Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

BIO Logical Agents: Norms, Beliefs, Intentions in Defeasible Logic

In this paper we follow the BOID (Belief, Obligation, Intention, Desire) architecture to describe agents and agent types in Defeasible Logic. We argue, in particular, that the introduction of obligations can provide a new reading of the concepts of intention and intentionality. Then we examine the notion of social agent (i.e., an agent where obligations prevail over intentions) and discuss some computational and philosophical issues related to it. We show that the notion of social agent either requires more complex computations or has some philosophical drawbacks.

Interaction between Normative Systems and Cognitive Agents in Temporal Modal Defeasible Logic

While some recent frameworks on cognitive agents addressed the combination of mental attitudes with deontic concepts, they commonly ignore the representation of time. An exception is [1]that manages also some temporal aspects both with respect to cognition and normative provisions. We propose in this paper an extension of the logic presented in [1]with temporal intervals.

Contextual Deliberation of Cognitive Agents in Defeasible Logic

This article extends Defeasible Logic to deal with the contextual deliberation process of cognitive agents. First, we introduce meta-rules to reason with rules. Meta-rules are rules that have as a consequent rules for motivational components, such as obligations, intentions and desires. In other words, they include nested rules. Second, we introduce explicit preferences among rules. They deal with complex structures where nested rules can be involved.

Effect of smoke-free policies on the behaviour of social smokers

Objective-To test the hypothesis that proposed amendments to the Occupational Safety and Health Act making all enclosed workplaces in Western Australia smoke free would result in a decrease in cigarette consumption by patrons at nightclubs, pubs, and restaurants without adversely affecting attendance. Design-Cross sectional structured interview survey. Participants and setting-Patrons of several inner city pubs and nightclubs in Perth were interviewed while queuing for admission to these venues. Outcome measures-Current social habits, smoking habits, and how these might be affected by the proposed regulations. Persons who did not smoke daily were classified as social smokers. Results-Half (50%) of the 374 patrons interviewed were male, 51% currently did not smoke at all, 34.3% smoked every day, and the remaining 15.7% smoked, brat not every day. A clear majority (62.5%) of all 374 respondents anticipated no change to the frequency of their patronage of hospitality venues if smoke-free policies became mandatory One in five (19.3%) indicated that they would,ao out more often, and 18.2% said they would go out less often. Half (52%) of daily smokers anticipated no change to their cigarette consumption, while 44.5% of daily smokers anticipated a reduction in consumption. A majority of social smokers (54%) predicted a reduction in their cigarette consumption, with 42% of these anticipating quitting. Conclusions-One in nine (11.5%) of smokers say that adoption of smoke-Pi ee policies would prompt them to quit smoking entirely without a significant decrease in attendance at pubs and nightclubs. There can be few other initiatives as simple, cheap, and popular that would achieve so much for public health.

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (pi)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-alpha-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from pi-A curves applying the additivity rule by calculating the excess free energy of mixture (Delta G(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes. (C) 2010 Elsevier B.V. All rights reserved.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGF beta 1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. (C) 2009 UICC

Insights on PRAME and osteosarcoma by means of gene expression profiling

Osteosarcoma (OS) is the most frequent bone tumor in children and adolescents. Tumor antigens are encoded by genes that are expressed in many types of solid tumors but are silent in normal tissues, with the exception of placenta and male germ-line cells. It has been proposed that antigen tumors are potential tumor markers. The premise of this study is that the identification of novel OS-associated transcripts will lead to a better understanding of the events involved in OS pathogenesis and biology. We analyzed the expression of a panel of seven tumor antigens in OS samples to identify possible tumor markers. After selecting the tumor antigen expressed in most samples of the panel, gene expression profiling was used to identify osteosarcoma-associated molecular alterations. A microarray was employed because of its ability to accurately produce comprehensive expression profiles. PRAME was identified as the tumor antigen expressed in most OS samples; it was detected in 68% of the cases. Microarray results showed differences in expression for genes functioning in cell signaling and adhesion as well as extracellular matrix-related genes, implying that such tumors could indeed differ in regard to distinct patterns of tumorigenesis. The hypothesis inferred in this study was gathered mostly from available data concerning other kinds of tumors. There is circumstantial evidence that PRAME expression might be related to distinct patterns of tumorigenesis. Further investigation is needed to validate the differential expression of genes belonging to tumorigenesis-related pathways in PRAME-positive and PRAME-negative tumors.

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

A test of the ash-free dry weight technique on the developmental stages of Patiriella spp. (Echinodermata : Asteroidea)

Determination of the ash-free dry weight (AFDW) of marine specimens requires samples to be rinsed, soaked, and centrifuged. Problems associated with this technique were examined with the developmental stages of seastar species (Patiriella) with different modes of development. The influence of three rinsing solutions (ammonium formate [AF], filtered seawater [FSW], and reverse osmosis water [RO]) was assessed. The hypothesis that the AFDW technique is a measure of organic material was addressed by drying inorganic salts. Developmental stages of Patiriella calcar rinsed in FSW were twice as heavy as those rinsed in RO or AE indicating that samples should be rinsed in RO or AF before weighing. Soaking treatments had a significant effect on the AFDW of samples of P. calcar (planktonic developer), indicating that the rinsing period should be brief. Zygotes of Patiriella re gularis (planktonic developer) were significantly heavier than ova or gastrulae, regardless of treatment. In contrast, there were no significant differences in the AFDW of any stages or treatments of Patiriella exigua (benthic developer). This may be due to the presence of a modified fertilization envelope, which protects these benthic embryos. Inorganic salts with water of crystallization and FSW lost 20-75% and 14% of their dry weight, respectively, after ashing. We propose that salt ions may retain water, which does not evaporate during drying but is lost during ashing, resulting in the overestimation of sample AFDW. If a similar process occurs in the developmental stages of marine invertebrates, changes in the intracellular ionic composition through development may result in inaccurate estimates of biomass.

Extratos vegetais e Bacillus thuringiensis visando o manejo de Duponchelia fovealis Zeller (Lepidoptera: Crambidae)

A lagarta Duponchelia fovealis Zeller (Lepidoptera: Crambidae) foi relatada em plantios comerciais do morangueiro no estado do Espírito Santo ocasionando grandes problemas à cultura. Porém, por ser uma praga recente, não há registro de produtos para o seu controle. A cada dia aumenta a demanda por alimentos e outros produtos livres de resíduos, além da necessidade de uma agricultura mais desenvolvida e sustentável. Pesquisas com agentes de controle biológico e extratos vegetais surgem como alternativa para o manejo desse inseto-praga. Este trabalho teve como objetivo avaliar a eficiência de formulados comerciais à base de Bacillus thuringiensis e a atividade do uso dos extratos aquosos de alho e fumo, visando sua adoção como métodos alternativos de controle de D. fovealis. Nos bioensaios para avaliar a patogenicidade e virulência de duas formulações comerciais à base de B. thuringiensis, Agree® e Dipel WP®, sobre a dieta artificial adaptada à base de farelo de soja, germe de trigo e açúcar, proposta por King e Hartley (1985) para Diatraea saccharalis (Lepidoptera: Crambidae), foram inoculados 70 μL de cada formulado comercial, na concentração 1 x 108 esporos·mL-1. Em seguida, avaliou-se a virulência dos respectivos formulados, isso através da estimativa da concentração letal (CL50) para o estádio de maior suscetibilidade. Em virtude dos resultados encontrados, observou-se que o estágio 1 de desenvolvimento apresentou 95,88% e 86,76% de mortalidades para os produtos Agree® e Dipel WP®, respectivamente, demonstrando patogenicidade e virulência à D. fovealis. No bioensaio para avaliar a atividade dos extratos aquosos de alho e fumo, estes foram aplicados na concentração 10% (m/v). Todos os tratamentos foram pulverizados com Torre de Potter, calibrada a pressão de 15 lb/pol². Posteriormente estimou-se a concentração letal (CL50) do extrato aquoso de fumo, o qual apresentou 95% de mortalidade no teste de suscetibilidade. Desta forma, com os resultados obtidos na presente pesquisa, concluiu-se que a utilização de formulados comerciais à base de B. thuringiensis e extrato de fumo podem ser uma alternativa no manejo fitossanitário de D. fovealis.

Seletividade de acaricidas utilizados em cafeeiro para larvas de crisopídeos

Os crisopídeos são encontrados em agroecossistema cafeeiro alimentando-se de várias pragas. Objetivou-se avaliar a seletividade de acaricidas utilizados na cafeeiculta sobre a biologia de Chrysoperla externa (Hagen, 1861). Os tratamentos foram: testemunha (água), espirodiclofeno (0,12 g i.a./L), fenpropatrina (0,15 e 0,30 g i.a./L), enxofre (4,0 e 8,0 g i.a./L) e abamectina (0,0067 e 0,0225 g i.a./L). Pulverizaram-se os produtos sobre larvas de primeiro, segundo e terceiro instares de C. externa por meio da torre de Potter. Avaliaram-se a duração do instar, sobrevivência das larvas e pupas e viabilidade dos ovos produzidos pelos adultos provenientes das larvas tratadas. Os produtos foram classificados em classes de toxicidade. Fenpropatrina foi nocivo e espirodiclofeno e abamectina foram moderadamente nocivos, necessitando de novos estudos em condições de casa de vegetação e campo para confirmação ou não de suas toxicidades. Em função da baixa toxicidade apresentada pelo enxofre ao predador C. externa, pode ser recomendado em programas de manejo de pragas do cafeeiro visando compatibilizar os métodos químico e biológico por meio dessa espécie de crisopídeo.

Divergência genética em cultivares de morangueiro, baseada em caracteres morfoagronômicos

Os caracteres morfoagronômicos são tradicionalmente usados na caracterização de cultivares e no estudo da divergência genética, contribuindo na definição de estratégias para o melhoramento genético. Este trabalho teve por objetivo avaliar a divergência genética por meio de caracteres morfoagronômicos de 11 cultivares de morangueiro (Aromas, Camarosa, Camino Real, Campinas, Diamante, Dover, Oso Grande, Sweet Charlie, Toyonoka, Tudla e Ventana), nas condições climáticas da região Centro-Sul do Paraná. Foram analisados 29 caracteres morfoagronômicos relacionados com a planta, folha, flor, fruto e aquênios do morangueiro. As similaridades genéticas foram calculadas por meio de análise multivariada e, os cultivares, agrupados com base na matriz de similaridade genética, usando-se UPGMA. Dentre os 29 caracteres morfoagronômicos avaliados, oito apresentaram diferenças não significativas (p < 0,05). A similaridade média foi de 38%, variando de 19 (aromas e camino real) a 62% (Camino Real e Camarosa; Aromas e Sweet Charlie). O dendrograma alocou os cultivares em quatro grupos, contudo, essa divisão não foi coerente com a origem e genealogia dos cultivares. O cultivar Tudla apresenta elevado potencial "per se" para utilização em programas de melhoramento. O cruzamento mais promissor com base nos caracteres morfoagronômicos é entre os cultivares Camarosa e Campinas.