Yeast RNA polymerase I: A eukaryotic zinc metalloenzyme

Microwave excitation spectrometry and metal binding inhibition studies show that zinc is a catlytically essential component of the highly purified RNA polymerase I from yeast, the first eukaryotic RNA polymerase I available in quantities sufficient for such studies. It contains 2.4 g-atom of zinc based on a molecular weight of 6.5 × 10⁵ (8). Copper, iron, manganese and magnesium are absent, i.e., below the limits of detection, 10^?13 to 10^?14 g-atoms. A number of derivatives of 1,10-phenanthroline reversibly inhibit the polymerase catalyzed reaction, apparently by forming a ternary polymerase·Zn·OP complex while the nonchelating isomer, 1,7-phenanthroline, is ineffective.     

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.     

Potential Early Predictors for Outcomes of Experimental Hemorrhagic Shock Induced by Uncontrolled Internal Bleeding in Rats

Uncontrolled hemorrhage, resulting from traumatic injuries, continues to be the leading cause of death in civilian and military environments. Hemorrhagic deaths usually occur within the first 6 hours of admission to hospital; therefore, early prehospital identification of patients who are at risk for developing shock may improve survival. The aims of the current study were: 1. To establish and characterize a unique model of uncontrolled internal hemorrhage induced by massive renal injury (MRI), of different degrees (20-35% unilateral nephrectomy) in rats, 2. To identify early biomarkers those best predict the outcome of severe internal hemorrhage. For this purpose, male Sprague Dawley rats were anesthetized and cannulas were inserted into the trachea and carotid artery. After abdominal laparotomy, the lower pole of the kidney was excised. During 120 minutes, hematocrit, pO₂, pCO₂, base excess, potassium, lactate and glucose were measured from blood samples, and mean arterial pressure (MAP) was measured through arterial tracing. After 120 minutes, blood loss was determined. Statistical prediction models of mortality and amount of blood loss were performed. In this model, the lowest blood loss and mortality rate were observed in the group with 20% nephrectomy. Escalation of the extent of nephrectomy to 25% and 30% significantly increased blood loss and mortality rate. Two phases of hemodynamic and biochemical response to MRI were noticed: the primary phase, occurring during the first 15 minutes after injury, and the secondary phase, beginning 30 minutes after the induction of bleeding. A Significant correlation between early blood loss and mean arterial pressure (MAP) decrements and survival were noted. Our data also indicate that prediction of outcome was attainable in the very early stages of blood loss, over the first 15 minutes after the injury, and that blood loss and MAP were the strongest predictors of mortality.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

Unique genetic variation revealed by a microsatellite polymorphism survey in ten wild-derived inbred strains

Here we report on a genome polymorphism survey using 254 microsatellite markers in ten recently wild-derived inbred strains. Allele size analysis showed that the rate of polymorphism of these wild-derived mouse strains when compared with any of the common laboratory strains is on average 79.8%. We found 632 wild-derived alleles that were not present in the common laboratory strains, representing a 61% increase over the genetic variation observed in the laboratory strains. We also found that on average 14.5% of the microsatellite alleles of any given wild-derived inbred strain were unique. Our results indicate that the recently wild-derived mouse strains represent repositories of unique naturally occurring genetic variability and may prove invaluable for the study of complex phenotypes and in the construction of new mouse models of human disease.     

PhyTB: Phylogenetic tree visualisation and sample positioning for M. tuberculosis

Background

Phylogenetic-based classification of M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.

Results

We have developed a web-based tool called PhyTB (http://pathogenseq.lshtm.ac.uk/phytblive/index.php) to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.

Conclusion

PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (http://sourceforge.net/projects/phylotrack).     

Circadian cortisol secretion and circadian adrenal responses to ACTH are maintained in dexamethasone suppressed capuchin monkeys (Cebus apella)

We tested the hypothesis that the capuchin monkey adrenal (Cebus apella) gland has oscillatory properties that are independent of adrenocorticotropic hormone (ACTH) by exploring under ACTH suppression by dexamethasone: (i) maintenance of a circadian rhythm of plasma cortisol and (ii) clock time dependency of plasma cortisol response to exogenous ACTH. The capuchin monkey had a clear ACTH and plasma cortisol rhythm. Dexamethasone treatment resulted in low non-rhythmic ACTH levels and decreased cortisol to 1/10 of control values; nevertheless, the circadian rhythm of plasma cortisol persisted. We found that cortisol response to exogenous ACTH was clock time-dependent. The maximal response to ACTH occurred at the acrophase of the cortisol rhythm (0800 h). These results suggest that the capuchin monkey adrenal cortex may possess intrinsic oscillatory properties that participate in the circadian rhythm of adrenal cortisol secretion and in the circadian cortisol response to ACTH.     

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.