Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.     

Prevalence of hepatitis B and C virus infection among leprosy patients in a leprosy-endemic region of central Brazil

Leprosy and hepatitis B virus (HBV) are highly endemic in some regions of the state of Mato Grosso, in central Brazil. The association of leprosy with HBV and hepatitis C virus (HCV) was assessed using a seroprevalence study and 191 leprosy outpatients were included. Demographic data and the clinical classification of leprosy were recorded. Evidence of previous HBV infection was present in 53 patients (27.7%, 95% confidence interval: 21.9-34.5) and two (1%) were HBsAg positive. Five (2.6%) had antibodies to HCV. The prevalence of previous exposure to HBV was higher than expected for an adult population in central Brazil. In contrast, the prevalence of anti-HCV antibodies was not much higher regarding the age range of participants. HBV markers were associated with a higher number of sex partners and the use of injections without proper sterilisation of the syringes. The number of HBV carriers was small, suggesting that there was no increased likelihood of chronification among these patients.     

The relative impact of lichen symbiotic partners to repeated copper uptake

The relative impact of lichen photobiont and mycobiont was evaluated by submitting nine lichen species with: (i) different photobiont types; (ii) different lichen growth forms; and (iii) different nutrients, pH, humidity preferences; to a range of Cu concentrations (μM) supplied in repeated cycles to simulate the natural process of uptake under field conditions. The physiological performance of the photosystem II photochemical reactions was measured using F_v/F_m and the metabolic activity of the mycobiont was evaluated using ergosterol and intracellular K-loss as indicators. Lichens with higher cation exchange capacity showed higher intracellular Cu uptake and their ecology seemed to be associated with low-nutrient environments. Thus the wall and external matrix, mainly characteristic of the mycobiont partner, cannot be ignored as the first site of interaction of metals with lichens. No common intracellular Cu concentration threshold was found for the physiological impacts observed in the different species. Most physiological effects of Cu uptake in sensitive lichens occurred for intracellular Cu below 200 μg/g dw whereas more tolerant species were able to cope with intracellular Cu at least 3 times higher. Cyanobacterial lichens showed to be more sensitive to Cu uptake than green-algal lichens. Within the Trebouxia lichens, different species showed different sensitivities to Cu uptake, suggesting that the mycobiont may change the microenvironment close to the photobiont partner providing different degrees of protection. Despite the fact that the photobiont is the productive partner, the metabolic activity of the mycobiont of lichen species adapted to environments rich in nutrients, showed to be more sensitive to Cu uptake than the photochemical performance of the photobiont.     

Taxonomic and Functional Metagenomic Profiling of the Microbial Community in the Anoxic Sediment of a Sub-saline Shallow Lake (Laguna de Carrizo, Central Spain)

The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4)?×?2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1?Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.     

Haplotypes of the bovine IgG2 heavy gamma chain in tick-resistant and tick-susceptible breeds of cattle

Bovines present contrasting, heritable phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus. Tick salivary glands produce IgG-binding proteins (IGBPs) as a mechanism for escaping from host antibodies that these ectoparasites ingest during blood meals. Allotypes that occur in the constant region of IgG may differ in their capacity to bind with tick IGBPs; this may be reflected by the distribution of distinct allotypes according to phenotypes of tick infestations. In order to test this hypothesis, we investigated the frequency of haplotypes of bovine IgG2 among tick-resistant and tick-susceptible breeds of bovines. Sequencing of the gene coding for the heavy chain of IgG2 from 114 tick-resistant (Bos taurus indicus, Nelore breed) and tick-susceptible (B. t. taurus, Holstein breed) bovines revealed SNPs that generated 13 different haplotypes, of which 11 were novel and 5 were exclusive of Holstein and 3 of Nelore breeds. Alignment and modeling of coded haplotypes for hinge regions of the bovine IgG2 showed that they differ in the distribution of polar and hydrophobic amino acids and in shape according to the distribution of these amino acids. We also found that there was an association between genotypes of the constant region of the IgG2 heavy chain with phenotypes of tick infestations. These findings open the possibility of investigating if certain IgG allotypes hinder the function of tick IGBPs. If so, they may be markers for breeding for resistance against tick infestations.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.     

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.