Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Sugar metabolism and developmental stages of rubber tree (Hevea brasiliensis L.) seeds

Changes in the concentration of sugars and sucrose metabolism enzymes can characterize the developmental stages of a seed. In recalcitrant species such as Hevea brasiliensis L., little is known about these changes. We aimed to evaluate the three main stages of development of rubber tree seeds – histodifferentiation, cell elongation and accumulation of reserves. The activities of acid and neutral invertases (E.C. 3.2.1.26) and sucrose synthase (EC 2.4.1.13), and the concentrations of reducing sugars (RS), total soluble sugars (TSS) and sucrose (Suc) were determined concomitantly with the histochemical and anatomical evaluation of seed structure. Histodifferentiation in rubber tree seeds occurs up to 75 days after anthesis (DAA). The concentration of RS is high and of Suc is low during seed histodifferentiation, which occurs along with a visible increase in the number of cell divisions. After that period, there is an increase in the concentration of Suc (mg g^?1) and in the number and size of starch granules, and a decrease in the concentration of RS (mg g^?1). At that point, cell elongation occurs. At 135 DAA, there is an inversion in the concentration of these two sugars and an increase in reserve accumulation. Thus, in seeds of the evaluated clone, the period up to 75 DAA is characterized as the histodifferentiation stage, while from that time up to 120 DAA the cell elongation stage takes place. The final stage of seed maturation and reserve accumulation begins at 135 DAA, and the seed, including the embryo, is completely formed at 175 DAA.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

MicroRNAs: new players in heart failure

MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the most exciting areas of modern medical science. miRNAs modulate a large and complex regulatory network of gene expression of the majority of the protein-coding genes. Currently, evidences suggest that miRNAs play a crucial role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and miR-208a are highly expressed in the heart and strongly associated with the development of cardiac hypertrophy. Recent data indicate that these miRNAs as well as miR-206 change their expression quickly in response to physical activity. The differential regulation of miRNAs in response to exercise suggests a potential value of circulating miRNAs (c-miRNAs) as biomarkers of physiological mediators of the cardiovascular adaptation induced by exercise. Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of miRNAs levels using techniques such as ‘miR mimics’ and ‘antagomiRs’ is becoming evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.     

Effects of Anabolic Steroids and High-Intensity Aerobic Exercise on Skeletal Muscle of Transgenic Mice

In an attempt to shorten recovery time and improve performance, strength and endurance athletes occasionally turn to the illicit use of anabolic-androgenic steroids (AAS). This study evaluated the effects of AAS treatment on the muscle mass and phenotypic characteristics of transgenic mice subjected to a high-intensity, aerobic training program (5d/wk for 6 weeks). The transgenic mice (CETP^+/-LDLr^-/+) were engineered to exhibit a lipid profile closer to humans. Animals were divided into groups of sedentary (Sed) and/or training (Ex) mice (each treated orally with AAS or gum arabic/vehicle: Sed-C, Sed-M, ex-C, ex-M). The effects of AAS (mesterolone: M) on specific phenotypic adaptations (muscle wet weight, cross-sectional area, and fiber type composition) in three hindlimb muscles (soleus:SOL, tibialis anterior:TA and gastrocnemius:GAS) were assessed. In order to detect subtle changes in fiber type profile, the entire range of fiber types (I, IC, IIAC, IIA, IIAD, IID, IIDB, IIB) was delineated using mATPase histochemistry. Body weight gain occurred throughout the study for all groups. However, the body weight gain was significantly minimized with exercise. This effect was blunted with mesterolone treatment. Both AAS treatment (Sed-M) and high-intensity, aerobic training (ex-C) increased the wet weights of all three muscles and induced differential hypertrophy of pure and hybrid fibers. Combination of AAS and training (ex-M) resulted in enhanced hypertrophy. In the SOL, mesterolone treatment (Sed-M and ex-M) caused dramatic increases in the percentages of fiber types IC, IIAC, IIAD, IID, with concomitant decrease in IIA, but had minimal impact on fiber type percentages in the predominantly fast muscles. Overall, the AAS-induced differential adaptive changes amounted to significant fiber type transformations in the fast-to-slow direction in SOL. AAS treatment had a significant effect on muscle weights and fiber type composition in SOL, TA and GAS which was even maximized in animals subjected to metabolically high-intensity aerobic exercise.     

Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera.

Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.     

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis

BackgroundThis study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population.MethodsA cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34).FindingsIn total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms “anesthetized skin area” and “pain in nerves” were most commonly mentioned in the LG while “tingling, numbness in the hands/feet”, “sensation of pricks and needles”, “pain in nerves” and “spots on the skin” responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients.InterpretationOur findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.     

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi’s virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.     

Cytotaxonomy of Eurypyga helias (Gruiformes,Eurypygidae): First Karyotypic Description and Phylogenetic Proximity with Rynochetidae

The sunbittern (Eurypyga helias) is a South American Gruiformes, the only member of Family Eurypigidae. In most phylogenetic proposals, it is placed in a more distant position than other families of the so-called “core Gruiformes”. Different studies based on molecular, morphological and biogeographical data suggest that the Eurypigidae is closely related to the kagu (Rhynochetos jubatus), the only species in Rynochetidae, another family not included in the core Gruiformes. Here, the karyotype of the sunbittern is described for the first time, by classical and molecular cytogenetics, using whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. We found a diploid number of 80, with only one pair of biarmed autosomal macrochromosomes, similar to that observed in the kagu. Chromosome painting revealed that most syntenies found in the avian putative ancestral karyotype (PAK) were conserved in the sunbittern. However, PAK1, PAK2, and PAK5 corresponded to two chromosome pairs each. Probes derived from L. albicollis confirm that fissions in PAK1 and PAK2 were centric, whereas in PAK5 the fission is interstitial. In addition, there is fusion of segments homologous to PAK2q and PAK5. From a phylogenetic point of view, comparisons of our results with two other Gruiformes belonging to family Rallidae suggest that the PAK5q fission might be a synapomorphy for Gruiformes. Fissions in PAK1 and PAK2 are found only in Eurypigidae, and might also occur in Rynochetidae, in view of the similar chromosomal morphology between the sunbittern and the kagu. This suggests a close phylogenetic relationship between Eurypigidae and Rynochetidae, whose common ancestor was separated by the Gondwana vicariancy in South America and New Caledonia, respectively.