Mitochondrial respiration and genomic analysis provide insight into the influence of the symbiotic bacterium on host trypanosomatid oxygen consumption.

Certain trypanosomatids co-evolve with an endosymbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges. Symbionts were able to respire for up to 4 h after isolation from Angomonas deanei. FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) similarly increased respiration in wild-type and aposymbiotic protozoa, though a higher maximal O2 consumption capacity was observed in the symbiont-containing cells. Rotenone, a complex I inhibitor, did not affect A. deanei respiration, whereas TTFA (thenoyltrifluoroacetone), a complex II activity inhibitor, completely blocked respiration in both strains. Antimycin A and cyanide, inhibitors of complexes III and IV, respectively, abolished O2 consumption, but the aposymbiotic protozoa were more sensitive to both compounds. Oligomycin did not affect cell respiration, whereas carboxyatractyloside (CAT), an inhibitor of the ADP-ATP translocator, slightly reduced O2 consumption. In the A. deanei genome, sequences encoding most proteins of the respiratory chain are present. The symbiont genome lost part of the electron transport system (ETS), but complex I, a cytochrome d oxidase, and FoF1-ATP synthase remain. In conclusion, this work suggests that the symbiont influences the mitochondrial respiration of the host protozoan.

The presence of a symbiotic bacterium in Strigomonas culicis is related to differential ecto-phosphatase activity and influences the mosquito-protozoa interaction.

Strigomonas culicis is a monoxenous trypanosomatid that co-evolves with a symbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges between both partners. S. culicis infects and colonizes the Aedes aegypti mosquito midgut, reaches its hemocoel and then invades the salivary glands. An artificial aposymbiotic strain is unable to colonize insects, reinforcing the idea that the bacterium influences the protozoan surface composition and cell interaction. Here, we report the characterization of the hydrolytic activity of ecto-phosphatases evaluated in symbiont-bearing and aposymbiotic strains of S. culicis by incubating the protozoa with p-nitrophenyl phosphate (pNPP) at different pH levels, in the presence of phosphatase inhibitors, and with several divalent metals. The symbiont-bearing and aposymbiotic cells differ in their ecto-phosphatase enzymes, based on their activities and specificities. Furthermore, the ability of the protozoan to bind to the mosquito midgut and salivary glands was impaired by ecto-phosphatase inhibition. Taken together, our data suggest that the symbiont influences the host protozoan ecto-phosphatase activity and indicate a possible role of this enzyme during mosquito tissue colonization by S. culicis.

Functional changes in Duchenne muscular dystrophy: a 12-month longitudinal cohort study.

OBJECTIVE: The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment. METHODS: The study is a longitudinal multicentric cohort study. A total of 106 ambulant patients with DMD were assessed using the 6-minute walk test (6MWT) and North Star Ambulatory Assessment (NSAA) at baseline and 12 months. Clinical data including age and steroid treatment were collected. RESULTS: During the 12 months of the study, we observed a mean decline of 25.8 meters in the 6MWT with a SD of 74.3 meters. On NSAA, the mean decline was 2.2 points with a SD of 3.7. Not all the boys with DMD in our cohort showed a decline over the 12 months, with young boys showing some improvement in their 6MWT and NSAA scores up to the age of 7. NSAA and the 6MWT had the highest correlation (r = 0.52, p < 0.001). CONCLUSIONS: This study provides longitudinal data of NSAA and 6MWT over a 12-month period. These data can be useful when designing a clinical trial.

Access to tuberculosis diagnosis in Itaboraí City, Rio de Janeiro, Brazil: the patient's point of view.

SETTING: Itaboraí Municipality in Rio de Janeiro, Brazil. OBJECTIVE: To evaluate access to tuberculosis (TB) diagnosis for users of the Family Health Program (FHP) and Reference Ambulatory Units (RAUs). DESIGN: A cross-sectional study was conducted in Itaboraí City, Rio de Janeiro, Brazil. Between July and October 2007, a sample of 100 TB patients registered consecutively with the TB Control Program was interviewed using the primary care assessment tool. The two highest scores, describing 'almost always' and 'always', or 'good' and 'very good', were used as a cut-off point to define high quality access to diagnosis. RESULTS: FHP patients were older and had less education than RAU interviewees. Sex and overcrowding did not differ in the two groups. Patient groups did not differ with regard to the number of times care was sought at a unit, transport problems, cost of attending units and availability of consultation within 24 h. Adequate access to diagnosis was identified by 62% of the FHP patients and 53% of the RAU patients (P = 0.01). CONCLUSION: In Itaboraí, Rio de Janeiro, TB patients believe that the FHP units provide greater access to TB diagnosis than RAUs. These findings will be used by the Department of Health to improve access to diagnosis in Itaboraí.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys.

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys.

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Construction, characterization and immunogenicity of recombinant yellow fever 17D-dengue type 2 viruses.

Chimeric yellow fever (YF)-dengue type 2 (Den 2) viruses were constructed by replacing the premembrane (prM) and envelope (E) genes of YF 17D virus with those from Den 2 virus strains of south-east Asian genotype. Whereas viable chimeric viruses were successfully recovered when the YF 17D C gene and the Den 2 prM gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. Unlike YF virus that replicated in all the cell lines tested and similar to the Den 2 virus, the recombinant viruses did not replicate in vaccine-production certified CEF and MRC5 cells. Besides, chimeric 17D/Den 2 viruses and their parental viruses reached similar growth titers in Vero and C6/36 cell cultures. Analysis of mouse neurovirulence, performed by intracerebral inoculation, demonstrated that the 17D/Den 2 chimera is more attenuated in this system than the YF 17DD virus. Immunization of mice with this chimera induced a neutralizing antibody response associated with a partial protection against an otherwise lethal dose of mouse neurovirulent Den 2 NGC virus. Overall, these results provide further support for the use of chimeric viruses as an attractive methodology for the development of new live flavivirus vaccines.

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms.

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.

Endosymbiosis in protozoa of the Trypanosomatidae family.

A small number of trypanosomatids present bacterium endosymbionts in the cytoplasm, which divide synchronously with the host cell. Crithidia oncopleti, Crithidia deanei. Crithidia desouzai, Blastocrithidia culicis and Herpetomonas roitmani are the best characterized species. The endosymbiont is surrounded by two membranes separated from each other by an electron-lucent space. The presence of the endosymbiont led to the appearance of morphological changes which include the lack of the paraflagellar rod associated to the axoneme, the morphology of the kinetoplast and the association of the sub-pellicular microtubules with portions of the protozoan plasma membrane. Aposymbiotic strains could be obtained by antibiotic treatment, opening the possibility to make comparative analysis of endosymbiont-containing an endosymbiont-free populations of the same species. It is clear that metabolic cycles are established between the prokaryiont and the host cell. The results obtained show that endosymbiont-containing species of trypanosomatids constitute an excellent model to study basic processes on the endosymbiont-host cell relationship and the origin of new organelles.

Interactions of the CCAAT-binding trimer NF-Y with nucleosomes.

NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.

In vitro reconstitution of Artemia satellite chromatin.

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.

Ultrastructural and biochemical analysis of the relationship of Crithidia deanei with its endosymbiont.

Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.

Serum concentration and increased temperature alter Mayaro virus RNA and protein synthesis in Aedes albopictus (mosquito)-infected cells.

We have previously shown the inhibition of Mayaro virus multiplication in Aedes albopictus-infected cells maintained at a supraoptimal temperature for growth (37 degrees C) and a stimulation of virus production in response to high serum concentrations in the incubation medium. In the present study, we addressed the question of how the effect of continuous heat stress and high serum concentration soon after infection interfere with virus macromolecule synthesis. Cells maintained at 28 degrees C in the presence of 2% serum synthesized a viral genomic RNA of 12 kb and a subgenomic RNA of 5.2 kb 6 h postinfection. Analysis of the protein profile showed the presence of the viral nucleocapsid protein of 34 kDa (P34). However, if infected cells were maintained at 37 degrees C, a smear starting immediately below the 5.2-kb RNA was noticed and the viral P34 was not detected by SDS-PAGE. Addition of 10% serum to the growth medium of infected cells maintained at 37 degrees C results in a viral RNA profile and protein synthesis similar to those observed in cultures kept at 28 degrees C, i.e., the smear was not observed and the P34 protein was detected. The results suggest that the inhibition of virus multiplication by temperature may be related to the inhibition of viral nonstructural protein synthesis early during infection. The presence of high serum levels in the incubation medium protects macromolecule synthesis against heat stress.

Serum concentration and increased temperature alter Mayaro virus RNA and protein synthesis in Aedes albopictus (mosquito)-infected cells

We have previously shown the inhibition of Mayaro virus multiplication in Aedes albopictus-infected cells maintained at a supraoptimal temperature for growth (37§C) and a stimulation of virus production in response to high serum concentrations in the incubation medium. In the present study, we addressed the question of how the effect of continuous heat stress and high serum concentration soon after infection interfere with virus macromolecule synthesis. Cells maintained at 28§C in the presence of 2 percent serum synthesized a viral genomic RNA of 12 kb and a subgenomic RNA of 5.2 kb 6 h post-infection. Analysis of the protein profile showed the presence of the viral nucleocapsid protein of 34 kDa (P34). However, if infected cells were maintained at 37§C, a smear starting immediately below the 5.2-kb RNA was noticed and the viral P34 was not detected by SDS-PAGE. Addition of 10 percent serum to the growth medium of infected cells maintained at 37§C results in a viral RNA profile and proteins synthesis similar to those observed in cultures kept at 28§C, i.e., the smear was not observed and the P34 protein was detected. The results suggest that the inhibition of virus multiplication by temperature may be related to the inhibition of viral nonstructural protein synthesis early during infection. The presence of high serum levels in the incubation medium protects macromolecule synthesis against heat stress

Intracellular lectin-binding sites in symbiont-bearing Crithidia species.

Crithidia oncopelti, C. deanei, and C. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectins Ulex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, and Canavalia ensiformis, which recognize alpha-L-fucose, alpha- and beta-N-acetylglucosamine, beta-galactose and beta-N-acetylgalactosamine, beta-galactose, alpha-galactose, beta-N-acetylgalactosamine, sialic acid and alpha-D-mannose, and alpha-D-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.

Herpetomonas roitmani (Fiorini et al., 1989) n. comb.: a trypanosomatid with a bacterium-like endosymbiont in the cytoplasm.

The trypanosomatid previously described as Crithidia roitmani is characterized here at the ultrastructural and biochemical levels. The data indicates that the parasite belongs to the Herpetomonas genus, and we therefore suggest the flagellate to be denominated as Herpetomonas roitmani n. comb. Cladistic analysis of isoenzyme data generated by eight different enzymes showed that the parasite presented a distinct banding pattern and could be grouped with some Herpetomonas spp., but not with Crithidia spp., used as reference strains. Accordingly, when the parasites were grown for longer periods in Roitman's defined medium, expontaneous differentiation from promastigotes to opisthomastigotes (typical of the Herpetomonas genus) occurred. Transmission electron microscopy revealed the presence of bacterium-like endosymbionts in the cytoplasm of all evolutive forms of the parasite. All morphological alterations characteristic of endosymbiont-bearing trypanosomatids could be observed.

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.