An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

Mechanisms of diarrhea in lymphocytic colitis and therapeutic effect of Budesonide

In der hier vorliegenden Arbeit wurden an humanen Biopsien aus dem Colon sigmoideum die pathophysiologischen Mechanismen der Diarrhoe bei der lymphozytären Kolitis sowie der Einfluss von Budesonid auf die Diarrhoe untersucht. Ziel war es, die Transport- und Barriereeigenschaften des Darmes näher zu erforschen. Dabei wurden insbesondere der aktive elektrogene Natrium- Transport und die epitheliale Barriere untersucht. In einem Ussing-Experiment wurde die aktive elektrogene Natrium-Resorption des Dickdarmepithels als Ausdruck der elektrogenen Aktivität des Amilorid-sensitiven Natrium-Kanals (ENaC) bestimmt. Bei der lymphozytären Kolitis liegt eine Reduktion des Kurzschlussstroms um 68% im Vergleich zum Kontrollkollektiv vor. Diese gestörte ENaC-Funktion ist hier Ausdruck einer veränderten Induktion der mRNA- Expression der ENaC-Untereinheiten, die mit einer Herabregulation vor allem der γ-Untereinheit um 86% im Vergleich zu den Kontrollen einhergeht, während die α- und β-Untereinheiten unverändert bleiben. Zur Untersuchung der epithelialen Barriere kam die Wechselstrom-Impedanz-spektroskopie zum Einsatz. Bei der lymphozytären Kolitis beobachteten wir eine Minderung des epithelialen Widerstandes um 41% von 39 auf 23 Ω•cm2. Als Korrelat der Epithelwiderstandsreduktion stellten wir bei der Kolitis einen um 66% höheren parazellulären Flux als bei Kontrollen fest. In einer Expressionsanalyse der Tight Junction-Proteine zeigte sich eine verminderte Expression von Claudin-4 (71 ± 8%), Claudin-5 (72 ± 4%) und am stärksten von Claudin-8 (27 ± 10%). Die immunhistochemische Lokalisationsanalyse von Claudin-5 und Claudin-8 zeigte bei der lymphozytären Kolitis eine zelluläre Umverteilung dieser Tigh Junction-Proteine. Eine erhöhte Apoptoserate als einen weiteren Grund für eine strukturelle Barrierestörung des Epithels konnten wir bei der lymphozytären Kolitis nicht nachweisen. Eine Therapie mit Budesonid bewirkte eine klinische und histologische Befundbesserung, beeinflusste jedoch weder die ENaC-Funktion noch die epitheliale Barriere. Im zweiten Teil der Arbeit wurden an einem Rattenmodell die Transporteigenschaften des ENaC unter dem Einfluss der bei der mikroskopischen Kolitis erhöhten Zytokine TNFα, IFNγ und IL-15 untersucht. Wir konnten zeigen, dass der ENaC im Kolon der Ratte durch die Präinkubation mit TNFα um 51% gehemmt werden kann, während die alleinige Inkubation mit IL-15 keinen Einfluss auf seine Funktion hat. Ein synergistischer Effekt von TNFα und IL-15 bzw. TNFα, IFNγ und IL-15 konnte nicht gezeigt werden. Die Diarrhoe bei der lymphozytären Kolitis entsteht durch das Zusammenwirken mehrerer Pathomechanismen. Ein verminderter aktiver elektrogener Natrium- Transport, im Sinne einer Malabsorption mit einer gestörten ENaC-Funktion und eine epitheliale Barrierestörung mit verminderter Expression von abdichtenden Tight Junction-Proteinen mit daraus resultierender Leckflux-Diarrhoe sind hierbei die entscheidenden Diarrhoemechanismen.This study analyses the pathogenic mechanisms of diarrhea in lymphocytic colitis and the therapeutic effect of Budesonide. Therefore measurements on colonic specimens of patients with confirmed lymphocytic colitis were performed and compared with control patients. The aim was to examine the transport and the barrier functions of the epithelium. In short-circuit experiments we determined the electrogenic sodium absorption through the epithelial sodium channel (ENaC). In lymphocytic colitis the electrogenic sodium absorption was decreased by 68% compared to the values of controls. This impaired ENaC function was attributed to an altered induction of the ENaC-mRNA with decreased expression by 86% of the γ-ENaC subunit while the α- and β-subunits remain unchanged to controls. Alternating current impedance analysis was performed to estimate the epithelial barrier function. In lymphocytic colitis the epithelial resistance was decreased by 41% from 39 to 23 Ω∙cm2. At the same time we measured an increase by 66% of the paracellular flow of controls. In Western blot analysis and subsequent densitometry the tight junction proteins claudin-4 (71 ± 8%), claudin-5 (72 ± 4%) and claudin-8 (27 ± 10%) were decreased. In an immunohistochemistry staining a cellular redistribution of claufin-5 and claudin-8 could be shown. Beyond tight junction changes, epithelial apoptosis can also contribute to barrier dysfunction. However, we could not demonstrate an increased rate of apoptosis in lymphocytic colitis. Budesonide induced clinical and histological improvement however, it had no effect on ENaC function as well as on the epithelial barrier. The second part of the study included a rat model for the investigation of the effect of the cytokines TNFα, IFNγ and IL-15 which are elevated in microscopic colitis on the ENaC. We could show a decreased electrogenic sodium absorption by 51% after incubation with TNFα. There is no effect on the ENaC after incubating with IL-15 or the simultaneous incubation with TNFα and IL-15 or TNFα, IFNγ and IL-15. Diarrhea in lymphocytic colitis occurs as a synergy of sodium malabsorption due to an impaired ENaC and of barrier dysfunction due to decreased expression of tightening proteins leading to a leak flux

Genome Organization and Localization of the pufLM Genes of the Photosynthesis Reaction Center in Phylogenetically Diverse Marine Alphaproteobacteria

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria

Pseudomonas extremaustralis sp. nov. a polyhydroxybutyrate-producer isolated from Antarctic environments

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3T is closely related to Pseudomonas veronii DSM 11331T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3T has been deposited as DSM 17835T and as CIP 109839T.Fil: López, Nancy Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Stackebrandt, Erko. Deutsche Sammlung von Mikroorganismen und Zellkulturen; AlemaniaFil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Põtter, Markus. Universität Münster; AlemaniaFil: Steinbüchel, Alexander. Universität Münster; AlemaniaFil: Mendez, Beatriz Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis

Background: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na+ absorption and regulatory inputs in patients with LC by examining the epithelial Na+ channel (ENaC) function as the predominant Na+ transport system in human distal colon. Methods: Epithelial Na+ channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. Results: Epithelial Na+ channel-mediated electrogenic Na+ transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in gamma-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNF alpha, IFN gamma, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. Conclusions: In the sigmoid colon of patients with LC, the key effector cytokines TNF alpha, IFN gamma, and IL-15 inhibited gamma-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na+ malabsorption which contributes to diarrhea

Epithelial barrier dysfunction in lymphocytic colitis through cytokine-dependent internalization of claudin-5 and-8

Background Watery diarrhea is the cardinal symptom of lymphocytic colitis (LC). We have previously shown that colonic Na malabsorption is one of the major pathologic alterations of LC and found evidence for an epithelial barrier defect. On these grounds, this study aimed to identify the inherent mechanisms of this epithelial barrier dysfunction and its regulatory features. Methods Epithelial resistance (R-epi) was determined by one-path impedance spectroscopy and H-3-mannitol fluxes were performed on biopsies from sigmoid colon in miniaturized Ussing chambers. Tight junction proteins were analyzed by Western blot and confocal microscopy. Inflammatory signaling was characterized in HT-29/B6 cells. Apoptosis and mucosal surface parameters were quantified morphologically. Results Repi was reduced to 53% and H-3-mannitol fluxes increased 1.7-fold in LC due to lower expression of claudin-4, -5, and -8 and altered subcellular claudin-5 and -8 distributions off the tight junction. TNF alpha and IFN gamma could mimic subcellular redistribution in HT-29/B6 cells, a process which was independent on MLCK activation. Epithelial apoptosis did not contribute to barrier dysfunction in LC and mucosal surface area was unchanged. Conclusions Epithelial barrier dysfunction in LC occurs through downregulation of claudin-4, -5, and -8, and redistribution of claudin-5 and -8 off the tight junction, which contributes to diarrhea by a leak-flux mechanism. The key effector cytokines TNF alpha and IFNc gamma turned out to be the trigger for redistribution of claudin-5 and -8. Thus, alongside sodium malabsorption, leak-flux is yet another important diarrheal mechanism in LC

ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis

Background: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na+ absorption and regulatory inputs in patients with LC by examining the epithelial Na+ channel (ENaC) function as the predominant Na+ transport system in human distal colon. Methods: Epithelial Na+ channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. Results: Epithelial Na+ channel-mediated electrogenic Na+ transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in gamma-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNF alpha, IFN gamma, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. Conclusions: In the sigmoid colon of patients with LC, the key effector cytokines TNF alpha, IFN gamma, and IL-15 inhibited gamma-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na+ malabsorption which contributes to diarrhea