Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin

The objective of this work was to study the pattern of salivation of triatomines during feeding in mouse skin. Rhodnius prolixus was fed with a solution of the dye acridine orange or fluorescein. The saliva was efficiently labelled with acridine orange, probably due to the difference in pH between the salivary gland (6.0) and the hemolymph (6.5-7.0). This procedure was not effective at labelling the saliva of Triatoma infestans, however, fluorescent labelling of R. prolixus saliva allowed us to demonstrate that salivation occurs during entire feeding process. The saliva is released soon after the bite. In the probing phase, saliva is pumped continuously in the host skin, including around the blood vessels. During the engorgement phase, saliva is observed in a bolus within the blood vessel and some of it is sucked up by the insect, together with blood. The frequency of saliva emission inside the vessels was low (0.51+/-0.18 Hz). The saliva deposition in the microcirculation is continuous and modulated by the frequency of the cibarial pump because, when functioning at high frequency, cibarial pump sucks almost all saliva to the insect gut. This mechanism would determine the quantity of saliva deposited in the microcirculation as necessary, and consequently minimizing the host's immune response to salivary antigens.     

Comparison of methods for determination of vitamin B12 in microbial material

Three different methods for the measurement of vitamin B12 were compared: two spectrophotometric methods and an HPLC one. When the pure vitamin was used, the results obtained using all three methods were similar, but when samples from microbial material were used, the results were different. The HPLC method could distinguish the true vitamin B12 from the different vitamin B12 analogues whereas the spectrophotometric methods could not.     

Liquid vs Solid Culture Medium to Evaluate Proportion and Time to Change in Management of Suspects of Tuberculosis—A Pragmatic Randomized Trial in Secondary and Tertiary Health Care Units in Brazil

Background

The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes.

Methods and Findings

A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44–.96, p = 0.002, NNT 16, 95% CI 10–39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6–36.4, p = 0.0001). No other important differences were observed.

Conclusions

This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB.

Trial Registration

Controlled-Trials.com ISRCTN79888843     

CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil

Helicobacter pylori infection is one of the most common infectionsworldwide and is associated with gastric diseases. Virulence factors such as VacA andCagA have been shown to increase the risk of these diseases. Studies have suggested acausal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shownto be geographically diverse. We investigated the number of CagA EPIYA motifs and thevacA i genotypes in H. pylori strains fromasymptomatic children. We included samples from 40 infected children (18 females and22 males), extracted DNA directly from the gastric mucus/juice (obtained using thestring procedure) and analysed the DNA using polymerase chain reaction and DNAsequencing. The vacA i1 genotype was present in 30 (75%) samples,the i2 allele was present in nine (22.5%) samples and both alleles were present inone (2.5%) sample. The cagA-positive samples showed distinctpatterns in the 3’ variable region of cagA and 18 of the 30 (60%)strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-Cmotifs. We confirmed that the studied population was colonised early by the mostvirulent H. pylori strains, as demonstrated by the high frequency ofthe vacA i1 allele and the high number of EPIYA-C motifs. Therefore,asymptomatic children from an urban community in Fortaleza in northeastern Brazil arefrequently colonised with the most virulent H. pyloristrains.     

The Spatiotemporal Program of DNA Replication Is Associated with Specific Combinations of Chromatin Marks in Human Cells

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.     

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells,Growth, Photosynthetic Capacity,and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.     

Abstracts

16th Smyte (Small Meeting on Yeast Transport and Energetics) held in Častá-Papiernička, Slovakia September 23–27, 1998

Abstracts     

DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI,WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.1

Skeletonema costatum (Grev.) Cleve emend. Zingone et Sarno and S. grevillei Sarno et Zingone were known only from the type material collected from Hong Kong waters more than a century ago. Both species have now been collected as live material, and their morphology and phylogenetic position are investigated in this study. Eight Skeletonema strains isolated from Florida, USA; Uruguay; and Brazil are attributed to S. costatum, while one strain from Oman is ascribed to S. grevillei based on morphological similarity to the type material of these species. In addition, a new Skeletonema species, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Australian waters. Skeletonema ardens has terminal fultoportula processes ending in a tapered, undulate protrusion and long intercalary fultoportulae with 1:1 junctions. The rimoportula of terminal valves is located at the margin of the valve face. No major morphological variations were observed within S. grevillei and S. ardens along a salinity gradient, whereas in S. costatum, the processes shortened and the valves came into close contact at low salinities, as already described for S. subsalsum (Cleve) Bethge. Consistent with their morphology, Skeletonema costatum and Skeletonema subsalsum also had similar rDNA sequences. Skeletonema grevillei and S. ardens were distinct in the large subunit (LSU) phylogeny. Skeletonema ardens exhibited consistent intraspecific genetic differences in both the LSU and small subunit (SSU) rDNA.     

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with magnesium and GDP

Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1alpha in complex with GDP (SsEF-1alpha.GDP) does not bind Mg(2+), when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1alpha and to check the ability of SsEF-1alpha.GDP to bind the ion, we have determined the crystal structure of SsEF-1alpha.GDP in the presence of a nonphysiological concentration (100 mM) of Mg(2+). The analysis of the coordination of Mg(2+) unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1alpha, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1alpha.GDP for Mg(2+) is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1alpha, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1alpha sequences hitherto reported.