Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro.

BACKGROUND: Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE: Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS: L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS: Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION: Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells.

Evaluation of target sequences for the polymerase chain reaction-based detection of Salmonella in artificially contaminated beef.

Salmonella is a major cause of foodborne diseases worldwide, which has fueled the demand for the development and evaluation of sensitive, specific, and rapid detection methodologies, such as polymerase chain reaction (PCR). In this study, six primer pairs for the detection of Salmonella were evaluated by PCR with isolates of Salmonella spp. (115) and other bacteria (104). The primers designed for the sifB gene provided the best performance regarding specificity and sensitivity (100%). These primers were selected and used to develop a PCR assay for Salmonella detection during the enrichment steps of the conventional detection method in spiked beef samples. The enrichment steps were: buffered peptone water (BPW), Rappaport-Vassiliadis soya broth (RVS) and at the Müller-Kauffmann tetrathionate novobiocin broth (MKTTn), after 18 h (BPW) and 24 h (RVS and MKTTn) of incubation. The initial concentrations of the Salmonella inocula were 10¹, 10², and 10³ colony-forming units/25 g. The protocol was able to detect Salmonella at all concentrations in the enrichment steps, but not in the nonenriched samples. These results indicated that the proposed protocol was suitable to detect Salmonella in beef during the intermediate stages of the conventional isolation protocol, substantially reducing the time required to obtain the final results.

Enamino-oxindole HIV protease inhibitors.

We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP.

Structures of PmSOD1 and PmSOD2, two superoxide dismutases from the protozoan parasite Perkinsus marinus.

Perkinsus marinus, a facultative intracellular parasite of the eastern oyster Crassostrea virginica, is responsible for mass mortalities of oyster populations. P. marinus trophozoites survive and proliferate within oyster hemocytes, invading most tissues and fluids, thus causing a systemic infection that eventually kills the host. The phagocytosis of P. marinus trophozoites lacks a respiratory burst, suggesting that the parasite has mechanisms that actively abrogate the host's oxidative defense responses. One mechanism and the first line of defense against oxidative damage is the dismutation of superoxide radical to molecular oxygen and hydrogen peroxide by superoxide dismutases (SODs). P. marinus possesses two iron-cofactored SODs, PmSOD1 and PmSOD2. Here, the crystallization and X-ray structures of both PmSOD1 and PmSOD2 are presented.

Novel uncomplexed and complexed structures of plasmepsin II, an aspartic protease from Plasmodium falciparum.

Malaria remains a human disease of global significance and a major cause of high infant mortality in endemic nations. Parasites of the genus Plasmodium cause the disease by degrading human hemoglobin as a source of amino acids for their growth and maturation. Hemoglobin degradation is initiated by aspartic proteases, termed plasmepsins, with a cleavage at the alpha-chain between residues Phe33 and Leu34. Plasmepsin II is one of the four catalytically active plasmepsins that has been identified in the food vacuole of Plasmodium falciparum. Novel crystal structures of uncomplexed plasmepsin II as well as the complex with a potent inhibitor have been refined with data extending to resolution limits of 1.9A and 2.7A, and to R factors of 17% and 18%, respectively. The inhibitor, N-(3-[(2-benzo[1,3]dioxol-5-yl-ethyl)[3-(1-methyl-3-oxo-1,3-dihydro-isoindol-2-yl)-propionyl]-amino]-1-benzyl-2-(hydroxypropyl)-4-benzyloxy-3,5-dimethoxy-benzamide, belongs to a family of potent non-peptidic inhibitors that have large P1' groups. Such inhibitors could not be modeled into the binding cavity of the structure of plasmepsin II in complex with pepstatin A. Our structures reveal that the binding cavities of the new complex and uncomplexed plasmepsin II are considerably more open than that of the pepstatin A complex, allowing for larger heterocyclic groups in the P1', P2' and P2 positions. Both complexed and uncomplexed plasmepsin II crystallized in space group P2, with one monomer in the asymmetric unit. The structures show extensive interlocking of monomers around the crystallographic axis of symmetry, with areas in excess of 2300A(2) buried at the interface, and a loop of one monomer interacting with the binding cavity of the 2-fold related monomer. Electron density for this loop is only fully ordered in the complexed structure.

Gene organization and homology modeling of two iron superoxide dismutases of the early branching protist Perkinsus marinus.

The facultative intracellular oyster parasite, Perkinsus marinus, taxonomically related to both dinoflagellates and apicomplexans, possesses at least two distinct genes (PmSOD1 and PmSOD2) predicted to encode iron-containing superoxide dismutases (FeSOD). DNA blots and sequence analysis suggest that both PmSOD1 and PmSOD2 are single copy and are unlinked. PmSOD1 and PmSOD2 are composed of five and six exons, respectively. All introns are delimited by canonical GT/AG boundaries, and have some features more similar to apicomplexan than dinoflagellate introns. Interestingly, exon 1 of PmSOD2 encodes putative transmembrane and spacer domains with no homology to FeSODs, while exon 2 begins with a methionine codon and is homologous to the N-terminus of FeSODs. The position of introns is not highly conserved between PmSOD1 and PmSOD2, although one intron is in a similar location. Comparison of the intron positions of PmSOD1 and PmSOD2 to those of available apicomplexan FeSODs shows that the intron position shared by PmSOD1 and PmSOD2 is also observed in the FeSOD of Toxoplasma gondii. Comparison of the untranscribed regions 5' and 3' of the coding regions for PmSOD1 and PmSOD2 reveals few motifs in common. Instead, each gene possesses a distinct set of putative upstream transcription factor binding sites. Although the proteins encoded by PmSOD1 and PmSOD2 are only 38% identical to each other, homology modeling indicates that they have nearly identical active site structures. The divergent genomic organizations of two FeSOD genes in the same organism illustrates the complexity of the antioxidant system of even simple, early-branching protists such as P. marinus.

Influence of lactic acid and acetic acid on Salmonella spp. growth and expression of acid tolerance-related genes.

Salmonella spp. is an important foodborne pathogen, often associated with meat products. This pathogen presents a complex tolerance mechanism in the presence of organic acids, which is regulated by a diversity of genes, including rpoS, nlpD and clpP. The present study aimed to measure the expression of such genes by Salmonella strains subjected to acid stress conditions, and associate these data with microbial growth. A culture collection composed of 79 strains of Salmonella spp. obtained from bovine and swine production chains was subjected to PFGE using XbaI, and 3 strains (serovars Derby, Typhimurium and Meleagridis) were selected for acid tolerance trials. The selected strains were inoculated in meat extract broth (MEB) added to lactic or acetic acids at a final pH of 4.0, 5.0 or 6.0, and incubated at 37°C for 6, 12, 24 and 48h. As controls, Salmonella strains were inoculated in MEB at pH7.0, and incubated in the same conditions. Bacterial populations were monitored by direct plating and gene expression using qPCR. Salmonella presented similar populations to controls and evident expression of rpoS at pH5.0 and 6.0. However, Salmonella populations were not detectable after 6h at pH4.0. The adaptability of Salmonella to pH5.0 and 6.0 emphasizes the importance of adequate monitoring of pH reduction during cleaning procedures in food industries, such as organic acid spraying in bovine carcasses. The data obtained demonstrated the relevance of rpoS in the acid tolerance mechanism of Salmonella strains, prompting further studies to investigate its expression in meat systems.

Reparación de la insuficiencia tricuspídea: resultados a largo plazo en 114 pacientes / Repair of tricuspid valve insufficiency: long-term results in 114 patients

Resumen Antecedentes la reparación de la insuficiencia tricuspídea funcional está indicada en casos de insuficiencia cardíaca, generalmente, asociada a enfermedades de las válvulas cardíacas izquierdas y en algunos casos de enfermedad coronaria o cardiopatías congénitas. Objetivo : evaluar los resultados a largo plazo de la reparación tricuspídea en pacientes operados en la región centro sur de Chile. Pacientes y métodos : estudio retrospectivo de registros clínicos y operatorios en 114 pacientes con edad promedio 57.8 (DE 13) años (72 mujeres) sometidos a reparación tricuspídea asociada a otros procedimientos quirúrgicos entre 2009 y 2017. Resultados : la etiología de la insuficiencia tricuspídea fue debida a enfermedad reumática inactiva en 45% y a endocarditis infecciosa en 2.6%. 63% estaban en fibrilación auricular y 75% en capacidad funcional NYHC III. 15.8% tenían cirugía valvular cardíaca previa. El ecocardiograma mostró insuficiencia severa en 56% de los casos y había hipertensión pulmonar severa en 39.5%. La cirugía consistió en la implantación de un anillo semi-rígido en forma concomitante a reemplazo o reparación de lesión valvular mitral, reemplazo valvular aórtico, cierre de comunicación interauricular, cirugía coronaria o resección de tumor intra cardíaco. La mortalidad post operatoria global fue 16%, debido a falla multisistémica en 6%, insuficiencia cardíaca en 5% y hemorragia cerebral en 4.3%. El seguimiento promedio fue 78.8 (DS 7.2) meses. La supervivencia actuarial fue 74% a los 60 y 68% a los 96 meses. Conclusión : La cirugía de reparación de la insuficiencia tricuspídea moderada o severa, asociada a otras enfermedades cardíacas provee una buena recuperación sintomática, con excelente sobrevida alejada.

Abstract Background : Surgery for functional tricuspid valve insufficiency is indicated in cases of congestive heart failure usually associated to left heart valve diseases or as a concomitant surgery for other causes such as congenital or coronary disease. Aim : To assess and report the long-term results of tricuspid valve repair in adult patients in a regional cardiac surgery center in south Chile. Patients and Methods : Retrospective review of clinical and operative records of 114 patients aged 57.8 +/- 13 years (72 women) subjected to tricuspid reparative surgery concomitant with other cardiac procedures between 2009 to 2017. Results : In 45% of cases etiology was due to inactive rheumatic disease and 2.6% was due to endocarditis. 63% presented with atrial fibrillation and 75% were in NYHC CFIII. 15.8% had a previous cardiac valve surgery. Preoperative echocardiography showed severe tricuspid insufficiency in 56% of cases and pulmonary hypertension was severe in 39.5%. In all cases tricuspid repair was performed through the insertion of a semi rigid ring as a concomitant procedure for mitral repair/replacement in most cases, aortic valve replacement, surgical closure of an ASD, CABG surgery and the resection of cardiac tumors. Overall postoperative mortality was 16% due to multi-organic dysfunction in 6%, cardiac failure in 5% and cerebral hemorrhage in 4.3%. Mean long term follow up was 78.8+/- 7.2 months. Actuarial survival was 74% at 60 and 68% at 96 months. Conclusion: Surgical tricuspid valve repair for moderate to severe tricuspid insufficiency isolated or associated to other cardiac diseases provides a good symptomatic recovery, with an excellent long term survival.

Tracking the molecular epidemiology of Brazilian Infectious bursal disease virus (IBDV) isolates.

Infectious bursal disease is a highly contagious disease of young chickens caused by Infectious bursal disease virus (IBDV). Genome segment A encodes the capsid protein (VP2), while segment B encodes the RNA-dependent RNA polymerase (VP1). In the present study, we trace the molecular epidemiology of IBDV in Brazil by analyzing 29 isolates collected in the major regions of poultry production. To genetically characterize the isolates, phylogenetic and population dynamic analyses were conducted using 68 VP1 (2634 nt) and 102 VP2 (1356 nt) coding sequences from IBDV isolates from different regions of the world. Furthermore, the evolution of IBDV was analyzed by characterizing the selective forces that operated during the diversification of viral isolates. We show that IBDV isolates were introduced into Brazil mainly from the Netherlands and the USA. These introductions were associated with all Brazilian poultry production regions analyzed in this work. In addition, we show that the evolution of IBDV has been shaped by a combination of very low recombination rates and relatively high rates of nucleotide substitution (2.988×10(-4) for VP1 and 3.2937×10(-4) for VP2), which themselves are a function of purifying selection operating on VP1 and VP2. Furthermore, our extended Bayesian skyline plot suggests that the increase in the effective population size of isolates of IBDV is consistent with its epidemiological history, with a large increase during the emergence of acute outbreaks of IBD in the 1980s.

Campanulotes compar (Burmeister, 1838) (Phthiraptera: Ischnocera) in chickens (Gallus gallus domesticus) from Rio Grande do Norte State, Brazil. The reemergence of an ectoparasite?

The importance of ectoparasites in the transmission of pathogens, as well as the variability of species from one region to another, motivated this notification of the ectoparasite lice Campanulotes compar in range chickens (Gallus gallus domesticus L.) reared in an extensive system in the city of Apodi, Rio Grande do Norte state, in the Northeast region of Brazil. The examined birds were infested with ten males and six females of C. compar. Thus, C. compar is recorded as parasitizing chickens in the state of Rio Grande do Norte after 77 years from its unique citation in the Southeast region of Brazil. We further discuss the possible risks of this finding.

Lantibiotics biosynthesis genes and bacteriocinogenic activity of Lactobacillus spp. isolated from raw milk and cheese.

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.

Using nested PCR to detect the hlyA gene of Listeria monocytogenes in Minas Frescal cow's milk cheese.

To evaluate a nested PCR protocol for Listeria monocytogenes detection, Minas Frescal cow's milk cheeses were produced, artificially inoculated with this pathogen at concentrations ranging from 1 to 1,000 CFU/g, and stored at 4°C for 10 days. The International Organization for Standardization (ISO) standardized method 11290-1/A1 was used to detect L. monocytogenes in the inoculated samples, and DNA was extracted from aliquots (1, 5, and 10 ml) of 1:10 dilution, followed by a nested PCR protocol for the hlyA gene. The ISO standardized reference method and nested PCR both detected L. monocytogenes at all concentrations and during all storage periods; McNemar's test showed no significant difference (P > 0.05). The results indicated that the nested PCR protocol can be used as a screening test to detect L. monocytogenes in Minas Frescal cheese, allowing earlier detection of the pathogen that can later be confirmed by the ISO standardized reference method.

Síndrome de Austrian: reporte de caso / Austrian syndrome: report of one case

Austrian syndrome is a triad characterized by pneumonia, meningitis and endocarditis, as a result of a Streptococcus pneumoniae bacteremia. We report a previously healthy 49 year-old male, who consulted at the emergency care unit with a history of one week of pleuritic pain, fever leading to an altered level of consciousness and seizures. A diagnosis of community-acquired pneumonia and meningitis was reached, isolating Streptococcus pneumoniae in the cerebrospinal fluid and blood cultures. Antibiotic treatment was started but the patient had an unsatisfactory response. During hospitalization a new heart murmur was found in the physical examination. An echocardiography was performed and a massive aortic valve insufficiency was found along with vegetations and a perforation of the same valve. The valve was replaced by a prosthetic one and the patient responded satisfactorily to the surgical and antibiotic treatment, without complications.

[Myocardial revascularization of the anterior descending coronary artery with left internal mammary artery by means of extracorporeal circulation: 10 years follow-up]. / Revascularización miocárdica de la arteria descendente anterior con arteria mamaria interna izquierda con circulación extracorpórea: seguimiento a 10 años.

BACKGROUND: The use of left internal mammary artery (LIMA) as a graft to anterior descending artery (LAD) has been associated with better long term results in coronary surgery. AIM: To assess and report the long-term results of LIMA to LAD bypass grafting for isolated LDA lesions. PATIENTS AND METHODS: Retrospective analysis of the medical records and surgical protocols of 40 patients (aged 60+/-10 years, 28 male) subjected to coronary surgery between 1992 and 2002. RESULTS: Thirty-four patients presented with unstable angina. On angiography, the LAD had a proximal obstruction in 35 patients. Sixteen presented with a myocardial infarction of the LAD territory. Six were managed previously with angioplasty; four had a new critical obstruction, 1 was catalogued as a procedure failure, and one was totally occluded. There was no operative mortality, myocardial infarction, stroke or need for re operation. There were two late deaths, caused by an advanced cardiac failure at 120 months in one patient, and chronic renal failure at 61 months of follow-up in another. Actuarial survival probability was 100%, 93% and 75% at 1, 5 and 10 years. Probability of freedom from angina was 98%, and freedom of suffering a new myocardial infarction was 100% at more than 10 years. The probability of no need for a new coronary procedure (angioplasty or surgery) also was 100% at more than 10 years. CONCLUSIONS: The use of LIMA as a coronary bypass graft to LAD is a safe surgical technique, with an excellent duration and permeability and also provides a prolonged time free from cardiac events as mortality, angina, myocardial infarction, and the need of a new coronary procedure.

Utility of (His)6 tag for purification and refolding of proplasmepsin-2 and mutants with altered activation properties.

Plasmepsin-2 is a malarial aspartic proteinase that has been implicated in the initial steps of hemoglobin degradation in parasites and thus represents an attractive antimalarial target. Escherichia coli expressed proplasmepsin-2 is capable of activation at acidic pH by autocatalytic cleavage of the pro part region, which results in products of different length. We designed a 10-amino-acid deletion in the pro part region that allows faster generation of homogeneous enzyme upon activation. Incorporation of a (His)6 tag onto the N-terminus of the pro part enables on-column refolding of proplasmepsin-2 and simplifies proenzyme purification and pro part separation after activation. The proposed purification procedure results in highly pure and easily crystallizable enzyme.

Structures of Ser205 mutant plasmepsin II from Plasmodium falciparum at 1.8 A in complex with the inhibitors rs367 and rs370.

Plasmepsin II is one of the four catalytically active plasmepsins found in the food vacuole of Plasmodium falciparum. These enzymes initiate hemoglobin degradation by cleavage at the alpha-chain between Phe33 and Leu34. The crystal structures of Ser205 mutant plasmepsin II from P. falciparum in complex with two inhibitors have been refined at a resolution of 1.8 A in the space group I222 and to R factors of 19.9 and 19.5%. Each crystal contains one monomer in the asymmetric unit. Both inhibitors have a Phe-Leu core and incorporate tetrahedral transition-state mimetic hydroxypropylamine. The inhibitor rs367 possesses a 2,6-dimethylphenyloxyacetyl group at the P2 position and 3-aminobenzamide at the P2' position, while rs370 has the same P2 group but 4-aminobenzamide in the P2' position. These complexes reveal key conserved hydrogen bonds between the inhibitor and the binding-cavity residues, notably with the flap residues Val78 and Ser79, the catalytic dyad Asp34 and Asp214 and the residues Ser218 and Gly36 that are in proximity to the catalytic dyad. The structures also show unexpected conformational variability of the binding cavity of plasmepsin II and may reflect the mode of binding of the hemoglobin alpha-chain for cleavage.

Ajuda entre passageiros de ônibus / Helping behavior among passengers

Comportamentos de ajuda entre passageiros de ônibus podem contribuir para a qualidade do transporte público. Durante 40 viagens e observações sistemáticas em duas linhas de ônibus registrou-se sob quais condições passageiros sentados auxiliariam quem viajava de pé, oferecendo-se para carregar as bagagens. De maneira geral, ajuda foi recebida em 60 porcento das situações criadas. Significativamente mais ajuda foi oferecida numa linha para a periferia da cidade do que numa linha para o centro da cidade. Mulheres estiveram mais dispostas a ajudar e verificou-se uma tendência de oferecer mais ajuda em situações de contato face a face. Os dados sugerem que se pode encontrar comportamentos pró-sociais entre passageiros que favoreçam a qualidade no transporte público da cidade. A implementação de avisos educativos no interior dos ônibus e nos terminais rodoviários pode incentivar a ajuda entre passageiros, além de outros comportamentos desejáveis