Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom.

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a ß-neurotoxin phospholipase A2-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.

Diallel crosses for resistance to Macrophomina phaseolina and Thanatephorus cucumeris on cowpea.

This study aimed at identifying and selecting through partial diallel analysis, segregating populations of cowpea resistant to Macrophomina phaseolina and Thanatephorus cucumeris, based on the evaluation of general (GCA) and specific combining ability (SCA), involved in the genetic control of resistance. For this reason, 19 grouped cowpea genotypes, considering the resistance to these pathogens, were crossed in partial diallel scheme 14 x 5, during 2013 and 2014. The 70 F2 populations and the 19 parents were evaluated in a greenhouse as the reaction to pathogens, separately. The diallel analysis was performed according to the model of partial diallel proposed by Geraldi and Miranda Filho (1988). The additive effects predominated in the genetic control of the traits severity of charcoal rot (SEV) and area under the disease progress curve (AUDPC) to web blight, enabling the achievement of genetic gain with selection of resistant strains. Analyzing the effects of GCA, the parents BR 14-Mulato, BRS Tumucumaque and BRS Guariba, have a higher concentration of favorable alleles, highlighting, according to the values of SCA, the combinations BR 14-Mulato x MNC02-675F-4-10, BRS Tumucumaque x IT98K-1092-1, BRS Tumucumaque x MNC02-675F-4-10, BRS Tumucumaque x MNC02-675F-9-2, BRS Guariba x IT98K-1092-1, BRS Guariba x MNC02-675F-4-9, and BRS Guariba x MNC02-675F-4-10, as the most promising and indicated to obtain lines resistant to M. phaseolina and T. cucumeris in cowpea, simultaneously.

Genotypic gain with simultaneous selection of production, nutrition, and culinary traits in cowpea crosses and backcrosses using mixed models.

The aim of this study was to estimate the genotypic gain with simultaneous selection of production, nutrition, and culinary traits in cowpea crosses and backcrosses and to compare different selection indexes. Eleven cowpea populations were evaluated in a randomized complete block design with four replications. Fourteen traits were evaluated, and the following parameters were estimated: genotypic variation coefficient, genotypic determination coefficient, experimental quality indicator and selection reliability, estimated genotypic values ​​- BLUE, genotypic correlation coefficient among traits, and genotypic gain with simultaneous selection of all traits. The genotypic gain was estimated based on tree selection indexes: classical, multiplicative, and the sum of ranks. The genotypic variation coefficient was higher than the environmental variation coefficient for the number of days to start flowering, plant type, the weight of one hundred grains, grain index, and protein concentration. The majority of the traits presented genotypic determination coefficient from medium to high magnitude. The identification of increases in the production components is associated with decreases in protein concentration, and the increase in precocity leads to decreases in protein concentration and cooking time. The index based on the sum of ranks was the best alternative for simultaneous selection of traits in the cowpea segregating populations resulting from the crosses and backcrosses evaluated, with emphasis on the F4BC12, F4C21, and F4C12 populations, which had the highest genotypic gains.

Analysis of a fully penetrant spinocerebellar ataxia type 8 Brazilian family.

Spinocerebellar ataxia type 8 (SCA8) is a progressive neurological disorder caused by the expanded repeat CTA/CTG of two overlapping genes, ATXN8OS and ATXN8, expressed bidirectionally. Normal alleles have 15-50 repeats, and pathogenic alleles range from 71 to 1300 repeats. The disorder is relatively rare, accounting for about 2%-5% of the autosomal dominant forms of hereditary ataxia worldwide. However, the prevalence of disease-causing ATXN8OS/ATXN8 expansions is higher than the disease because of the reduced penetrance of the expanded allele. The aim of this study was to describe the first fully penetrant SCA8 family showing mixed Brazilian African and Amerindian origin. Eight members of this family were evaluated-the mother and seven offspring-through a complete neurological examination conducted at the Neurogenetics Clinic, HCFMRP-USP in Brazil. The number of CTA/CTG repeats was obtained after polymerase chain reaction (PCR) and fragment analysis. The haplotype analysis was conducted using a microsatellite marker, D13S1296, and four single nucleotide polymorphisms (SNPs), rs1831189, rs8002227, rs11841483, and rs72284461, all spanning a 70.1 Mb region on chromosome 13q21.3. The molecular analysis showed that the expansions ranged from 104 to 109 CTA/CTG repeats in the six affected individuals and were absent in two asymptomatic daughters (aged 53 and 40 years). Three SNPs cosegregate with the expanded alleles, confirming the connection between expansion and disease in this family. As the SCA8 diagnosis demands careful interpretation, we suggest the use of linkage analysis to observe segregation of the mutation, making more accurate its genotyping.

Diallelic analysis to obtain cowpea (Vigna unguiculata L. Walp.) populations tolerant to water deficit.

The purpose of this study was to identify parents and obtain segregating populations of cowpea (Vigna unguiculata L. Walp.) with the potential for tolerance to water deficit. A full diallel was performed with six cowpea genotypes, and two experiments were conducted in Teresina, PI, Brazil in 2011 to evaluate 30 F2 populations and their parents, one under water deficit and the other under full irrigation. A triple-lattice experimental design was used, with six 2-m-long rows in each plot. Sixteen plants were sampled per plot. The data were subjected to analysis of variance, and general and specific combining ability estimates were obtained based on the means. Additive effects were more important than non-additive effects, and maternal inheritance had occurred. The genotypes BRS Xiquexique, Pingo de Ouro-1-2, and MNC99-510F-16-1 were the most promising for use in selection programs aimed at water deficit tolerance. The hybrid combinations Pingo de Ouro-1-2 x BRS Xiquexique, BRS Xiquexique x Santo Inácio, CNCx 698-128G x MNC99-510F-16-1, Santo Inácio x CNCx 698-128G, MNC99-510F-16-1 x BRS Paraguaçu, MNC99- 510F-16-1 x Pingo de Ouro-1-2, and MNC99-510F-16-1 x BRS Xiquexique have the potential to increase grain production and tolerate water deficit.

Selection of cowpea progenies with enhanced drought-tolerance traits using principal component analysis.

Vigna unguiculata (L.) Walp (cowpea) is a food crop with high nutritional value that is cultivated throughout tropical and subtropical regions of the world. The main constraint on high productivity of cowpea is water deficit, caused by the long periods of drought that occur in these regions. The aim of the present study was to select elite cowpea genotypes with enhanced drought tolerance, by applying principal component analysis to 219 first-cycle progenies obtained in a recurrent selection program. The experimental design comprised a simple 15 x 15 lattice with 450 plots, each of two rows of 10 plants. Plants were grown under water-deficit conditions by applying a water depth of 205 mm representing one-half of that required by cowpea. Variables assessed were flowering, maturation, pod length, number and mass of beans/pod, mass of 100 beans, and productivity/plot. Ten elite cowpea genotypes were selected, in which principal components 1 and 2 encompassed variables related to yield (pod length, beans/pod, and productivity/plot) and life precocity (flowering and maturation), respectively.

Redescription of the Monotypic Neotropical Genus Crepititermes Emerson (Termitidae: Termitinae).

Based on a reexamination of specimens of Crepititermes Emerson deposited in the Museu de Zoologia da Universidade de São Paulo, São Paulo, Brasil (MZUSP), we characterize the morphology and coiling in situ of the digestive tube of workers of Crepititermes verruculosus Emerson for the first time. We provide additional notes on the imago and soldier and present digital images and illustrations for all castes. We also update the currently known geographical distribution of C. verruculosus, adding some biological remarks.

An alternative micromethod to access the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom.

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies largely on traditional rodent lethality assay (LD50). However, adequately validated in vitro tests should be introduced for assessing antivenom neutralizing capacity in plasma of immunized horses as well as for in-process quality control. The dynamic of fibrin formation in recalcified avian plasma samples is extremely slow, when compared to that presented by mammalian plasmas. In this study, we present one new coagulant assay, by performing dose-response curve after plotting the clotting time (CT) parameter of the ROTEM profile of recalcified chicken plasma samples (target) against semi-logarithmic doses of Bothrops jararaca venom (agonist), either in absence or in presence of the semi-logarithmic doses of anti-bothropic serum (ABS) (antagonist). The mean coagulant dose 50% (CD50) was defined as the quantity of venom (in µg) which reduces CT to 900 s, between minimum and maximum responses. The CT induced by 5CD50 of the venom was used as the control for calculating the effective dose (ED) of each batch of ABS. ED was defined as the ABS dose (nanoliters, nL) at which CT induced by one amount of venom corresponding to 5CD50 is displaced to the maximum threshold (1800 s). Five batches of the ABS, previously assayed for their lethality neutralizing activity (ED50) were assayed. The correlation coefficient (r) between both in vitro (ED) and in vivo (ED50) values was 0.87 (p value < 0.05). We propose this micro method as highly sensitive for characterization and quantification of possible procoagulant activity of small doses of snake venoms (nanograms) and for detecting small doses (nanoliters) of specific antibodies against this effect in little volume samples of biological fluids.

Genetic divergence among African cowpea lines based on morphoagronomic traits.

The aim of this study was to estimate the genetic divergence between African cowpea lines from the Cowpea Germplasm Bank of Embrapa Meio-Norte, Brazil. The morphoagronomic diversity of 57 cowpea lines was assessed using multivariate analysis. The germplasm was evaluated in August 2009 using a randomized block design with three replications based on the following traits: number of pods per peduncle, pod length (PL), number of grains per pod (NGP), grain length (GRL), grain width, 100-grain weight (W100G), and yield. The heritability values of the traits PL, NGP, GRL, and W100G were all higher than 70%, indicating the possibility of genetic progress with selection. The crosses between the lines IT82D-889 and IT89KD-245, IT85F-1380 and IT89KD-245, and IT89KD-245 and IT98K-1092-1 could result in promising genetic combinations. The characteristics that contributed most to genetic divergence were W100G (49.7%), PL (16.7%), GRL (12.0%), and NGP (9.7%).

Behavioural/emotional problems in Brazilian children: findings from parents' reports on the Child Behavior Checklist.

Background. To compare Child Behavior Checklist (CBCL) findings for a large Brazilian general population sample with those for US children considering: (a) mean problem item ratings; (b) fit of the US-derived CBCL 8-syndrome model; (c) scale internal consistency measured by Cronbach's alphas; (d) effects of society, age, gender on CBCL problem scores; and (e) ability to discriminate referred from non-referred children. Methods. Parents of 1228 non-referred 6-to-11-year-olds from three different regions of Brazil and 247 referred 6-to-11-year-olds from one clinic rated their children's behavioural and emotional problems using the CBCL/6-18. Results. Results for mean item ratings and scale internal consistencies were very similar to those found in the US and in Uruguay. Confirmatory factor analysis indicated that Brazilian data showed the best fit to the US 8-syndrome model of all countries studied to date. Gender patterns were comparable to those reported in other societies, but mean problem scores for non-referred Brazilian children were higher than those for US children. Therefore, the CBCL discriminated less well between non-referred and referred children in Brazil than in the US. Conclusions. Overall, our findings replicated those reported in international comparisons of CBCL scores for 31 societies, thereby providing support for the multicultural robustness of the CBCL in Brazil.

Maintaining rear-fanged snakes for venom production: an evaluation of mortality and survival rates for Philodryas olfersii and P. patagoniensis in captivity

This study evaluates the mortality and average survival rates of captive female Philodryas olfersii and Philodryas patagoniensis snakes maintained for venom production. Also, two factors likely to reduce captive survival were studied - body condition at admission and seasonality. Mortality peaks occurred during the second month in captivity. More than half the individuals were dead at the end of the third month. This suggests that the first three months in captivity are the most critical in terms of survival and adaptation. Females collected and admitted during spring and summer lived less time than those collected in autumn and winter. As gravidity and egg-laying occur during spring and summer, we suggest that the lower survival rates in these seasons may be due to high costs and stress involved in these reproductive events. Unexpectedly, body mass and body condition were poor predictors of survival in captivity. Our results have important implications in maintaining snakes for venom production. We propose some prophylactic measures to minimize the deleterious impacts of captivity during the adaptation period.(AU)

Studies on the stripping voltammetric determination and speciation of chromium at a rotating-disc bismuth film electrode.

An adsorptive stripping voltammetric protocol coupled with a rotating-disc bismuth film electrode for the determination and speciation of chromium (III) and chromium (VI) in the presence of diethylenetriaminepentaacetic acid (DTPA) is presented. The developed methodology involves a mass-transport controlled preconcentration step, during which a Cr(III)-DTPA complex is adsorbed onto a pre-plated rotating-disc bismuth film electrode held at -0.4V, followed by a reductive square wave stripping scan. At -1.07 V vs. Ag/AgCl, a peak is recorded due to the catalytic reduction of Cr(III)-DTPA to Cr(II)-DTPA. As a result of different chemical behaviours of Cr(III) and Cr(VI) in the presence of DTPA, the corresponding voltammetric signals presented different stabilities in time. A univariate optimization study was performed with several experimental parameters as variables. For Cr(VI) and total chromium, Cr(III)+Cr(VI), an accumulation time of 60s at -0.4V vs. Ag/AgCl resulted in detection limits of 0.336 and 0.414 nM and quantification limits of 1.12 and 1.40 nM, respectively. The relative standard deviation for 10 measurements of 5.0 nM chromate was 2.4%. Interference of other electroactive trace metals and surfactants was considered. A simple speciation scheme was proposed and satisfactorily applied to Cr(III) and Cr(VI) determinations in river water samples.

Analysis of the ontogenetic variation in the venom proteome/peptidome of bothrops jararaca reveals different strategies to deal with prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Molecular genotyping and epidemiology of Mycobacterium tuberculosis isolates obtained from inmates of correctional institutions of Campinas, Southeast Brazil

The objective of this study was to investigate the possible transmission of tuberculosis among 39 inmates with positive Mycobacterium tuberculosis smears in four correctional institutions located in Campinas City, SP, Brazil over a 19-month period. Fifty-one M. tuberculosis isolates from these inmates were characterized according to the number of IS6110 insertion elements present in their genomic DNA. The number of insertion elements in M. tuberculosis isolates varied from two to twelve. The dendrogram of similarity resulted in the grouping the isolates in six main clusters. These results, associated to epidemiological data, suggested the transmission of tuberculosis among inmates of the same and different institutions inmates. Univariate analysis of epidemiological data (total delay for beginning of treatment, previous treatment, and HIV status) and clustering occurrence showed that only "previous treatment" (OR = 7.65, p = 0.032) was associated with the possible transmission of tuberculosis in the studied prisons.