Studies on nucleotide levels and electron transport genes of Clostridium acetobutylicum P262

Clostridium acetobutylicum P262 is an endospore-forming Gram positive anaerobic bacterium, and for many years this organism has been used in the industrial fermentation for the production of acetone and butanol from carbohydrate substrates. The aims of this thesis included studies on small phosphorylated molecules involved in energy metabolism and cell differentiation, and an investigation into the genetics and molecular biology of C. acetobutylicum electron transport genes. To facilitate quantitation of nucleoside triphosphates in extracts of C. acetobutylicum, a chromatographic data acquisition and analysis system was constructed. Samples were prepared from C. acetobutylicum cultures by treatment with formic acid, and nucleotides contained in these extracts were separated by strong anion exchange HPLC. The developed manual integration system features the ability to collect and store chromatographic data, allowing for multiple integration using different calibration curves. Nucleoside triphosphate profiles were obtained from batch fermentations of the C. acetobutylicum P262 wild type, sporulation deficient (spo-1) and solvent deficient (ds-1) strains. The nucleoside triphosphate profiles of the wild type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. Exponential phase nucleotide levels in the cls-1 mutant were comparable to those observed in the wild type and spo-1 mutant. Unlike the wild type and spo-1 strains, the cls-1 mutant, which does not switch to solventogenesis, did not demonstrate an increase in nucleotide levels after the cessation of cell division. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation. The antibacterial agent metronidazole, was used as a tool for the isolation of C. acetobutylicum electron transport genes. Since it was desired to clone these genes in Escherichia coli, and investigation into the activation of metronidazole by E. coli strains was necessary. E. coli strains with lesions in their DNA repair systems were more susceptible to metronidazole than wild type strains. However, it has been reported that DNA repair deficient strains of E. coli that also had a diminished ability to reduce chlorates and nitrates were no more susceptible to metronidazole than their wild type parents (Jackson et al., 1984; Yeung et al., 1984). To isolate a suitable E. coli cloning host for the selection of C. acetobutylicum electron transport genes which activated metronidazole, transposon mutagenesis of the recA E. coli strain CC118 with TnphoA, was used to construct the recA, metronidazole resistant E. coli strain Fl9. F19 was shown to have diminished nitroreductase activity, which was presumed to be responsible for the metronidazole resistant phenotype. However, the recA mutation renders E. coli F19 highly susceptible to the reduced toxic intermediates of metronidazole. The E. coli F19 recA, nitroreductase deficient mutant was used for the isolation of C. acetobutylicum genes on recombinant plasmids which activated metronidazole. Twenty-five E. coli F19 clones which contained different recombinant plasmids were isolated. The clones were tested for nitroreductase, pyruvate-Fd­oxidoreductase and hydrogenase activities. Nitroreductase and pyruvate-Fd­oxidoreductase activity was not demonstrated in any of the isolated clones, and only one clone tested positive for hydrogenase activity. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1 kb chromosomal fragment. It was determined that this 11.1 kb fragment contained at least two regions responsible for activating metronidazole. The one gene responsible for making E. coli F19 extremely sensitive to metronidazole was localized to a 2 kb region. The nucleotide sequence of this 2 kb region was determined and two truncated open reading frames and one complete open reading were present. The complete open reading frame was shown to be responsible for activating metronidazole. The deduced amino acid sequence of this open reading frame was determined to be 160 amino acids in length, and database searches showed good similarity to flavodoxin proteins from many organisms. Based on alignments to the amino acid sequences of these flavodoxins, as well as the fact that Chen and Blanchard (1979) reported that reduced flavodoxin can transfer electrons to metronidazole, the sequence corresponding to this C. acetobutylicum metronidazole activating gene was identified as coding for a flavodoxin gene. The role of flavodoxin in C. acetobutylicum and other organisms is presented. Possible relationships between the cloned C. acetobutylicum flavodoxin gene and metronidazole sensitivity are discussed

The Seroprevalence and Seroincidence of Enterovirus71 Infection in Infants and Children in Ho Chi Minh City, Viet Nam

Enterovirus 71 (EV71)-associated hand, foot and mouth disease has emerged as a serious public health problem in South East Asia over the last decade. To better understand the prevalence of EV71 infection, we determined EV71 seroprevalence and seroincidence amongst healthy infants and children in Ho Chi Minh City, Viet Nam. In a cohort of 200 newborns, 55% of cord blood samples contained EV71 neutralizing antibodies and these decayed to undetectable levels by 6 months of age in 98% of infants. The EV71 neutralizing antibody seroconversion rate was 5.6% in the first year and 14% in the second year of life. In children 5–15 yrs of age, seroprevalence of EV71 neutralizing antibodies was 84% and in cord blood it was 55%. Taken together, these data suggest EV71 force of infection is high and highlights the need for more research into its epidemiology and pathogenesis in high disease burden countries

Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection

Towards an understanding of induced-charge electrokinetics at large applied voltages in concentrated solutions

The venerable theory of electrokinetic phenomena rests on the hypothesis of a dilute solution of point-like ions in quasi-equilibrium with a weakly charged surface, whose potential relative to the bulk is of order the thermal voltage (kT/e ≈ 25 mV at room temperature). In nonlinear electrokinetic phenomena, such as AC or induced-charge electro-osmosis (ACEO, ICEO) and induced-charge electrophoresis (ICEP), several V ≈ 100 kT/e are applied to polarizable surfaces in microscopic geometries, and the resulting electric fields and induced surface charges are large enough to violate the assumptions of the classical theory. In this article, we review the experimental and theoretical literatures, highlight discrepancies between theory and experiment, introduce possible modifications of the theory, and analyze their consequences. We argue that, in response to a large applied voltage, the “compact layer” and “shear plane” effectively advance into the liquid, due to the crowding of counterions. Using simple continuum models, we predict two general trends at large voltages: (i) ionic crowding against a blocking surface expands the diffuse double layer and thus decreases its differential capacitance, and (ii) a charge-induced viscosity increase near the surface reduces the electro-osmotic mobility; each trend is enhanced by dielectric saturation. The first effect is able to predict high-frequency flow reversal in ACEO pumps, while the second may explain the decay of ICEO flow with increasing salt concentration. Through several colloidal examples, such as ICEP of an uncharged metal sphere in an asymmetric electrolyte, we show that nonlinear electrokinetic phenomena are generally ion-specific. Similar theoretical issues arise in nanofluidics (due to confinement) and ionic liquids (due to the lack of solvent), so the paper concludes with a general framework of modified electrokinetic equations for finite-sized ions.National Science Foundation (U.S.) (contract DMS-0707641

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Competitive outcome of Daphnia-Simocephalus experimental microcosms: salinity versus priority effects

Competition is a major driving force in freshwaters, especially given the cyclic nature and dynamics of pelagic food webs. Competition is especially important in the initial species assortment during colonization and re-colonization events, which depends strongly on the environmental context. Subtle changes, such as saline intrusion, may disrupt competitive relationships and, thus, influence community composition. Bearing this in mind, our objective was to assess whether low salinity levels (using NaCl as a proxy) alter the competitive outcome (measured as the rate of population biomass increase) of Daphnia-Simocephalus experimental microcosms, taking into account interactions with priority effects (sequential species arrival order). With this approach, we aimed to experimentally demonstrate a putative mechanism of differential species sorting in brackish environments or in freshwaters facing secondary salinization. Experiments considered three salinity levels, regarding NaCl added (0.00, 0.75 and 1.50 g L(-1)), crossed with three competition scenarios (no priority, priority of Daphnia over Simocephalus, and vice-versa). At lower NaCl concentrations (0.00 and 0.75 g L(-1)), Daphnia was a significantly superior competitor, irrespective of the species inoculation order, suggesting negligible priority effects. However, the strong decrease in Daphnia population growth at 1.50 g L(-1) alleviated the competitive pressure on Simocephalus, causing an inversion of the competitive outcome in favour of Simocephalus. The intensity of this inversion depended on the competition scenario. This salinity-mediated disruption of the competitive outcome demonstrates that subtle environmental changes produce indirect effects in key ecological mechanisms, thus altering community composition, which may lead to serious implications in terms of ecosystem functioning (e.g. lake regime shifts due to reduced grazing) and biodiversity

Rates, distribution and implications of postzygotic mosaic mutations in autism spectrum disorder

We systematically analyzed postzygotic mutations (PZMs) in whole-exome sequences from the largest collection of trios (5,947) with autism spectrum disorder (ASD) available, including 282 unpublished trios, and performed resequencing using multiple independent technologies. We identified 7.5% of de novo mutations as PZMs, 83.3% of which were not described in previous studies. Damaging, nonsynonymous PZMs within critical exons of prenatally expressed genes were more common in ASD probands than controls (P < 1 Ã 10-6), and genes carrying these PZMs were enriched for expression in the amygdala (P = 5.4 Ã 10-3). Two genes (KLF16 and MSANTD2) were significantly enriched for PZMs genome-wide, and other PZMs involved genes (SCN2A, HNRNPU and SMARCA4) whose mutation is known to cause ASD or other neurodevelopmental disorders. PZMs constitute a significant proportion of de novo mutations and contribute importantly to ASD risk

International AIDS Society global scientific strategy: towards an HIV cure 2016

Antiretroviral therapy is not curative. Given the challenges in providing lifelong therapy to a global population of more than 35 million people living with HIV, there is intense interest in developing a cure for HIV infection. The International AIDS Society convened a group of international experts to develop a scientific strategy for research towards an HIV cure. This Perspective summarizes the group's strategy