A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Trypanosoma cruzi: ancestral genomes and population structure

Although the genome of Trypanosoma cruzi has been completely sequenced, little is known about its population structure and evolution. Since 1999, two major evolutionary lineages presenting distinct epidemiological characteristics have been recognised: T. cruzi I and T. cruzi II. We describe new and important aspects of the population structure of the parasite, and unequivocally characterise a third ancestral lineage that we propose to name T. cruzi III. Through a careful analysis of haplotypes (blocks of genes that are stably transmitted from generation to generation of the parasite), we inferred at least two hybridisation events between the parental lineages T. cruzi II and T. cruzi III. The strain CL Brener, whose genome was sequenced, is one such hybrid. Based on these results, we propose a simple evolutionary model based on three ancestral genomes, T. cruzi I, T. cruzi II and T. cruzi III. At least two hybridisation events produced evolutionarily viable progeny, and T. cruzi III was the cytoplasmic donor for the resulting offspring (as identified by the mitochondrial clade of the hybrid strains) in both events. This model should be useful to inform evolutionary and pathogenetic hypotheses regarding T. cruzi.

Medidas preventivas e exposição a agrotóxicos em Uberaba – MG / Preventive measures and exposure to agrotoxics in Uberaba – MG

Foram estudados elementos do processo de trabalho que expõem indivíduos ao risco de intoxicação por agrotóxicos nas culturas de feijão, milho, soja, arroz, laranja, café e hortaliças na zona rural de Uberaba, Minas Gerais. Dentre 46 trabalhadores examinados, 6(13,2%) apresentaram atividade da acetilcolinesterase alterada. Durante a aplicação dos produtos químicos, 56,6% dos trabalhadores não usam botas ou botinas; 60,9% não usam máscaras; 67,5% não usam luvas e 32,6% não usam qualquer tipo de equipamento de proteção individual. As culturas de mandioca, soja, tomate, jiló, laranja, quiabo, abóbora e café requisitam períodos de pulverização diária. Pimentão, algodão, abacate, abobrinha, abacaxi, alface, repolho, couve, salsa, milho, chuchu, arroz, limão, mamão, goiaba, maracujá, morango e a criação de bovinos requisitam pulverização semanal de produtos tóxicos. Banana, pimenta e criação de aves solicitam pulverização ou aplicação mensal. O tempo diário de pulverização varia de menos de uma hora (21,7%) a doze horas ininterruptas (2,1%). Os sintomas mais referidos pelos aplicadores de agrotóxicos foram cefaléia (17,4%); tontura (15,2%) e alteração visual (4,4%).

Elements pertaining to the work process that bring about the liability of poisoning by agrotoxics in the bean, corn, soya, rice, orange, coffee and vegetable plantations in the rural zone of Uberaba, Minas Gerais were studied. Among 46 workers who were examined, 6(13.2%) showed an altered activity of acetylcolinesterase. During the application of the chemical products, 56.6% of the workers do not wear short nor long-legged boots; 60.9% do not wear masks; 67.5% do not wear gloves and 32.6% do not wear any type of individual protective equipment. The manioc, soya, tomato, 'jiló', orange, okra, squash and coffee plantations require daily periods of pulverisation. Green pepper, cotton, avocado, zucchini, pineapple, lettuce, cabbage, kale, parsley, corn, chayote, rice, lemon, papaya, guava, passion fruit and strawberry plantations, along with the cattle herds, require a weekly pulverisation of toxic products. Bananas, peppers and chicken breeding require a monthly pulverisation or application. The daily pulverisation period varies from less than one hour (21.7%) to twelve uninterrupted hours (2.1%). The symptoms which are most mentioned by those who handle the agrotoxics are: severe headaches (17.4%); dizziness (15.2%) and alterations to sight (4.4%).