how S. cerevisiae maintain mitochondrial DNA (mtDNA) could lend valuable insight into mitochondrial genome maintenance in higher eukaryotes (Perocchi et al. 2008).The necessary process of electron transport during respiration can cause damage to proteins, lipids, and nucleic acids through the formation of reactive oxygen species (ROS) (Longo et al. 1996). Because mtDNA exists in this harsh environment, it is thought that it is especially prone to oxidative damage (Bohr 2002). Damaged bases can be mutagenic by misincorporation opposite the damage by the replicative polymerase or by translesion synthesis beyond the damaged base. Therefore, the repair of oxidative lesions is essential for the stability of the mitochondrial genome.An important mechanism for repair of oxidative DNA damage is the base excision repair (BER) pathway (Croteau and Bohr 1997; Nilsen and Krokan 2001; Bohr 2002). This pathway is well studied in the nucleus of many organisms, and isoforms of several key components have been shown to localize to the mitochondrial compartment (Rosenquist et al. 1997; You et al. 1999; Vongsamphanh et al. 2001). However, despite their extensive nuclear and biochemical characterization, the role of these isoforms in the repair of mtDNA is poorly understood.BER is initiated when an N-glycosylase recognizes a damaged base and cleaves the glycosidic bond between it and the sugar-phosphate backbone, creating an apurinic/apyrimidinic (AP) site that can be repaired by one of two BER pathways. In short patch BER, the AP site is processed by an AP endonuclease on the 5′ side of the damaged base and by the AP lyase activity of a glycosylase, or polymerase β, on the 3′ side of the damage, to create a single-strand gap (Wilson et al. 1998). This gap is filled by a DNA polymerase and then ligated to complete the repair. In the alternative method of long-patch BER, the DNA is again cleaved by an AP endonuclease to generate an available 3′-end for synthesis by a DNA polymerase at the nick, displacing the existing sequence containing the abasic site and creating a 5′ flap. This flap is cleaved by a flap endonuclease, and the resulting nick is sealed by DNA ligase, completing the repair. Biochemical studies suggest that both short-patch and long-patch pathways are active in mitochondria (Akbari et al. 2008; Liu et al. 2008; Szczesny et al. 2008).In this study, we examine the mitochondrial roles of Apn1p, Ntg1p, and Ogg1p, three well-studied BER components. The N-glycosylase Ogg1p is important for the repair of oxidatively damaged DNA, and studies of ogg1-Δ strains have found an increase in point mutations in both nuclear and mitochondrial DNA (Thomas et al. 1997; Singh et al. 2001). In yeast, it was previously demonstrated that a deletion of the N-glycosylase NTG1, or the AP endonuclease APN1, leads to a decrease in mitochondrial mutations as measured by rates of erythromycin resistance, suggesting that the actions of Ntg1p and Apn1p create mutagenic intermediates in mtDNA during repair (Phadnis et al. 2006). This stands in contrast to the increases seen for nuclear DNA mutation rates in the presence of these deletion alleles, indicating that it is not always possible to extrapolate the mitochondrial function of BER proteins on the basis of their nuclear functions, thus making mitochondrial-specific studies necessary (Ramotar et al. 1991, 1993; Alseth et al. 1999; Bennett 1999). In addition, there are likely to be mitochondrial-specific players in the pathway. Here we show that the mismatch repair homolog Msh1p plays multiple roles in mitochondrial BER.Msh1p is the only one of six yeast homologs of MutS, the bacterial mismatch repair protein, which has been found localized to the mitochondria (Reenan and Kolodner 1992; Chi and Kolodner 1994). Msh1p is essential for mitochondrial function and maintenance of mtDNA, necessitating the use of partial function mutants to study the role of Msh1p in mtDNA maintenance (Mookerjee et al. 2005). Although the effects of its disruption have been examined in multiple studies, the mechanism by which Msh1p acts to carry out its essential functions remains unclear (Reenan and Kolodner 1992; Koprowski et al. 2002; Mookerjee et al. 2005; Mookerjee and Sia 2006). Its role as a mitochondrial mismatch repair protein has been disputed, particularly since there are no other mismatch repair proteins that localize to the mitochondria. However, since mtDNA has such a high potential requirement for BER, it is possible that this pathway in the mitochondria may utilize Msh1p. Previous studies have shown genetic interactions between Msh1p and the BER proteins Ogg1p, Apn1p, and Ntg1p (Dzierzbicki et al. 2004; Kaniak et al. 2009). Using msh1 alleles disrupted in conserved DNA binding and ATPase domains, we have examined the frequency and spectrum of mutations responsible for the different mutation rates seen with each allele.     

Paternity analysis using microsatellite markers to identify pollen donors in an olive grove

Olive (Olea europaea L.) is a wind-pollinated, allogamous species that is generally not considered to be self-compatible. In addition, cross-incompatibilities exist between cultivars that can result in low fruit set if compatible pollinisers are not planted nearby. In this study, microsatellite markers were used to identify 17 genotypes that were potential pollen donors in a commercial olive orchard. DNA typing with the same primers was also applied to 800 olive embryos collected from five cultivars in the grove over 2 years of study. Pollen donors for the cultivars Barnea, Corregiola, Kalamata, Koroneiki, and Mission were estimated by paternity analysis, based on the parental contribution of alleles in the genotypes of the embryos. The exclusion probability for the marker set was 0.998 and paternity was assigned on the basis of the ‘most likely method’. Different pollen donors were identified for each of the maternal cultivars indicating that cross-compatibilities and incompatibilities varied between the genotypes studied. Cross-pollination was the principal method of fertilization, as selfing was only observed in two of the embryos studied and both of these were from the cultivar Mission. This is the first report where these techniques have been applied to survey the pollination patterns in an olive grove. The results indicate that careful planning in orchard design is required for efficient pollination between olive cultivars.