HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi

The activity of a Hexameric M17 Metallo-Aminopeptidase is associated with survival of Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metalloaminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs

Leishmania infantum and Leishmania braziliensis : differences and similarities to evade the innate immune system

Visceral leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self-healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human DCs’ biology for better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expres-sion when DCs were infected for 24 h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of DCs after 24 h. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology

AGENTE COMUNITÁRIO DE SAÚDE: PERCEPÇÕES NA ESTRATÉGIA DA SAÚDE DA FAMÍLIA

El estudio tuvo como objetivo comprender el significado de ser un agente comunitario de salud - ACS en unPrograma de Salud de la Familia (PSF). Investigación descriptiva, cualitativa, con inspiración fenomenológica que buscó laesencia del discurso del ACS en la ciudad de Sete Lagoas-MG. Las entrevistas fueron grabadas con siete ACS a partir decuestión norteadora: ¿cuál es su percepción como un Agente Comunitario de Salud en la Estrategia de Salud de la familia? Elanálisis comprensivo de los discursos posibilitó la construcción de cinco categorías: ACS, enlace entre el equipo multiprofesionaly la comunidad; valorización profesional; el papel del ACS en el equipo multiprofesional y su contribución para la mudanzadel modelo de atención básica; formación profesional; condiciones de trabajo insalubres. Se demostró que el Agente de Saludde la Familia tiene conocimiento y conciencia de su papel dentro del equipo multiprofesional, se sienten despreparados paraejercer todas sus atribuciones, desvalorizados en relación a la comunidad y a otros miembros del equipo. Percibieron losriesgos ocupacionales de la profesión y la falta de acciones para minimización de estos, además de baja remuneración.This study’s objective was to understand the meaning of being a Community Health Agent (ACS) in aFamily Health Program (PSF). Descriptive, qualitative, with phenomenological inspiration research that sought the essenceof the ACS’ speech, and all them work in the city of Sete Lagoas-MG. The interviews were recorded with 7 ACS, guided bythe question “what is your perception about family health strategy, as a Community Health Agent?” The comprehensiveanalysis of speech allowed the construction of five categories: ACS, link between the team and the community; professionalevaluation; the ACS role in the team and their contribution to the change of basic care model; training; unhealthy workconditions. It showed that the Agents of Family Health are aware and conscious of their role within the team, feelunprepared to exercise all their tasks, devalued in relation to community and other team members. Also, they perceive theoccupational hazards of the profession and lack of actions to minimize them, beyond the low payment.O estudo teve como objetivo compreender o significado de ser um Agente Comunitário de Saúde-ACS em umPrograma Saúde da Família. Pesquisa descritiva, qualitativa, com inspiração fenomenológica que buscou a essência dodiscurso do ACS do município de Sete Lagoas-MG. As entrevistas foram gravadas com sete ACS a partir da questãonorteadora: Qual a sua percepção como Agente Comunitário de Saúde na Estratégia Saúde da Família? A análise compreensivadas falas possibilitou a construção de cinco categorias: ACS, elo entre a equipe multiprofissional e a comunidade;valorização profissional; papel do ACS na equipe multiprofissional e sua contribuição para a mudança do modelo deatenção básica; formação profissional; condições de trabalho insalubres. Evidenciou-se que os Agentes de Saúde daFamília têm conhecimento e consciência do seu papel dentro da equipe multiprofissional, sentem-se despreparados paraexercer todas as suas atribuições, desvalorizados em relação à comunidade e a outros membros da equipe. Percebem osriscos ocupacionais da profissão e a falta de ações para minimização destes, além da baixa remuneração

Current millennium biotechniques for biomedical research on parasites and host-parasite interactions

The development of biotechnology in the last three decades has generated the feeling that the newest scientific achievements will deliver high standard quality of life through abundance of food and means for successfully combating diseases. Where the new biotechnologies give access to genetic information, there is a common belief that physiological and pathological processes result from subtle modifications of gene expression. Trustfully, modern genetics has produced genetic maps, physical maps and complete nucleotide sequences from 141 viruses, 51 organelles, two eubacteria, one archeon and one eukaryote (Saccharomices cerevisiae). In addition, during the Centennial Commemoration of the Oswaldo Cruz Institute the nearly complete human genome map was proudly announced, whereas the latest Brazilian key stone contribution to science was the publication of the Shillela fastidiosa genomic sequence highlythed on a Nature cover issue. There exists a belief among the populace that further scientific accomplishments will rapidly lead to new drugs and methodological approaches to cure genetic diseases and other incurable ailments. Yet, much evidence has been accumulated, showing that a large information gap exists between the knowledge of genome sequence and our knowledge of genome function. Now that many genome maps are available, people wish to know what are we going to do with them. Certainly, all these scientific accomplishments will shed light on many more secrets of life. Nevertheless, parsimony in the weekly announcements of promising scientific achievements is necessary. We also need many more creative experimental biologists to discover new, as yet un-envisaged biotechnological approaches, and the basic resource needed for carrying out mile stone research necessary for leading us to that "promised land"often proclaimed by the mass media

Essential oils : in vitro activity against Leishmania amazonensis, cytotoxicity and chemical composition

Background: The current chemotherapy for cutaneous leishmaniosis (CL) has a series of drug limitations such as toxic side effects, long duration, high costs and drug resistance, which requires the development of new drugs or effective alternatives to the CL treatment. Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against protozoa. Thus, this study aims to evaluate the biological activity of different essential oils (EOs) on Leishmania (L.) amazonensis promastigotes forms, as well as their cytotoxicity on mammalian cells and chemical composition. Methods: Sixteen EOs were evaluated by mean of IC50/24 h and cytotoxicity against L6 cells (CC50/24 h) using Resazurin assay. Only those EOs that presented better results for IC50/24 h were submitted to GC–MS analysis to determine their chemical constitution. Results: The EO from Cinnamodendron dinisii, Matricaria chamomilla, Myroxylon peruiferum, Salvia sclarea, Bulnesia sarmientoi, Ferula galbaniflua, Siparuna guianensis and Melissa officinalis were the most active against L. amazonensis with IC50/24 h ranging from 54.05 to 162.25 μg/mL. Analysis of EOs by GC–MS showed mainly the presence of β-farnesene (52.73 %) and bisabolol oxide (12.09 %) for M. chamomilla; α-copaene (13.41 %), safrole (8.35 %) and δ-cadinene (7.08 %) for M. peruiferum; linalool (28.80 %) and linalyl acetate (60.08 %) for S. sclarea; guaiol (48.29 %) and 2-undecanone (19.49 %) for B. sarmientoi; ethyl phthalate (13.09 %) and methyl-8-pimaren-18-oate (41.82 %) for F. galbaniflua; and neral (37.18 %) and citral (5.02 %) for M. officinalis. Conclusion: The EO from F. galbaniflua showed to be effective against L. amazonensis promastigotes forms and presented low cytotoxic activity against L6 cells. Thus, it represents a strong candidate for future studies aiming its molecular activity on these pathogenic parasites

Triatomines: Trypanosomatids, Bacteria, and Viruses Potential Vectors?

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatomines may be competent vectors for pathogens such as Serratia marcescens, Bartonella, and Mycobacterium leprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

Triatomines: Trypanosomatids, Bacteria, and Viruses Potential Vectors? [Review]

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatomines may be competent vectors for pathogens such as Serratia marcescens, Bartonella, and Mycobacterium leprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

EFEITOS DA LACTULOSE NA SAÚDE GASTROINTESTINAL DE FRANGOS DE CORTE EXPERIMENTALMENTE INOCULADOS COM Salmonella entérica SOROVAR Typhimurium

Avaliaram-se os efeitos da lactulose na saúde gastrointestinal de frangos de corte pela aferição do pH e enumeração de unidades formadoras de colônias (UFCs) de Salmonella Typhimurium e Escherichia coli no inglúvio e ceco de aves inoculadas experimentalmente, via oral, na dose de 5,0 X 102 UFC /0,5mL com Salmonella Typhimurium. O delineamento adotado foi o inteiramente casualizado, utilizando-se 630 pintos, machos, os quais foram distribuídos em seis tratamentos, com sete repetições e 15 aves por unidade experimental. O tratamento 1: grupo controle (placebo); tratamento 2: grupo que recebeu somente a lactulose na água; tratamento 3: grupo que recebeu somente Salmonella Typhimurium; tratamento 4: grupo que recebeu a lactulose e Salmonella Typhimurium simultaneamente no primeiro dia de vida [L (1d) + ST (1d)]; tratamento 5: grupo que recebeu a lactulose 48 horas antes de serem inoculadas com Salmonella Typhimurium [L (1d) + ST (48h)] e tratamento 6: grupo que foi inoculado com Salmonella Typhimurium no primeiro dia e 48 horas depois receberam a lactulose [ST (1d) + L (48h)]. Aos dias sete, 14, 21 e 28 uma ave por parcela foi sacrificada e os conteúdos do inglúvio e do ceco foram coletados para a aferição do pH e contagem de Salmonella Typhimurium e Escherichia coli. Constatou-se que a lactulose determinou redução nos valores (P<0,05) de pH nos conteúdos do trato digestório aos sete dias de vida, e esta redução se manteve até 28 dias somente para o inglúvio nos tratamentos que receberam a lactulose, independente do período de inoculação do patógeno. Verificou-se, também, que a lactulose reduziu (P<0,05) as UFCs de Escherichia coli e de Salmonella Typhimurium no inglúvio aos 21 e 28 dias de vida nos tratamentos em que se administrou a lactulose antes do patógeno. Pode-se concluir que a lactulose altera os valores de pH do inglúvio e reduza colonização de Salmonella Typhimurium no ceco e as UFCs de Escherichia coli no inglúvio em todo o período experimental. PALAVRAS-CHAVE: controle, Escherichia coli, pH, unidades formadoras de colônias