Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena

Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B. sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans. All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process. This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control. Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B. sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.

Time course of in vitro maturation of intra-erythrocytic malaria parasite: a comparison between Plasmodium falciparum and Plasmodium knowlesi

The schizont maturation assay for in vitro drug sensitivity tests has been a standard method employed in the global baseline assessment and monitoring of drug response in Plasmodium falciparum. This test is limited in its application to synchronous plasmodial infections because it evaluates the effect of drug on the maturation of parasite especially from ring to schizont stage and therefore synchronized P. falciparum cultures are required. On the other hand, P. knowlesi, a simian malaria parasite has a unique 24-h periodicity and maintains high natural synchronicity in monkeys. The present report presents the results of a comparative study on the course of in vitro maturation of sorbitol synchronized P. falciparum and naturally synchronous P. knowlesi. Ring stage parasites were incubated in RPMI medium supplemented with 10-15% pooled homologous serum in flat-bottomed 96-well micro plates using a candle jar at 37°C. The results suggest that the ideal time for harvesting the micro-assay plates for in vitro drug sensitivity test for sorbitol-synchronized P. falciparum and naturally synchronous P. knowlesi are from 26 to 30 h and from 22 to 25 h, respectively. The advantages of using P. knowlesi in chemotherapeutic studies are discussed.

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Sharing competences in strategic alliances: a case study of the Cosan and Shell biofuel venture

In a competitive world, the way a firm establishes its organizational arrangements may determine the enhancement of its core competences and the possibility of reaching new markets. Firms that find their skills to be applicable in just one type of market encounter constraints in expanding their markets, and through alliances may find a competitive form of value capture. Hybrid forms of organization appear primarily as an alternative to capturing value and managing joint assets when the market and hierarchy modes do not present any yields for the firm's competitiveness. As a result, this form may present other challenging issues, such as the allocation of rights and principal-agent problems. The biofuel market has presented a strong pattern of changes over the last 10 years. New intra-firm arrangements have appeared as a path to participate or survive among global competition. Given the need for capital to achieve better results, there has been a consistent movement of mergers and acquisitions in the Biofuel sector, especially since the 2008 financial crisis. In 2011 there were five major groups in Brazil with a grinding capacity of more than 15 million tons per year: Raízen (joint venture formed by Cosan and Shell), Louis Dreyfus, Tereos Petrobras, ETH, and Bunge. Major oil companies have implemented the strategy of diversification as a hedge against the rising cost of oil. Using the alliance of Cosan and Shell in the Brazilian biofuel market as a case study, this paper analyses the governance mode and challenging issues raised by strategic alliances when firms aim to reach new markets through the sharing of core competences with local firms. The article is based on documentary research and interviews with Cosan's Investor Relations staff, and examines the main questions involving hybrid forms through the lens of the Transaction Cost Economics (TCE), Agency Theory, Resource Based View (RBV), and dynamic capabilities theoretical approaches. One focal point is knowledge "appropriability" and the specific assets originated by the joint venture. Once the alliance is formed, it is expected that competences will be shared and new capabilities will expand the limits of the firm. In the case studied, Cosan and Shell shared a number of strategic assets related to their competences. Raízen was formed with economizing incentives, as well to continue marshalling internal resources to enhance the company's presence in the world energy sector. Therefore, some challenges might be related to the control and monitoring agents' behavior, considering the two-part organism formed by distinctive organizational culture, tacit knowledge, and long-term incentives. The case study analyzed illustrates the hybrid arrangement as a middle form for organizing the transaction: neither in the market nor in the hierarchy mode, but rather a more flexible commitment agreement with a strategic central authority. The corporate governance devices are also a challenge, since the alignment between the parent companies in the joint ventures is far more complex. These characteristics have led to an organism with bilateral dependence, offering favorable conditions for developing dynamic capabilities. However, these conditions might rely on the partners' long-term interest in the joint venture.

Hipotermia no período intra-operatório: é possível evitá-la?

O estudo teve como objetivo identificar as medidas adotadas para a prevenção de hipotermia no período intra-operatório. Trata-se de um estudo com delineamento de pesquisa não experimental, tipo descritivo-exploratório, prospectivo. Para tal elaborou-se um instrumento de coleta de dados, o qual foi submetido à validação aparente e de conteúdo. A amostra foi constituída de 70 pacientes. A medida mais empregada na sala de operação foi o método passivo de aquecimento cutâneo como o uso de lençol de algodão (11,4%) e o enfaixamento dos membros inferiores (14,3%). A utilização de um método ativo de aquecimento cutâneo ocorreu uma única vez (1,4%). Os resultados evidenciados demonstraram a necessidade de implementação de intervenções eficazes para a prevenção da hipotermia e o enfermeiro tem papel importante neste contexto, uma vez que a segurança do paciente e a redução de complicações decorrentes do procedimento anestésico cirúrgico são metas do cuidado de enfermagem.

Lesões de pele no intra-operatório de cirurgia cardíaca: incidência e caracterização

Neste estudo exploratório, descritivo e de coorte o objetivo principal foi verificar a incidência de pacientes submetidos à cirurgia cardíaca que desenvolveram lesões de pele no período intra-operatório, caracterizar as lesões. A coleta de dados foi realizada no Centro Cirúrgico (CC) de um hospital público de ensino, de atenção terciária à saúde, predominantemente cirúrgico, especializado em cardiologia no Município de São Paulo. A amostra do estudo foi de 182 pacientes. O estudo foi feito com um p significativo (<0,05) frente aos testes estatísticos não-paramétricos. Quanto à incidência de pacientes submetidos à cirurgia cardíaca, que desenvolveram lesões de pele em decorrência do período intra-operatório, obteve-se incidência de 20,9%. Tivemos que 19,2% das lesões apresentaram-se como Úlceras por Pressão (UP) no estágio I; 1,1% das lesões caracterizaram-se como abrasão; 1,1% feridas incisas; 0,5% laceração; 0,5% queimadura elétrica superficial e 0,5% UP no estágio II.

Influência da competição intra-específica entre fêmeas e da ausência de hospedeiro no parasitismo de Telenomus remus Nixon (Hymenoptera, Scelionidae) sobre ovos de Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae)

A competição intraespecífica e a ausência de hospedeiros podem comprometer o sucesso de programas de controle biológico. Assim, este trabalho objetivou avaliar o efeito de diferentes densidades de fêmeas (1, 3, 6 e 9 fêmeas/ 100 ovos) e da ausência de hospedeiro no parasitismo de Telenomus remus Nixon (Hymenoptera, Scelionidae) em ovos de Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae). Observou-se aumento no parasitismo de ovos de S. frugiperda por T. remus a partir da densidade 3 fêmeas/ 100 ovos tanto em condições de laboratório (18%) quanto em campo (32%), embora o parasitismo causado por densidades maiores que 1 fêmea/100 ovos não foi significativamente diferente. Todavia, isso não causou superparasitismo. Por outro lado, o número de fêmeas produzidas na progênie diminuiu (39%) com a maior densidade de fêmeas sob condições de laboratório. Contudo, não verificou-se diferença significativa sob condições de campo. Mesmo após 9 dias de ausência de hospedeiro não houve redução nos índices de parasitismo, permanecendo em torno de 95 ovos parasitados/fêmea/dia. Todavia, no décimo dia pode-se observar redução (40%) no número médio de ovos parasitados. Desse modo, a liberação de três fêmeas por 100 ovos é adequada para se obter elevados níveis de parasitismo.

Intra-puparial development of the females of Chrysomya albiceps (Wiedemann) (Diptera, Calliphoridae)

Intra-puparial development of the females of Chrysomya albiceps (Wiedemann) (Diptera, Calliphoridae). The chronology and morphological changes that take place during intra-puparial development of Chrysomya albiceps is described based on 254 specimens reared in the laboratory. Larvae were obtained from the eggs laid by a single female. The pre-pupae were separated according to the reduction of larval length and the degree of pigmentation and sclerotization of the cuticle. After pupation, 10 individuals were fixed in Carnoy's solution and preserved in 70% ethanol, 10 individuals were fixed every 3 hours up to complete the first 24 hours (n = 80), the remaining individuals were fixed every six hours up to the 90th hour (n = 110) when 54 females emerged. The pupae were immersed in 5% formic acid for 48 hours and maintained in 70% ethanol, and then dissected and analyzed. C. albiceps shows four intra-puparial stages, each of which were described and compared with those described for Musca domestica, Calliphora erythrocephala, Sarcophaga bullata, Cuterebra tenebrosa, Oestrus ovis and Dermatobia hominis. Four developmental stages may be described: (1) the larva-pupa apolysis, after three hours; (2) the criptocephalic pupa, after six hours; (3) the phanerocephalic pupa, after nine hours; (4) the pharate pupa, after nine hours. The pharate adult is completely formed after 81 hours.

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert. The spatial distribution of the immature stages of the leaf miner Angelabella tecomae Vargas & Parra, 2005 was determined at two intra-plant levels (shoot and leaflet) on the shrub Tecoma fulva fulva (Cav.) D. Don (Bignoniaceae) in the Azapa valley, northern Chilean Atacama Desert. An aggregated spatial pattern was detected for all the immature stages along the shoot, with an age dependent relative position: eggs and first instar larvae were clumped at apex; second, third and fourth instar larvae were mostly found at intermediate positions; meanwhile the spinning larva and pupa were clumped at basis. This pattern suggests that the females select new, actively growing leaflets for egg laying. At the leaflet level, the immature stages were found more frequently at underside. Furthermore, survivorship was higher for larvae from underside mines. All these results highlight the importance of an accurate selection of egg laying site in the life history of this highly specialized leaf miner. By contrast, eventual wrong choices in the egg laying site selection may be associated with diminished larval survivorship. The importance of the continuous availability of new plant tissue in this highly human modified arid environment is discussed in relation with the observed patterns.

Propriedades espectrais da matéria orgânica leve-livre e leve intra-agregado de dois latossolos sob plantio direto e preparo convencional

Este trabalho teve como objetivo estudar a natureza química das frações leves-livres (FLL) e leves intra-agregado (FLI) da matéria orgânica do solo, obtidas pelo fracionamento físico do solo por densidade, por meio da espectroscopia na região do infravermelho, para verificar se tais frações constituem compartimentos distintos da matéria orgânica do solo. Foram analisadas amostras de Latossolos de dois estados do Brasil (RS e GO), submetidos a plantio direto e preparo convencional, em distintos sistemas de rotação de culturas. A análise por infravermelho revelou diferenças contrastantes entre os compartimentos orgânicos estudados. Os espectros de IV da fração leve-livre apresentaram configuração semelhante aos dos resíduos vegetais, indicando que ela se encontra em estádios iniciais de transformação. Não foram observadas diferenças estruturais na FLL entre os distintos sistemas de preparo e rotação de culturas. Os espectros de IV da FLI apresentaram bandas de absorção N-H e C-O de polissacarídeos menos intensas e em maior conjugação, em relação aos espectros da FLL, características de material mais humificado. Foi observada ainda uma maior transformação estrutural da fração leve intra-agregado em solos sob preparo convencional, quando comparada à FLI de solo sob vegetação natural e plantio direto. Os índices de hidrofobicidade (IH) e de condensação (IC), determinados a partir de relações entre as bandas de absorção de grupamentos - CH3 alifáticos, C-O de polissacarídeos e C=O conjugados, permitiram identificar as diferenças na recalcitrância e condensação das frações leves. Constatou-se que ambos os índices foram significativamente maiores para a matéria orgânica intra-agregado, por conseqüência de seu maior grau de humificação.

Resistência inter e intra-agregados em ensaios de cisalhamento direto de um nitossolo vermelho distrófico

Para solos agregados, a envoltória da resistência ao cisalhamento pode ser dividida em dois segmentos, com declividades e interceptos diferentes. Um primeiro segmento mais inclinado e com intercepto menor representaria a envoltória de ruptura definida pelo atrito e coesão interagregados, enquanto o segundo segmento, menos inclinado e com intercepto maior, seria a envoltória definida pelo atrito e coesão intra-agregados. O presente estudo foi realizado com o objetivo de avaliar se a envoltória de resistência ao cisalhamento de agregados do horizonte superficial de um Nitossolo Vermelho pode ser subdividida em segmentos distintos, com coeficientes angulares diferentes, e se isso está relacionado à existência de agregados nele. Inicialmente, amostras coletadas na superfície de um Nitossolo Vermelho distrófico latossólico de textura argilosa foram submetidas ao ensaio de cisalhamento direto com pressões normais de 24,4; 48,9; 98,2; 196,4; 294,6; 392,8 e 491,8 kPa. Posteriormente, o ensaio foi realizado com amostras de agregados de cinco classes de diâmetro: < 0,25; 0,25-0,50; 0,50-1,00; 1,00-2,00 e 2,00-4,00 mm, empregando-se pressões normais de 24,4; 48,9; 73,5; 147,3; 294,6; 441,9; 589,2 e 736,6 kPa. Em todos os ensaios, verificou-se que as envoltórias obtidas podem ser divididas em dois segmentos de reta. Os resultados indicam que a agregação deve ser considerada na definição das cargas normais a serem utilizadas no ensaio de cisalhamento direto, bem como na análise dos resultados, pois ela tem efeitos sobre a envoltória de resistência ao cisalhamento. Para o Nitossolo Vermelho distrófico usado neste estudo, constatou-se que, na umidade em que as amostras foram ensaiadas, os agregados definem a resistência ao cisalhamento do solo para cargas normais de até 294,6 kPa.