Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota).

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

Rapid quantification of rice root-associated bacteria by flow cytometry.

UNLABELLED: To understand the mechanism of plant-bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 10(9) CFU g(-1) ). However, flow cytometry gave a number of viable cells of rice-associated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of precise number of root-associated bacteria is critical for plant-bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.

Superoxide radical-generating compounds activate a predicted promoter site for paraquat-inducible genes of the Chromobacterium violaceum bacterium in a dose-dependent manner.

The purpose of the present study was to functionally evaluate the influence of superoxide radical-generating compounds on the heterologous induction of a predicted promoter region of open reading frames for paraquat-inducible genes (pqi genes) revealed during genome annotation analyses of the Chromobacterium violaceum bacterium. A 388-bp fragment corresponding to a pqi gene promoter of C. violaceum was amplified using specific primers and cloned into a conjugative vector containing the Escherichia coli lacZ gene without a promoter. Assessments of the expression of the ß-galactosidase enzyme were performed in the presence of menadione (MEN) and phenazine methosulfate (PMS) compounds at different final concentrations to evaluate the heterologous activation of the predicted promoter region of interest in C. violaceum induced by these substrates. Under these experimental conditions, the MEN reagent promoted highly significant increases in the expression of the ß-galactosidase enzyme modulated by activating the promoter region of the pqi genes at all concentrations tested. On the other hand, significantly higher levels in the expression of the ß-galactosidase enzyme were detected exclusively in the presence of the PMS reagent at a final concentration of 50 µg/mL. The findings described in the present study demonstrate that superoxide radical-generating compounds can activate a predicted promoter DNA motif for pqi genes of the C. violaceum bacterium in a dose-dependent manner.

Polymorphisms of the promoter and exon 3 of the receptor for advanced glycation end products (RAGE) in Euro- and Afro-Brazilians.

The receptor for advanced glycation end products (RAGE or AGER), a member of the immunoglobulin superfamily, is involved in pathologies such as atherosclerosis and diabetes. Over 50 SNPs were reported for RAGE, among which were the promoter region polymorphisms -429T>C (rs1800625), -374T>A (rs1800624) and a 63-bp deletion (-407 to -345 bp), all related to increased RAGE expression. Additionally, in the exon 3, a putative site of binding ligands, the missense variation G82S (rs2070600) was associated with skin disorders in patients with diabetes. We have determined allele, genotype and haplotype frequencies of RAGE polymorphisms -429T>C, -374T>A, 63-bp deletion and G82S in Euro-Brazilians (n = 108) and Afro-Brazilians (n = 91), characterized according to the predominant ancestry of the individuals. The allele frequencies for Euro- and Afro-Brazilians were as follows: -429C, 12.5% vs. 12.1% (P = 0.90); -374A, 31.5% vs. 26.2% (P = 0.25); 63del, 0.0% vs. 3.8% (P = 0.004); and 82S, 1.9% vs. 0.6% (P = 0.24). Absolute linkage disequilibrium was found between the promoter polymorphisms -429T>C and -374T>A plus the 63-bp deletion (D'=1.000; P < 0.0001). The haplotype frequencies differed (P = 0.003) between Euro- and Afro-Brazilians. Our results showed that the frequencies of the 63-bp deletion were higher in Afro-Brazilians, while the other analysed polymorphisms were similarly distributed in the studied populations. The -374T>A plus 63-bp deletion polymorphism captures more than 80% of the haplotypic variation in the studied population.

Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12-14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD(50) values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.

In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi.

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro.

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhea in Paraná State, Brazil.

AIMS: To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC. METHODS AND RESULTS: A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx-positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx(1)eae ehxA and stx(1) respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested. CONCLUSIONS: These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence. SIGNIFICANCE AND IMPACT OF STUDY: These results indicate that molecular methods are required to diagnosis of STEC infections.

Fluoride Ion Release of Self-Adhesive Resin Cements and Their Potential to Inhibit In Situ Enamel and Dentin Demineralization.

OBJECTIVE: This study evaluated in situ the potential of a glass ionomer and self-adhesive resin cements to inhibit enamel and dentin demineralization around indirect restorations exposed to cariogenic challenge. The cumulative fluoride release (CFR) of materials was measured in water and acid. METHODS: Seventy blocks cut from human molars received two indirect composite restorations (one in enamel and another in dentin) luted with Ketac Cem EasyMix (GIC, positive control), SeT (SeT), Maxcem Elite (Max), Smart Cem2 (Smart), and RelyX Unicem 2 (Unicem2). Fourteen volunteers wore palatal appliances containing five blocks exposed to a cariogenic challenge (20% sucrose solution, eight times per day, seven days). Knoop microhardness (KH) at two distances from the margins and three depths from the outer surface determined enamel and dentin demineralization. Disc-shape specimens of materials were immersed in daily-replaced deionized water or lactic acid solutions. KH and CFR data were analyzed by analysis of variance, Games-Howell test, and Tukey test (α=0.05). RESULTS: The overall KH ranking was GIC > SeT > Max > Smart = Unicem2 in both enamel and dentin (">" means p<0.05). SeT was the only resin cement that resulted in enamel and dentin KH comparable to that of GIC at most distances and depths. In water, CFR rank of materials was GIC > SeT = Max > Smart = Unicem2. In acid, the rank was similar, except that Set was significantly superior to Max. CONCLUSION: SeT inhibited demineralization in enamel and dentin quite comparably to GIC. All resin cements released lower cumulative amounts of fluoride than the glass ionomer cement.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked ß(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Inter-domain cross-talk controls the NifA protein activity of Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize. The activity of NifA, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through a mechanism involving its N-terminal domain. Here we show that this domain interacts specifically in vitro with the N-truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA-binding activities of the N-truncated NifA protein. We suggest that the N-terminal domain inhibits NifA-dependent transcriptional activation by an inter-domain cross-talk between the catalytic domain of the NifA protein and its regulatory N-terminal domain in response to fixed nitrogen.

Phenyl substitution of furamidine markedly potentiates its anti-parasitic activity against Trypanosoma cruzi and Leishmania amazonensis.

Furamidine (DB75) and related unfused aromatic diamidines have proven useful for the treatment of parasitic infections. These compounds were primarily developed to combat infections by Pneumocystis carinii and African trypanosomes but they are also active against other parasites. Here we have investigated the in vitro effects of DB75 and its phenyl-substituted analog DB569 on two kinetoplastid haemoflagellates Trypanosomatidae: Trypanosoma cruzi and Leishmania (L) amazonensis. The phenyl-amidine compound DB569 has equivalent DNA binding properties compared to DB75 but it was selected on the basis of its distinct tumor cell distribution properties. We found that DB569 is significantly more potent than DB75 at reducing the proliferation of the parasites, using either isolated parasites in cultures or with cardiomyocyte and macrophage host cells. DB569 is effective towards the intracellular forms of T. cruzi (IC(50) in the low-micromolar range) and it exhibits trypanocidal dose-dependent effects against trypomastigote forms of T. cruzi parasites obtained from the Y strain and Dm28c clone, which belong to two different biodemes. Fluorescence microscopy experiments indicated that both diamidines were mostly localized in the nucleus of the mammalian host cells and within the nuclei and kinetoplast of the parasites. Electron microscopy studies showed that the treatment of the parasites with DB75 and DB569 induces important alterations of the parasite nucleus and kinetoplast, at sites where their DNA target is localized. Altogether, the data suggest that the phenyl-substituted furamidine analogue DB569 is a potential new candidate for the treatment of the Chagas' disease and Leishmaniasis.

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum.

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.

Complete regression of melanocytic nevi: clues for proper diagnosis.

A histopathological study of a distinct papule isolated from a patient's neck is reported and compared to 80 melanocytic nevi. The histogenesis of this unique tumor indicates that it probably represents the residual of a melanocytic nevus in which nevi cells have disappeared but fat infiltration and fibrosis of the dermis and the typical aspects of other cutaneous elements persisted. The finding of focal acantholytic dyskeratosis in multiple foci of the lesion may indicate melanocytic cells regression. Lesions with the histopathological appearance of melanocytic nevi, even in the absence of nevi cells, may be discovered scrutinizing the associated alterations.

Phytate from an alternative dietary supplement has no effect on the calcium, iron and zinc status in undernourished rats.

A mixture of cereal bran, eggshells and cassava leaf powder, known as multimixture (MM), has been widely used in developing countries as a dietary supplement to combat malnutrition in children. The introduction of phytate from cereal bran in infant diets has generated serious controversy about MM due to the mineral chelating effect of phytate. This paper reports on a study to investigate the bioavailability of calcium, iron and zinc in rats fed with a deficient diet supplemented with MM. Undernourished rats were treated with a deficient diet (DD) to which MM containing different phytate and mineral concentrations was added. Body weight gains, Ca, Fe, Zn and phytate balances, blood hemoglobin concentration and the mineral content of tissue were determined. DD supplemented with 5% and 25% of MM increased the rats' hemoglobin blood concentration, fur regrowth, Ca concentration in the femur and promoted body weight gain 40 times higher than did the DD. Extra calcium, iron and zinc added to the diet with 25% of MM did not increase the rats' growth rates. Both the addition of NaCl, KF and KI in MM and the use of dephytinized bran in the MM composition led to a significant increase in the rats' growth (P < 0.0001); however, these changes failed to increase Ca, Fe and Zn bioavailability. Our findings suggest that the Ca, Fe and Zn bioavailability was not affected by the MM phytate content or by the concentrations of NaCl, KF and KI in the diet.

[Verrucous hemangioma (neviform verrucous acrohemangioma)]. / Hemangioma verrucoso (acrohemangioma verrucoso neviforme).

A case of verrucous haemangioma in a eighteen-years old patient, during her second pregnancy is reported. The lesions were localized on the dorsal aspect of the right foot and leg, simulating clinically the angiokeratoma circunscriptum naeviforme. The diagnosis was first firmed by the histologic features of the malformation on superficial, medial and deep dermis and a rough epidermic reactivity. It has been discussed the mean differential diagnosis and commented that those type of hemangiomatous lesions (macular and elevated lesions) plus varicosities, without hypertrophy of the limb, may represent a discrete form of Klippel-Trenaunay syndrome. The authors propose the name "Verrucous neviform acrohaemangioma" for those cases similar to this one and to make a difference from other type of haemangioma that became verrucous.

Trypanosoma cruzi: alpha-2-macroglobulin regulates host cell apoptosis induced by the parasite infection in vitro.

A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form-A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment.