Latent microsporidial infection in immunocompetent individuals - a longitudinal study

Background

Microsporidia (Fungi) have been repeatedly identified as the cause of opportunistic infections predominantly in immunodeficient individuals such as AIDS patients. However, the global epidemiology of human microsporidiosis is poorly understood and the ability of microsporidia to survive and multiply in immunocompetent hosts remains unsolved.

Aims

To determine the presence of latent microsporidia infections in apparently healthy humans in the Czech Republic, the authors tested sera, urine and stool originating from fifteen persons within a three month period examined on a weekly basis.

Methods

Sera, stool and urine samples originating from fifteen HIV-negative people at risk with occupational exposure to animals, aged 22–56 years, living in the Czech Republic were tested by indirect immunofluorescence assay (IFA) for the presence of specific anti-microsporidial antibodies, standard Calcofluor M2R staining for the detection of microsporidian spores in all urine sediments and stool smears and molecular methods for the microsporidial species determination.

Results

Specific anti-microsporidial antibodies were detected in fourteen individuals, asymptomatic Encephalitozoon spp. infection was found in thirteen and E. bieneusi infection was detected in seven of those examined. While E. hellem 1A and E. cuniculi II were the major causative agents identified, seven different genotypes of E. bieneusi were recorded.

Conclusions

These findings clearly show that exposure to microsporidia is common and chronic microsporidiosis is not linked to any clinical manifestation in healthy population. Moreover, our results indicate much higher incidence of microsporidial infections among an apparently healthy population than previously reported. These results open the question about the potential risk of reactivation of latent microsporidiosis in cases of immunosupression causing life-threatening disease.     

Carboxylate and aromatic active-site residues are determinants of high-affinity binding of ω-aminoaldehydes to plant aminoaldehyde dehydrogenases

The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet?al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.     

Drug metabolome of the simvastatin formed by human intestinal microbiota in vitro

The human colon contains a diverse microbial population which contributes to degradation and metabolism of food components. Drug metabolism in the colon is generally poorly understood. Metabolomics techniques and in vitro colon models are now available which afford detailed characterization of drug metabolites in the context of colon metabolism. The aim of this work was to identify novel drug metabolites of Simvastatin (SV) by using an anaerobic human in vitro colon model at body temperature coupled with systems biology platform, excluding the metabolism of the host liver and intestinal epithelia. Comprehensive two-dimensional gas chromatography with a time-of-flight mass spectrometry (GC×GC-TOFMS) was used for the metabolomic analysis. Metabolites showing the most significant differences in the active faecal suspension were elucidated in reference with SV fragmentation and compared with controls: inactive suspension or buffer with SV, or with active suspension alone. Finally, time courses of selected metabolites were investigated. Our data suggest that SV is degraded by hydrolytic cleavage of methylbutanoic acid from the SV backbone. Metabolism involves demethylation of dimethylbutanoic acid, hydroxylation/dehydroxylation and β-oxidation resulting in the production of 2-hydroxyisovaleric acid (3-methyl-2-hydroxybutanoic acid), 3-hydroxybutanoic acid and lactic acid (2-hydroxypropanoic acid), and finally re-cyclisation of heptanoic acid (possibly de-esterified and cleaved methylpyranyl arm) to produce cyclohexanecarboxylic acid. Our study elucidates a pathway of colonic microbial metabolism of SV as well as demonstrates the applicability of the in vitro colon model and metabolomics to the discovery of novel drug metabolites from drug response profiles.     

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism

Background

Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved.

Methodology/Principal Findings

In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux.

Conclusions/Significance

Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.     

Daily and seasonal variation in the activity of potential vector mosquitoes

In the course of season 2010 (May–September), three 2-day trappings of female mosquitoes were carried out at two sites in order to determine the daily activity of the common mosquito species (e.g. species from genus Culex, Aedes, Ochlerotatus, Anopheles, etc.) in the area. CDC light traps filled with CO₂ and placed at a height of 1 m were used to trap individuals, and were sampled every 2 h. A total of 19,604 female mosquitoes from 20 identifiable species were trapped: 7,549 at Sedlec and 12,055 at Kančí obora. The activity of the major species of mosquitoes in South Moravia differed throughout the course of the day. Calamity species of the genus Aedes and Ochlerotatus remained active throughout the day and night, but with different course. Aedes vexans, Ae. cinereus, and Ae. rossicus were most active in the late afternoon and highly active for most of the night. Ochlerotatus sticticus was captured most often in the afternoon, and its abundance decreased rapidly before sunset. The activity of Oc. cantans s.l. (Oc. cantans + Oc. annulipes) females varied little during the day and night. The daily activity for the main vectors of West Nile virus, Culex pipiens and Cx. modestus, were totally different from that of other species. Cx. pipiens females showed significant night activity, while Cx. modestus was most active in the evening. Nighttime activity was also observed in female mosquitoes of the genus Anopheles.     

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Background

The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA.

Principal Findings

As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located.

Conclusions

The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.     

Genetic differentiation of the Western Capercaillie highlights the importance of South-eastern Europe for understanding the species phylogeography

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.     

Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.     

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.