Helminth Parasites and Diet of Leptodactylus Petersii (Steindachner, 1864) (Anura: Leptodactylidae) from Amapá State, Eastern Amazon, Brazil.

Leptodactylus petersii is a species of anuran found in both terrestrial and aquatic habitats and occurs from South America to southern North America and the West Indies. Studies involving the fauna of anuran parasites offer complementary information related to ecology. Thus, since there are few studies on the natural history of this species, this research aims to analyze the diet and the presence of endoparasitic helminths of Leptodactylus petersii from the state of Amapá, Brazil. We found 10 different taxonomic categories of prey in stomach contents, with the categories Hymenoptera (Formicidae) with 32.26 % (n = 12) being the most representative. Among the 12 individuals of L. petersii that were analyzed for helminth parasites, 83.3 % were infected with at least one species of helminths allocated to Phylum Nematoda. Our results report a new occurrence site for Rhabdias breviensis, originally described for Leptodactylus petersii in the state of Pará, as well as the second report of Ortleppascaris sp. in Brazil.

Molecular detection and clinical aspects of porcine circovirus 3 infection in pigs from Brazil / [Detecção molecular e aspectos clínicos da infecção por circovírus suíno 3 em suínos do Brasil]

Porcine circovirus 3 (PCV-3) DNA has been detected in serum samples from apparently healthy pigs as well as pigs with different clinical conditions. Molecular detection of PCV-3 was observed in swine serum samples from Southeastern - Brazil using a nested PCR designed specifically for this study. The epidemiology and clinical aspects of PCV-3 infection were evaluated. The samples originated from 154 pigs of both genders from different production phases and with different clinical presentations, sampled from 31 pig farms visited between 2013 and 2018. In this study, PCV-3 was detected in 26.7% of samples from all populations across varying ages. Statistical association (P=0.0285) was observed only between animals with respiratory signs and PCV-3; no PCV-3-positive animal had diarrhea. No statistical association was observed between PCV-3 and age, or gender of the pigs. Because PCV-3 is a newly discovered virus, there is very little information about its epidemiology. We hope that these data can help in future studies investigating PCV-3 epidemiology.(AU)

O DNA do circovírus suíno 3 (PCV-3) foi detectado em amostras de soro de suínos aparentemente saudáveis, bem como em suínos com diferentes condições clínicas. A detecção molecular do PCV-3 foi observada em amostras de soro de suínos da região Sudeste do Brasil, com uma nested PCR desenhada especificamente para este estudo. A epidemiologia e os aspectos clínicos da infecção por PCV-3 foram avaliados. As amostras foram coletadas de 154 suínos de ambos os sexos, de diferentes fases de produção e com diferentes sinais clínicos. Os animais pertenciam a 31 granjas visitadas entre 2013 e 2018. Neste estudo, o PCV-3 foi detectado em 26,7% das amostras de animais saudáveis e de animais com variados sinais clínicos, de ambos os sexos e de idades variadas. Associação estatística (P=0,0285) foi observada apenas entre animais com sinais respiratórios e PCV-3; nenhum animal positivo para PCV-3 apresentava diarreia. Não foi observada associação estatística entre o PCV-3 e a idade ou o sexo dos suínos. Por se tratar de um vírus recém-descoberto, existem poucas informações sobre sua epidemiologia. Espera-se que os dados deste trabalho possam contribuir para futuros estudos sobre a epidemiologia do PCV-3.(AU)

Retrospective study of porcine circovirus 3 (PCV3) in swine tissue from Brazil (1967-2018).

In this study, we report the molecular diagnosis and retrospective study of porcine circovirus 3 (PCV3) in frozen and formalin-fixed paraffin-embedded swine tissues (FFPE) collected from 1967 to 2018 in southeastern Brazil (Espírito Santo and Rio de Janeiro states). Frozen tissues from 35 pigs and FFPE tissues from 143 pigs were tested by nested PCR, targeting the PCV3 partial capsid gene. Bidirectional sequencing of 16 positive samples was performed, followed by sequence analysis and haplotype networks. A total of 26/178 samples (14.6%) tested positive for PCV3: 14/35 (40%) frozen tissue and 12/143 (8.4%) FFPE tissue. PCV3 was detected in the 1960s, 1970s, 2000s, and 2010s with the characterization of types PCV3a and PCV3b. A star-like distribution was observed in the grid of haplotypes, with a low haplotype diversity and more recent dispersal of the virus. A total of 40% of asymptomatic animals considered fit for slaughter tested positive for PCV3. In conclusion, PCV3 DNA was detected over 51 years of study, prior to initial reports and, so far, the sample detected in 1967 is the oldest partial capsid sequence described. The circulation of two different genotypes was reported, suggesting more than one introduction event of this virus into Brazil. Moreover, taken together, our studies indicated an ancient origin of PCV3 and its circulation in asymptomatic animals in Brazilian herds.

Perfil clínico-angiográfico e tratamento de pacientes submetidos à exame diagnóstico de cineangiocoronariografia em centro terciário público de grande volume entre 2011 e 2018 / Clinical-angiographic profile and treatment of patients undergoing diagnostic examination of coronary angiography in a large public tertiary center between 2011 and 2018

INTRODUÇÃO: As doenças cardiovasculares estão entre as principais causas de mortalidade global e, dentre elas, destaca-se a doença arterial coronariana (DAC), cujo diagnóstico precoce e prevenção ainda continuam sendo a melhor forma de tratamento. O conhecimento do perfil clínico e angiográfico dos pacientes portadores de DAC é extremamente importante para a avaliação de risco, a partir da quantificação da extensão e gravidade da doença, assim como para o planejamento terapêutico e sucesso do tratamento. O nosso objetivo foi avaliar o perfil clínico e angiográfico dos pacientes submetidos a exame diagnóstico de cineangiocoronariografia em centro clínico de alto volume. MÉTODOS: Foi realizado um estudo retrospectivo e descritivo do perfil clínico-angiográfico dos pacientes >18 anos submetidos à cineangiocoronariografia diagnóstica no Instituto Dante Pazzanese de Cardiologia de janeiro de 2011 a dezembro de 2018, excluindo os pacientes com cirurgia de revascularização prévia e doença valvar associada. Os dados foram obtidos por meio de um banco de dados institucional. RESULTADOS: Um total de 25649 pacientes foram incluídos, sendo 59,4% do sexo masculino, com média das idades de 66,1 anos. Os fatores de risco cardiovasculares mais frequentemente encontrados foram: hipertensão arterial sistêmica (80,5%), dislipidemia (60,5%), diabetes mellitus (32,3%), tabagismo (40,9%), e doença renal crônica (21,5%). O quadro clínico era de angina estável em 32,5%, síndrome coronariana aguda (SCA) sem supra de ST em 20,4% incluindo SCA com supra de ST em 6,1%, e isquemia silenciosa/assintomáticos em 37,5%. Em relação ao perfil angiográfico, a doença arterial coronariana obstrutiva significativa (>50%) foi uni-, bi- ou tri arterial em, respectivamente, 25,7%, 17,5% e 12,22%, além do tronco da coronária esquerda em 1,4%. A partir do exame diagnóstico, a intervenção coronária percutânea (ICP) foi realizada em 14,7% dos pacientes, predominantemente naqueles com apresentação clínica de SCA. Os vasos coronários tratados mais frequentes foram descendente anterior em 40,9%, coronária direita em 29,5% e circunflexa em 23,2%. O sucesso angiográfico foi evidenciado em 99,2% dos casos. Em relação aos desfechos clínicos na fase intra-hospitalar, foi reportado a ocorrência de óbito em 0,3% dos pacientes. CONCLUSÃO: A população estudada apresentou elevada prevalência de fatores de risco cardiovasculares e de doença coronária obstrutiva significativa. Já os pacientes submetidos a ICP subsequente, apresentaram elevada taxa de sucesso no procedimento. (AU)

Valorization of sugarcane bagasse ash: producing glass-ceramic materials.

Some aluminosilicates, for example mullite and wollastonite, are very important in the ceramic and construction industries. The most significant glass-ceramic for building applications has wollastonite as the main crystal phase. In this work we report on the use of sugarcane bagasse ash (SCBA) to produce glass-ceramics with silicates as the major crystalline phases. The glasses (frits) were prepared by mixing ash, limestone (calcium and magnesium carbonates) and potassium carbonate as the fluxing agent. X-ray fluorescence was used to determine the chemical composition of the glasses and their crystallization was assessed by using thermal analysis (DTA/DSC/TGA) and X-ray diffraction. The results showed that glass-ceramic material can be produced with wollastonite as the major phase, at a temperature lower than 900 °C.

Reuse of sugarcane bagasse ash (SCBA) to produce ceramic materials.

Sugarcane bagasse ash (SCBA) is a residue resulting from the burning of bagasse in boilers in the sugarcane/alcohol industry. SCBA has a very high silica concentration and contains aluminum, iron, alkalis and alkaline earth oxides in smaller amounts. In this work, the properties of sintered ceramic bodies were evaluated based on the concentration of SCBA, which replaced non-plastic material. The ash was mixed (up to 60 wt%) with a clayed raw material that is used to produce roof tiles. Prismatic probes were pressed and sintered at different temperatures (up to 1200 °C). Technological tests of ceramic probes showed that the addition of ash has little influence on the ceramic properties up to 1000 °C. X-ray diffraction and thermal analysis data showed that, above this temperature the ash participates in the sintering process and in the formation of new important phases. The results reported show that the reuse of SCBA in the ceramic industry is feasible.

Alpha-thalassemia (3.7 kb deletion) in a population from the Brazilian Amazon region: Santarém, Pará State.

The ethnic composition of the Brazilian population favors high frequencies of the -alpha3.7 deletion, responsible for alpha-thalassemia, because this mutation is very common in African populations. In spite of its importance, this hemoglobinopathy has been poorly investigated in Brazil, especially at the molecular level. We investigated the prevalence of the -alpha3.7 mutation in 220 individuals attended at the Municipal Hospital of Santarém, in the state of Pará. These patients were distributed into three different groups: i) 103 individuals with anemia who had microcytosis and hypochromia, ii) 11 individuals without anemia who had microcytosis and hypochromia, and iii) 106 individuals with no hematological alterations. We examined the usefulness of investigating alpha-thalassemia carrier status for microcytosis. Among the 103 patients with anemia, 20 (19.4%) were heterozygotes (-alpha3.7/alphaalpha) and one (1.0%) was a homozygote (-alpha3.7/-alpha3.7). Among the 11 patients without anemia, one heterozygote (-alpha3.7/alphaalpha) was identified; in the third group, composed of normal individuals (106 samples), deletion -alpha3.7 was found in seven samples (6.6%), all of which were heterozygotes (-alpha/alphaalpha).These frequencies are within the expected range, given available data on the distribution of this hemoglobin disorder in human populations and the ethnic composition of the population of Santarém. We found that alpha-thalassemia is a common cause of microcytosis, given that a high proportion (19.2%) of the microcytic population carried alpha-globin gene deletions.

The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis.

RNA editing in the parasitic organism Trypanosoma brucei is characterised by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The processing reaction is a required pathway for the expression of most mitochondrial genes and proceeds by a cascade of enzyme-catalysed steps. RNA editing involves one or more macromolecular ribonucleoprotein complexes which are likely to interact with additional components as the reaction proceeds. Here we examined the involvement of the gRNA-binding polypeptide gBP21, a protein which has been demonstrated to be associated with active RNA editing complexes. We show that in vitro RNA editing can be suppressed by the addition of a gBP21-specific antibody or by immunodepletion of the protein. By creating a gBP21 knockout mutant we analysed the requirement for the protein in vivo. gBP21(-) trypanosomes are viable as bloodstream stage cells and contain edited mRNAs. However, the knockout mutant is not capable of differentiating from the bloodstream to the insect life cycle stage in vitro. Moreover, mutant cells are characterised by a low mitochondrial transcript abundance. Together, these data establish that gBP21 contributes a non-essential function to the RNA editing reaction and further suggest that the protein is involved in additional mitochondrial processes which impact a larger pool of mitochondrial transcripts.

The guide RNA database.

Guide RNAs (gRNAs) are small, metabolically stable RNA molecules which perform a pivotal, template-like function during the RNA editing process in kinetoplastid protozoa. The gRNA database currently contains 250 guide RNA sequences as well as secondary and tertiary structure models and other relevant information. The database is made available as a hypertext document accessible via the World Wide Web (WWW) at the URL: http://www.biochem.mpg.de/ goeringe/

Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.

The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences.

The cpb genes of Leishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

The guide RNA database.

The RNA editing process in protozoan parasites is controlled by small RNA molecules known as guide RNAs (gRNAs). The gRNA database is a comprehensive compilation of published guide RNA sequences from eight different kinetoplastid organisms. In addition to the RNA primary sequences, information on the gene localization, the experimental verification of the transcripts, and literature citations are provided. Accessory information includes the secondary structures of fourTrypanosoma bruceigRNAs as well as a computer modelled three dimensional gRNA structure. The database is made available as a hypertext document accessible via the World Wide Web (WWW) or from the authors in a printed form.

Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.

Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana.

The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expressed at highest levels in amastigotes. We recently characterised lmcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the amino terminal of the predicted mature proteinase. To investigate the role of LmCPa in L. mexicana, we used gene-targeting of promastigotes with hygromycin- and phleomycin-resistance markers to generate null mutants by disrupting sequentially both alleles of lmcpa. The promastigote null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics and the amastigote-like form, both of which could infect the J774G8 macrophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. By these criteria, therefore, LmCPa appears to be non-essential although there is the possibility that LmCPa could be required during development in the sandfly, a stage not analysed here. The apparent redundancy of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.

The alkane-inducible Candida maltosa ALI1 gene product is an NADH:ubiquinone oxidoreductase subunit homologue.

The ALI1 gene product in Candida maltosa was previously shown to be essential for n-alkane assimilation, possibly as a transcription factor [Hwang et al., Gene 106 (1991) 61-69]. We show that the predicted sequence is highly homologous to a subunit of respiratory complex I from another fungus, Neurospora crassa, and from Bos taurus. The predicted protein contains a motif conserved in this subunit from mitochondria, chloroplasts and bacteria. It also contains an N-terminal sequence that suggests a mitochondrial (mt) localization and a role for mt respiration in n-alkane assimilation.

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Characterization of a Trypanosoma brucei nuclear gene encoding a protein homologous to a subunit of bovine NADH:ubiquinone oxidoreductase (complex I).

A Trypanosoma brucei gene has been identified that encodes a protein predicted to be a component of the trypanosome homologue of mitochondrial NADH:ubiquinone oxidoreductase (complex I). High homology was found to a 20-kDa component of the iron-sulfur protein fraction of bovine mitochondrial NADH:ubiquinone oxidoreductase and the products of the ndhK locus of Paramecium tetraurelia mitochondria and the NQO6 locus of Paracoccus denitrificans. The homology extends to several other proteins predicted to function as part of electron transport systems, including the psbG/ndhK gene products of chloroplast and cyanobacterial genomes which are thought to be subunits of a NADH:plastoquinone oxidoreductase involved in chlororespiration. The T. brucei ndhK counterpart is nuclearly encoded. An extended amino terminus of the T. brucei ndhK with structural similarity to mitochondrial presequences indicates that its transfer into mitochondria is likely. Stumpy and slender bloodforms and procyclic forms all possess similar levels of ndhK transcripts despite previous reports of stage-regulated expression of complex I-like activity.

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.

Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria.

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be accounted for by RNA editing alone. Analyses of transcripts from 6 mitochondrial genes of Trypanosoma brucei indicates that the size variation is due to poly(A) tail length. A larger fraction of CYb, COI and COII transcripts have longer poly(A) tails in procyclic than in bloodstream forms. These transcripts are also more abundant in the procyclic forms. In contrast, a more substantial fraction of CR1 transcripts have longer poly(A) tails in bloodstream than in procyclic forms and these transcripts tend to be more abundant in bloodstream forms. Both ND4 and MURF1 transcripts show a similar size distribution of poly(A) tail lengths in these life cycle states although both transcripts are more abundant in bloodstream forms. Furthermore, genes with edited transcripts tend to have longer poly(A) tails than unedited transcripts. Transcript abundance is not strictly correlated with longer poly(A) tails. Thus, poly(A) length variation appears to be developmentally regulated in a transcript-specific fashion in T. brucei. This regulation of polyadenylation may influence mitochondrial gene expression as polyadenylation can regulate cytoplasmic gene expression in eukaryotes.

Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit.

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.