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BackgroundIn 2022, a global monkeypox virus (MPXV) clade II epidemic occurred mainly among men who have sex with men. Until early 1980s, European smallpox vaccination programmes were part of worldwide smallpox eradication efforts. Having received smallpox vaccine > 20 years ago may provide some cross-protection against MPXV.AimTo assess the effectiveness of historical smallpox vaccination against laboratory-confirmed mpox in 2022 in Europe.MethodsEuropean countries with sufficient data on case vaccination status and historical smallpox vaccination coverage were included. We selected mpox cases born in these countries during the height of the national smallpox vaccination campaigns (latest 1971), male, with date of onset before 1 August 2022. We estimated vaccine effectiveness (VE) and corresponding 95% CI for each country using logistic regression as per the Farrington screening method. We calculated a pooled estimate using a random effects model.ResultsIn Denmark, France, the Netherlands and Spain, historical smallpox vaccination coverage was high (80-90%) until the end of the 1960s. VE estimates varied widely (40-80%, I2 = 82%), possibly reflecting different booster strategies. The pooled VE estimate was 70% (95% CI: 23-89%).ConclusionOur findings suggest residual cross-protection by historical smallpox vaccination against mpox caused by MPXV clade II in men with high uncertainty and heterogeneity. Individuals at high-risk of exposure should be offered mpox vaccination, following national recommendations, regardless of prior smallpox vaccine history, until further evidence becomes available. There is an urgent need to conduct similar studies in sub-Saharan countries currently affected by the MPXV clade I outbreak.

Angiogenesis is a process that is controlled by a delicate combination of proangiogenic and antiangiogenic molecules and can be disrupted in various illnesses, including cancer. Non-cancerous diseases can also have an abnormal or insufficient vascular growth, inflammation and hypoxia, which exacerbate angiogenesis. These conditions include atherosclerosis, psoriasis, endometriosis, asthma, obesity and AIDS. Based on that, the present work assessed the in vitro and ex vivo antiangiogenic properties stemming from BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom, via the VEGF pathway. BthMP at a concentration of 5 and 40 μg/mL showed no toxicity to endothelial cells (HUVEC) in the MTT assay and was not able to induce necrosis and colony proliferation. Interestingly, BthMP inhibited adhesion, migration and invasion of HUVECs in Matrigel and arrested in vitro angiogenesis by reducing the average number of nodules in toxin-treated cells by 9.6 and 17.32 at 5 and 40 μg/mL, respectively, and the number of tubules by 15.9 at 5 μg/mL and 21.6 at 40 μg/mL in a VEGF-dependent way, an essential proangiogenic property. Furthermore, BthMP inhibited the occurrence of the angiogenic process in an ex vivo aortic ring test by decreasing new vessel formation by 52% at 5 μg/mL and by 66% at 40 μg/mL and by increasing the expression of an antiangiogenic gene, SFLT-1, and decreasing the expression of the proangiogenic genes VEGFA and ANGPT-1. Finally, this toxin reduces the production of nitric oxide, a marker that promotes angiogenesis and VEGF modulation, and decreases the protein expression of VEGFA in the supernatant of the HUVEC culture by about 30 %. These results suggest that BthMP has a promising antiangiogenic property and proves to be a biotechnological mechanism for understanding the antiangiogenic responses induced by snake venom metalloproteinases, which could be applied to a variety of diseases that exhibit an imbalance of angiogenesis mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora , Bothrops taeniatus , Bothrops bilineatus smaragdinus , Bothrops brazili , and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.

Tuberculosis remains a major public health problem and one of the top ten causes of death worldwide. The alarming increase in multidrug-resistant and extensively resistant variants (MDR, pre-XDR, and XDR) makes the disease more difficult to treat and control. New drugs that act against MDR/XDR strains are needed for programs to contain this major epidemic. The present study aimed to evaluate new compounds related to dihydro-sphingosine and ethambutol against sensitive and pre-XDR Mycobacterium strains, as well as to characterize the pharmacological activity through in vitro and in silico approaches in mmpL3 protein. Of the 48 compounds analyzed, 11 demonstrated good to moderate activity on sensitive and MDR Mycobacterium tuberculosis (Mtb), with a Minimum Inhibitory Concentration (MIC) ranging from 1.5 to 8 μM. They presented 2 to 14 times greater potency of activity when compared to ethambutol in pre-XDR strain, and demonstrated a selectivity index varying between 2.21 and 82.17. The substance 12b when combined with rifampicin, showed a synergistic effect (FICI = 0.5) on sensitive and MDR Mtb. It has also been shown to have a concentration-dependent intracellular bactericidal effect, and a time-dependent bactericidal effect in M. smegmatis and pre-XDR M. tuberculosis. The binding mode of the compounds in its cavity was identified through molecular docking and using a predicted structural model of mmpL3. Finally, we observed by transmission electron microscopy the induction of damage to the cell wall integrity of M. tuberculosis treated with the substance 12b. With these findings, we demonstrate the potential of a 2-aminoalkanol derivative to be a prototype substance and candidate for further optimization of molecular structure and anti-tubercular activity in preclinical studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

The 2022-2023 mpox outbreak predominantly affected adult men; 1.3% of reported cases were in children and adolescents <18 years of age. Analysis of global surveillance data showed 1 hospital intensive care unit admission and 0 deaths in that age group. Transmission routes and clinical manifestations varied across age subgroups.

Background: In May 2022, several countries with no history of sustained community transmission of mpox (formerly known as monkeypox) notified WHO of new mpox cases. These cases were soon followed by a large-scale outbreak, which unfolded across the world, driven by local, in-country transmission within previously unaffected countries. On July 23, 2022, WHO declared the outbreak a Public Health Emergency of International Concern. Here, we aim to describe the main epidemiological features of this outbreak, the largest reported to date., Methods: In this analysis of global surveillance data we analysed data for all confirmed mpox cases reported by WHO Member States through the global surveillance system from Jan 1, 2022, to Jan 29, 2023. Data included daily aggregated numbers of mpox cases by country and a case reporting form (CRF) containing information on demographics, clinical presentation, epidemiological exposure factors, and laboratory testing. We used the data to (1) describe the key epidemiological and clinical features of cases; (2) analyse risk factors for hospitalisation (by multivariable mixed-effects binary logistic regression); and (3) retrospectively analyse transmission trends. Sequencing data from GISAID and GenBank were used to analyse monkeypox virus (MPXV) genetic diversity., Findings: Data from 82 807 cases with submitted CRFs were included in the analysis. Cases were primarily due to clade IIb MPXV (mainly lineage B.1, followed by lineage A.2). The outbreak was driven by transmission among males (73 560 [96·4%] of 76 293 cases) who self-identify as men who have sex with men (25 938 [86·9%] of 29 854 cases). The most common reported route of transmission was sexual contact (14 941 [68·7%] of 21 749). 3927 (7·3%) of 54 117 cases were hospitalised, with increased odds for those aged younger than 5 years (adjusted odds ratio 2·12 [95% CI 1·32-3·40], p=0·0020), aged 65 years and older (1·54 [1·05-2·25], p=0·026), female cases (1·61 [1·35-1·91], p<0·0001), and for cases who are immunosuppressed either due to being HIV positive and immunosuppressed (2·00 [1·68-2·37], p<0·0001), or other immunocompromising conditions (3·47 [1·84-6·54], p=0·0001)., Interpretation: Continued global surveillance allowed WHO to monitor the epidemic, identify risk factors, and inform the public health response. The outbreak can be attributed to clade IIb MPXV spread by newly described modes of transmission., Funding: WHO Contingency Fund for Emergencies., Translations: For the French and Spanish translations of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests We declare no competing interest., (This is an Open Access article published under the CC BY 3.0 IGO license which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In any use of this article, there should be no suggestion that WHO endorses any specific organisation, products or services. The use of the WHO logo is not permitted. This notice should be preserved along with the article's original URL.)

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas , Chlorosoma and Xenoxybelis . The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas , while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis , respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Abdominal wall defects in calves are commonly diagnosed and treated via laparotomy. This technique has witnessed several advancements in the management of these disorders. This study aimed to create a study model and evaluate the feasibility of video-assisted percutaneous correction of abdominal wall defects in bovine fetuses (corpses) compared with the conventional technique. Sixteen bovine fetuses from pregnant cows slaughtered in slaughterhouses were included in this study. The fetuses were categorized into the control group (CG, n = 8), which was subjected to umbilical abdominorrhaphy via laparotomy, and the video-surgical group (VG, n = 8), which received video-assisted percutaneous sutures with two lateral accesses on the right flank. An abdominal wall defect was created in the VG group to generate a study model, which was corrected using the laparoscopic technique. The procedures were performed in two steps. The first step consisted of creating an abdominal wall defect in the umbilical region by laparoscopic approach in an iatrogenic manner (Step 1: E1). The second stage consisted of conventional abdominorrhaphy of the umbilical region wall defect in the CG group and video-assisted percutaneous suturing of the edges of the iatrogenic abdominal wall defect in the VG group, until reversal of the laparoscopic accesses (Step 2: E2). Step 1 showed no statistically significant difference between the two groups. However, a significant statistical difference (p < 0.0001) was observed between the two groups in step 2. The surgical time of step 2 was longer in the CG group (33.10 ± 0.43 minutes) than that in the VG group (10.13 ± 0.68 minutes, p < 0.0001), and the total surgical time was also longer in the CG group (38.48 ± 0.35 minutes) than that in the VG group (15.86 ± 0.67 minutes). The proposed laparoscopic technique allowed the creation of a study model for video-assisted percutaneous suturing with two portals and reduced the surgical time compared with the conventional technique. However, this method needs to be studied further in live animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Due to the complexity of ruminant digestion, cannulation of organs of the digestive tract has been carried out in order to advance the understanding of digestive physiology, nutrient degradability, gastrointestinal diseases and biotechnological research. The abomasal cannulation is interesting for nutritional studies, especially in suckling calves, to obtain fluid and abomasal content, evaluation of abomasal flow and function, and infusion of nutrients and drugs when it is intended to reach high concentrations in the organ. Conventionally, access and cannulation of digestive organs of ruminants has been performed by laparotomy, a method often criticized and classified as cruel by some sectors related to ethics and animal welfare. The aim of this present study is to describe and standardize a minimally invasive by laparoscopy assisted abomasal cannulation in bovine fetuses (cadavers), which had been previously slaughtered by accident and would be discarded in local slaughterhouses., Results: The abomasal cannulation technique was feasible, simple and did not present major difficulties. The surgical time for cannulation of the abomasum, from the insertion of the trocars to the completion of the technique with fixation of the organ to the abdominal wall, ranged from 9 to 27 min, with an average of 15.5 ± 6.62 min., Conclusions: The Laproscopic assisted abomasal cannulation in bovine fetuses was feasible and safe with minimal tissue injury to the abdominal wall and with short surgical time. More studies in the clinical routine related to minimally invasive abomasal content collection, abomasopexy and abomasotomy are required in order to demonstrate its impact and importance in bovine clinic., (© 2022. The Author(s).)

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Congenital adrenal hyperplasia (CAH), a genetic disease characterized by defective cortisol synthesis and excessive levels of sex hormones, can cause precocious puberty in both sexes in untreated individuals and virilization in female patients with a 46, XX karyotype. The female paraurethral (Skene's) gland has been reported as prostate analogous. Growth of prostate tissue is associated with androgen production; therefore, prostate-specific antigen (PSA) levels may represent a marker of virilization in 46, XX patients with CAH., Objectives: To describe PSA levels in 46, XX patients and evaluate whether higher PSA levels are associated with androgenization and the severity of the disease., Study Design: Sixty-six patients with CAH and a 46, XX karyotype were included, irrespective of age. Serum PSA, testosterone, 17-hydroxyprogesterone (17-OHP) and androstenedione levels were measured. Patients' age, age at diagnosis, forms of the disease, Prader classification, bone age assessment, sex of rearing, surgery, and the presence of clinical complications were obtained from their medical records., Results: The mean age of patients was 11.45 ± 10.74 years. Forty-three patients (65%) were diagnosed neonatally at a median of 0.08 years (mean 1.47 ± 2.34 years), with registers of 17-OHP measurements (Guthrie test) being available in 51%. Testosterone, 17-OHP and androstenedione were significantly high. PSA was detectable in 25% of cases (levels >0.01 ng/ml), with a mean of 0.03 ± 0.09 ng/ml, and only in patients over five years of age. A correlation was found between PSA and age (p < 0.001), age at diagnosis (p = 0.002), testosterone (p = 0.001) and androstenedione (p = 0.023). There was no correlation between PSA and the forms of CAH or Prader classification. A sub-analysis of the patients over five years of age in whom PSA was detectable also showed that there was a correlation between PSA (p < 0.05) and age at analysis, age at diagnosis, testosterone and androstenedione levels., Discussion: Limitations of this study include the small sample size due to the rareness of the disease, its retrospective nature, the absence of a control group, the fact that the sample was selected at two referral centers, which could have resulted in a selection bias, and the use of different reference values in the different laboratories conducting the PSA tests., Conclusions: PSA is detectable in 25% of 46, XX patients with CAH, only after five years of age. PSA level increases significantly with age, age at diagnosis, and testosterone and androstenedione levels, confirming a correlation between PSA levels and elevated androgen levels., Competing Interests: Conflicts of interest None., (Copyright © 2021. Published by Elsevier Ltd.)

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

Surgical intervention for umbilical diseases in calves, when indicated, is a complementary and indispensable therapeutic resource for the treatment of umbilical conditions and is commonly performed using celiotomy. However, laparoscopy has demonstrated feasibility in many diagnostic and therapeutic procedures. The aim of this study was to assess the feasibility of the techniques and the surgical time of laparoscopy and celiotomy used in intra-abdominal resection of the umbilical vein and urachus of bovine fetuses (cadavers). Resection of the umbilical vein and urachus using laparoscopy and celiotomy was performed in 26 anatomical specimens (bovine fetuses obtained from an official slaughterhouse). Resection of umbilical structures was feasible with both techniques, but shorter surgical time and minimal tissue damage were achieved using laparoscopy. Laparoscopy requires specialized training and appropriate instruments and is an important tool for diagnostic and therapeutic exploration of the umbilical structures, liver, bladder, and associated/adjacent structures.

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named snake venom metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an "SVMP-like" function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary noncatalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, whereas the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on nonhomologous genes, yielding alternate biochemical components., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A 2 (PLA 2 s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Ethnopharmacological Relevance: In the region of Western Pará, Amazonia, Brazil, Philodendron megalophyllum is widely used for the treatment of envenomations caused by bites from venomous snakes. The traditional use of plants is usually done through oral administration of an infusion (decoction) soon after the bite occurs. The efficiency of aqueous extracts of P. megalophyllum was demonstrated for blocking the activity of the venom of Bothrops sp., but only for a pre-incubation protocol (venom:extract), which fails to simulate the real form of use of this species. In this context, the objective of this research was to evaluate the anti-snakebite potential of the aqueous extract of P. megalophyllum to inhibit for the biological activity induced by Bothrops atrox venom (BaV) using traditional treatment methods., Material and Methods: Initially, an aqueous extract using the stem of P. megalophyllum (AEPm) was prepared following the standard procedure used by the residents of the rural area along the Tapajós River (Eixo Forte region) in Santarém, PA, Brazil. The phytochemical profile of AEPm was conducted using thin layer chromatography (TLC) and phenolic compounds were quantified through colorimetric trials. The cytotoxicity of AEPm was evaluated using the MRC-5 human fibroblast line, and the antioxidant potential was measured using DPPH methods and cell culture. AEPm antimicrobial action was evaluated by the 96-well plate microdilution and the minimum inhibitory concentration (MIC) methods using 18 types of microorganisms including bacteria that are present in the oral cavity of snakes. AEPm blocking potential was tested against BaV activity in vitro (fibrinolytic) and in vivo (defibrinating and hemorrhagic). In order to test for an interaction between BaV and AEPm SDS-PAGE electrophoresis was conducted., Results: The presence of coumarins, fatty acids, and hydrolysable tannins were detected in the AEPm. The colorimetric trials showed that AEPm had a high concentration of condensed tannins (20.1 ± 1.2%). The potential of AEPm for blocking of hemorrhagic and fibrinolytic activity of BaV showed a maximum reduction of 86.1% and 96.5%, respectively, for the pre-incubation protocol (1:10, venom:extract). However, when the extract was administered orally there was no significant blocking of these activities. The interaction of BaV and AEPm showed a modification of the profile of proteic bands when compared to the pattern of bands obtained from the BaV alone. The AEPm was not considered toxic, demonstrated antioxidant activity, and was capable of reducing the growth of 10 of the 18 studied microorganisms., Conclusion: Although the stem of P. megalophyllum is indicated by traditional medicine techniques as effective against snakebites, the extract, when tested orally was not able to significantly inhibit (p ˃ 0.05) hemorrhage and defibrinating activity induced by the B. atrox venom. On the other hand, the extract yielded a promising result with respect to antioxidant and antimicrobial potential, and after further studies it could be used as a complementary treatment for localized action and secondary infections that frequently occur with snakebites from the genus of Bothrops sp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients ( n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding ( n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding ( n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Bothrops atrox snakebites are a major public health problem in the Amazon region and also cause hemostatic disorders. In this study, we assessed the recovery from hemostatic disorders in Bothrops snakebite patients after being given antivenom therapy. Methods: This is a prospective study of Bothrops snakebite patients ( n  = 100) treated at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado , Manaus, Brazilian Amazon, between January 2016 and December 2017. Blood samples were taken for the measurement of venom concentrations, platelets, clotting time and factors of patients on admission, 12, 24 and 48 h after antivenom therapy, and taken again on discharge. The presence of systemic bleeding was recorded during the follow-up. Results: On admission, systemic bleeding was observed in 14% of the patients. Thrombocytopenia was noted in 10% of the patients. A total of 54% of the patients presented unclottable blood with low levels of fibrinogen and alpha 2-antiplasmin, and high levels of fibrin/fibrinogen degradation product (FDP) and D-dimers. Unclottable blood and systemic bleeding were overcome in most patients 12 h after the antivenom therapy. Three patients developed systemic bleeding 48 h after antivenom therapy. Levels of fibrinogen and alpha 2-antiplasmin, FDP and D-dimer returned to normal around 48 h after the treatment or on discharge. The frequency of thrombocytopenia with high mean platelet volume increased in the first 24 h after antivenom therapy, and decreased on discharge. Bothrops venom levels in patients decreased 12 h after antivenom therapy and were not correlated with coagulation and fibrinolytic parameters. There were no deaths. Conclusion: Laboratorial parameters of coagulopathy returned to normal values within 48 h after the antivenom therapy until discharge. A few patients still presented bleeding signs within 48 h after beginning antivenom therapy. However, the Brazilian antivenom was able to overcome the hemostatic disorders in these cases of envenomation.

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 μg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 10 6 with ATXL and 5 × 10 6 with BATXH. TNF-α was identified in the supernatant of BATXH-or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC-MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4-5 × 10 6 ) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways.

Many venom proteins have presumably been convergently recruited by taxa from diverse venomous lineages. These toxic proteins have characteristics that allow them to remain stable in solution and have a high propensity for toxic effects on prey and/or potential predators. Despite this well-established convergent toxin recruitment, some toxins seem to be lineage specific. To further investigate the toxic proteins found throughout venomous lineages, venom proteomics and venom-gland transcriptomics were performed on two individual red bark centipedes (Scolopocryptops sexspinosus). Combining the protein phenotype with the transcript genotype resulted in the first in-depth venom characterization of S. sexspinosus, including 72 venom components that were identified in both the transcriptome and proteome and 1468 nontoxin transcripts identified in the transcriptome. Ten different toxin families were represented in the venom and venom gland with the majority of the toxins belonging to metalloproteases, CAPS (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1 proteins), and β-pore-forming toxins. Nine of these toxin families shared a similar proteomic structure to venom proteins previously identified from other centipedes. However, the most highly expressed toxin family, the adamalysin-like metalloproteases, has until now only been observed in the venom of snakes. We confirmed adamalysin-like metalloprotease activity by means of in vivo functional assays. The recruitment of an adamalysin-like metalloprotease into centipede venom represents a striking case of convergent evolution., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

Botulism is a potentially fatal intoxication caused by botulinum neurotoxins (BoNTs) produced mainly by Clostridium botulinum . Vaccination against BoNT serotypes C and D is the main procedure to control cattle botulism. Current vaccines contain formaldehyde-inactivated native BoNTs, which have a time-consuming production process and pose safety risks. The development of non-toxic recombinant vaccines has helped to overcome these limitations. This study aims to evaluate the humoral immune response generated by cattle immunized with non-purified recombinant fragments of BoNTs C and D. Cattle were vaccinated in a two-dose scheme with 100, 200 and 400 µg of each antigen, with serum sampling on days 0, 56, 120, and 180 after vaccination. Animals who received either 200 or 400 μg of both antigens induced titers higher than the minimum required by the Brazilian ministry of Agriculture, Livestock and Food Supply and achieved 100% (8/8) seroconversion rate. Animals vaccinated with commercial toxoid vaccine had only a 75% (6/8) seroconversion rate for both toxins. Animals that received doses containing 400 µg of recombinant protein were the only ones to maintain titers above the required level up until day 120 post-vaccination, and to achieve 100% (8/8) seroconversion for both toxins. In conclusion, 400 µg the recombinant Escherichia coli cell lysates supernatant was demonstrated to be an affordable means of producing an effective and safe botulism vaccine for cattle.

Ethnopharmacological Relevance: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined., Materials and Methods: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts., Results: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line., Conclusion: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bothrops atrox is the snake responsible for the majority of snakebites in the Brazilian Amazon. Patients generally evolve to local manifestations such as edema, pain and ecchymoses. Systemic effects of B. atrox venom are usually restricted to blood incoagulability and spontaneous bleeding. However, in a few cases, bleeding in the central nervous system may occur, which can lead to sequels and deaths. Here, we report a case of a 59 year-old woman who presented edema, pain and ecchymoses on the right foot, headache, nausea, diarrhea, hypertension and blood incoagulability after the bite by Bothrops snake in the Brazilian Amazon. This case evolved with stroke resulting in death despite the antivenom and conservative therapy employed. In addition, we were able to identify the presence of venom in the patient's brain tissue after death. Direct action of toxins present in the snake's venom in the induction of systemic hemorrhage allied to blood incoagulability and hypertension presented by the patient could be involved in the mechanism of stroke in this case., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid venoms and hydrolyze BM components. However, hemorrhage is associated mostly with PIII-class SVMPs that contain non-catalytic domains responsible for the binding of SVMPs to BM proteins, facilitating enzyme accumulation in the tissue and enhancing its catalytic efficiency. Here we report on Atroxlysin-Ia, a PI-class SVMP that induces hemorrhagic lesions in levels comparable to those induced by Batroxrhagin (PIII-class), and a unique SVMP effect characterized by the rapid onset of dermonecrotic lesions. Atroxlysin-Ia was purified from B. atrox venom, and sequence analyses indicated that it is devoid of non-catalytic domains and unable to bind to BM proteins as collagen IV and laminin in vitro or in vivo. The presence of Atroxlysin-Ia was diffuse in mice skin, and localized mainly in the epidermis with no co-localization with BM components. Nevertheless, the skin lesions induced by Atroxlysin-Ia were comparable to those induced by Batroxrhagin, with induction of leukocyte infiltrates and hemorrhagic areas soon after toxin injection. Detachment of the epidermis was more intense in skin injected with Atroxlysin-Ia. Comparing the catalytic activity of both toxins, Batroxrhagin was more active in the hydrolysis of a peptide substrate while Atroxlysin-Ia hydrolyzed more efficiently fibrin, laminin, collagen IV and nidogen. Thus, the results suggest that Atroxlysin-Ia bypasses the binding step to BM proteins, essential for hemorrhagic lesions induced by PII- and P-III class SVMPs, causing a significantly fast onset of hemorrhage and dermonecrosis, due to its higher proteolytic capacity on BM components., Competing Interests: The authors declare no conflict of interest.

Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA 2 s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA 2 s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats., Biological Significance: In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Ethnobotanical studies have shown that Plathymenia reticulata Benth. (Fabaceae) has been widely used in cases of snake envenomation, particularly in Northern Brazil. In light of this, the aim of this study was to evaluate the inhibitory potential of the condensed-tannin-rich fraction obtained from the bark of P. reticulata against the main biological activities induced by Bothrops atrox venom (BaV)., Materials and Methods: The chemical composition of the aqueous extract of P. reticulata (AEPr) was first investigated by thin-layer chromatography (TLC) and the extract was then fractionated by column chromatography on Sephadex LH-20. This yielded five main fractions (Pr1, Pr2, Pr3, Pr4 and Pr5), which were analyzed by colorimetry to determine their concentrations of total phenolics, total tannins and condensed tannins and to assess their potential for blocking the phospholipase activity of BaV. The Pr5 fraction was defined as the fraction rich in condensed tannins (CTPr), and its inhibitory potential against the activities of the venom was evaluated. CTPr was evaluated in different in vivo and in vitro experimental protocols. The in vivo protocols consisted of (1) pre-incubation (venom:CTPr, w/w), (2) pre-treatment (orally administered) and (3) post-treatment (orally administered) to evaluate the effect on the hemorrhagic and edematogenic activities of BaV; in the in vitro protocol the effect on phospholipase and coagulant activity using pre-incubation in both tests was evaluated., Results: There was statistically significant inhibition (p<0.05) of hemorrhagic activity by CTPr when the pre-incubation protocol was used [55% (1:5, w/w) and 74% (1:10, w/w)] and when pre-treatment with doses of 50 and 100mg/kg was used (19% and 13%, respectively). However, for the concentrations tested, there was no statistically significant inhibition in the group subjected to post-treatment administered orally. CTPr blocked 100% of phospholipase activity and 63.3% (1:10, w/w) of coagulant activity when it was pre-incubated with BaV. There was a statistically significant reduction (p<0.05) in edema induced by BaV in the oral protocols. Maximum inhibition was 95% (pre-treatment)., Conclusion: Our findings indicate that CTPr could be a good source of natural inhibitors of the components of snake venom responsible for inducing local inflammation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Telehealth strategies have the potential to improve diabetes care, but there is a lack of evidence about the impact of these strategies in developing countries. Our objective was to analyze the feasibility, usability, and clinical impact of a decision support system (DSS) in Brazilian primary care diabetes patients., Materials and Methods: This was a quasi-experimental study that included type 2 diabetes primary care patients >40 years of age. Patients were assessed before (during 6 months) and after the implementation of the DSS application (4 months). The DSS application, used by health professionals, included clinical evaluations and blood glucose measurements and generated specific recommendations based on the data entered., Results: In total, 145 patients were included (mean age, 62.0 ± 9.9 years), 62.1% were female, and 70.0% had been diagnosed with diabetes more than 5 years ago. Overall, there was no decrease in median hemoglobin A1c (HbA1c), from 7.7% (range, 6.5-9.8%) to 7.4% (range, 6.5-9.2%) (P for slope = 0.347). Subgroup analysis showed that patients with an HbA1c level of ≥9% at baseline had a significant reduction in median HbA1c level, from 10.5% (range, 9.9-11.3%) to 10.0% (range, 8.9-10.9%) (P for difference of slope between subgroups = 0.004). The reduction occurred in the first phase of the study, before the DSS use. Healthcare practitioners considered the DSS easy to use (99%) and believed that it provided useful information for patient care (100%)., Conclusions: In this study the improvement of glycemic control before the application in more decompensated patients (HbA1c ≥9%) probably reflects the systematization of diabetes care. The DSS use did not improve the HbA1c level, possibly because of the short follow-up and/or infrequent use by the healthcare practitioners.

Comparisons between venoms from snakes kept under captivity or collected at the natural environment are of fundamental importance in order to obtain effective antivenoms to treat human victims of snakebites. In this study, we compared composition and biological activities of Bothrops atrox venom from snakes collected at Tapajós National Forest (Pará State, Brazil) or maintained for more than 10 years under captivity at Instituto Butantan herpetarium after have been collected mostly at Maranhão State, Brazil. Venoms from captive or wild snakes were similar except for small quantitative differences detected in peaks correspondent to phospholipases A2 (PLA2), snake venom metalloproteinases (SVMP) class PI and serine proteinases (SVSP), which did not correlate with fibrinolytic and coagulant activities (induced by PI-SVMPs and SVSPs). In both pools, the major toxic component corresponded to PIII-SVMPs, which were isolated and characterized. The characterization by mass spectrometry of both samples identified peptides that matched with a single PIII-SVMP cDNA characterized by transcriptomics, named Batroxrhagin. Sequence alignments show a strong similarity between Batroxrhagin and Jararhagin (96%). Batroxrhagin samples isolated from venoms of wild or captive snakes were not pro-coagulant, but inhibited collagen-induced platelet-aggregation, and induced hemorrhage and fibrin lysis with similar doses. Results suggest that in spite of environmental differences, venom variability was detected only among the less abundant components. In opposition, the most abundant toxin, which is a PIII-SVMP related to the key effects of the venom, is structurally conserved in the venoms. This observation is relevant for explaining the efficacy of antivenoms produced with venoms from captive snakes in human accidents inflicted at distinct natural environments., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Ethnopharmacological Relevance: The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities., Materials and Methods: Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom., Results: In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage., Conclusions: Traditional use of plants to treat snakebites is a common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

This study aimed to analyze ovary histology and quantify total protein in the hemolymph of Rhipicephalus (Boophilus) microplus females treated with hexane extracts from green fruits of Melia azedarach. Eight engorged females were immersed in the extract at 0.25% concentration, and eight in water containing 5% acetone (control). The females were dissected 72 hours after treatment, and the ovaries were weighed and subjected to standard histological techniques. The total protein concentration was measured in the hemolymph of 200 females, of which 100 were treated as described above and 100 served as a control. In the treated group, ovary weight reduction and predominance of immature oocytes were observed. In addition, there were decreases in the diameters of the cytoplasm and germ vesicle of the oocytes in the treated group, compared with the controls. The protein concentration in the hemolymph was higher in the treated group than in the controls. The morphological changes observed in the treated ovaries included: presence of vacuolization; alteration of oocyte morphology, which changed from rounded to elongated; deformation of the chorion; and disorganization of the yolk granules. These results demonstrate the action of M. azedarach fruit extracts on R. (B.) microplus oogenesis.

Clays are materials commonly used in the pharmaceutical industry, either as ingredients or as active ingredients. It was observed that when they are administered concurrently, they may interact with drugs reducing their absorption. Therefore, such interactions can be used to achieve technological and biopharmaceutical advantages, regarding the control of release. This review summarizes bibliographic (articles) and technological (patents) information on the use of systems containing clays and clay minerals in modified drug delivery. In this area, formulations such natural clay, commercial clay, synthetic clay, composites clay-polymers, nanocomposites clay-polymers, films and hidrogels composites clay-polymers are used to slow/extend or vectorize the release of drugs and consequently they increase their bioavailability. Finally, this review summarizes the fields of technology and biopharmaceutical applications, where clays are applied., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.

The objective of this study was to determine the prevalence of anemia and vitamin A deficiency and to analyze the association of food insecurity with hemoglobin levels and serum retinol in children under 5 years of age. This was a cross-sectional study with 501 families from Gameleira and 458 families from São João do Tigre. Food insecurity was assessed according to the Brazilian Food Insecurity Scale. Hemoglobin and serum retinol levels were classified according to the WHO. Socioeconomic and environmental conditions and biological indicators of children were analyzed as hemoglobin and serum retinol determinants. The prevalence of anemia and vitamin A was higher in Gameleira compared to São João do Tigre. Moderate and severe food insecurity of families was 75% in Gameleira and 64% in São João do Tigre. Maternal education, per capita family income, not having a color TV, untreated water supply, type of flooring (dirt), and child age (< 2 years) in Gameleira were associated with hemoglobin levels in the linear regression analysis. In São João do Tigre the association with hemoglobin levels was only for (no) motorcycle and child age. Variables per capita family income and sex in Gameleira and no mobile phone and lack of sanitation in São João do Tigre were statistically associated with serum retinol levels. The nutritional status of these populations is similar in terms of food insecurity but may present great differences in terms of the prevalence of specific nutritional deficits and their determinants.

Behavioral, electrophysiological, and anatomical assays of non-human primates have provided substantial evidence that the hippocampus and dentate gyrus are essential for memory consolidation. However, a single anatomical and stereological investigation of these regions has been done in New World primates to complement those assays. The aim of the present study was to describe the cyto-, myelo-, and histochemical architecture of the hippocampus and dentate gyrus, and to use the optical fractionator method to estimate the number of neurons in the hippocampal pyramidal and granular neurons in the dentate gyrus of the Cebus monkey. NeuN immunolabeling, lectin histochemical staining with Wisteria floribunda agglutinin (WFA), enzyme-histochemical detection of NADPH-diaphorase activity and Gallyas silver staining were used to define the layers and limits of the hippocampal fields and dentate gyrus. A comparative analysis of capuchin (Cebus apella) and Rhesus (Macaca mulatta) monkeys revealed similar structural organization of these regions but significant differences in the regional distribution of neurons. C. apella were found to have 1.3 times fewer pyramidal and 3.5 times fewer granular neurons than M. mulatta. Taken together the architectonic and stereological data of the present study suggest that hippocampal and dentate gyrus neural networks in the C. apella and M. mulatta may contribute to hippocampal-dentate gyrus-dependent tasks in different proportions., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

This study was developed to evaluate the repellent activity of N,N-diethyl-3-methylbenzamide (DEET) against Amblyomma cajennense nymphs. Two repellent bioassays were compared and the effective concentration and repellent time were calculated. The fingertip test was accomplished to evaluate in vivo four concentrations of the compound (0.200; 0.100; 0.050 and 0.025 mg.cm⁻²) and the filter-paper bioassay to evaluate in vitro the two highest concentrations.The compound provided repellence higher than 90% in all concentrations and at least 95% repellency in the highest concentration over 5 hours. The effective concentration against 50% of tested nymphs (EC50) was 0.006 mg.cm⁻² and the EC99 was 0.036 mg.cm⁻². Those concentrations were lower than the ones obtained against other tick species, denoting the effectiveness of DEET against A. cajennense. The repellency time against 50% of the ticks (RT50) was 4.8 hours and the RT90 was 2.7 hours. Both bioassays were adequate to evaluate A. cajennense repellency and provided similar results; however the in vivo test is more appropriate to estimate the effective concentration and repellency time.

Purpose: The 6-minute walk test (6MWT) is a simple and reliable method routinely used in the evaluation of many conditions including cardiac diseases. However, it is rarely performed in patients with pacemakers (PMs), and the applicability and safety in this group is not established. The purpose of this study was to verify the applicability and safety of the 6MWT in the evaluation of functional capacity of patients with PMs, considering the ergometry test (ET) as the criterion standard., Methods: Twenty-four patients with rate-responsive PMs underwent a treadmill ET, using the Chronotropic Assessment Exercise Protocol, and a 6MWT, performed twice with a rest interval of 20 minutes. Functional capacity, heart rate, and blood pressure in each test were recorded., Results: The distance covered during the 6MWT significantly correlated with the oxygen uptake estimated from the treadmill ET (r = 0.709, P < .001). Peak heart rates and systolic blood pressure levels reached during the 2 tests were also significantly correlated. No adverse events or malignant arrhythmias were observed during the 6MWT. The distance walked during the 6MWT accurately predicted the patient performance on the ET (area under the receiver operating characteristic curve = 0.921). The ability to walk more than 538 m in the 6MWT predicted, with a sensitivity of 90% and a specificity of 79%, an estimated oxygen uptake above 10 metabolic equivalents (METs) on the ET., Conclusions: The 6MWT can be considered safe to assess the submaximal functional capacity and can be used as an alternative test to evaluate the results of interventions in patients with permanent cardiac PMs.

Hexanic extracts gotten of the fruits of cinamomo (Melia azedarach) were tested on engorged females and larvae of Rhipicephalus (Boophilus) microplus. Four extracts were produced: one of unripe fruits and three of ripe fruits, being one with the fruits immediately processed, another one with fruits stored for five months and one tested after storage of the extract for 2 (two) years at 4 degrees C. The tests were made in triplicate, in 0.25% - 0.0156% dilutions. The effectiveness of extracts was evaluated through the comparison of the Reproduction Estimate of the groups "controlled and treated" for females, of mortality for the larvae, and of the calculation of the DL50 and the relative power for larvae and females. The extract of unripe fruits presented the effectiveness varying the 3.6% - 100% for females, and of ripe fruits it varied of 5.2% - 99.7%. The two extracts caused a mortality of 100% of the larvae in the highest concentrations. The extract of stored fruits presented effectiveness of 14% - 83% on females, while the extract stored in refrigerator presented effectiveness of 8.4% 100% and both did not present larvicidal effect. The results demonstrate a superiority of the unripe extract, with a minor DL50 and superior power 1.497 times to the extract of ripe fruits.

Objective: To translate, to make the cultural adaptation and to evaluate reproducibility and validity of the Portuguese version of the AQUAREL (Assessment of QUAlity of life and RELated events) questionnaire, which is a specific tool to assess quality of life in pacemaker patients., Methods: We evaluated 202 pacemaker patients: 63 patients during the cross-cultural adaptation stage and 139 during the reproducibility and validity evaluation stages. The questionnaire translation was reviewed repeatedly until > or = 85% of patients correctly understood the questions. Reproducibility of the final version was tested in 69 patients in whom the interview was performed twice by the same researcher. Validity was checked by the correlation between scores obtained in AQUAREL domains and those obtained in SF36 domains, in the functional class and the distance walked in the six-minute test., Results: The internal consistency of AQUAREL was adequate, with Cronbachs alpha coefficient varying between 0.59 and 0.85. Reproducibility was good, with high correlation coefficients (0.68-0.89) and random distribution of data in Bland and Altman plots, without systematic bias. A significant association was observed among AQUAREL domains and those obtained in SF36 domains and the functional class (p<0.01), although significant correlations with the distance walked in the six-minute test were not found., Conclusion: The Portuguese version of the AQUAREL questionnaire is easy and rapid to apply, and could be used as a specific questionnaire to assess quality of life in pacemaker patients.

Purpose: To investigate the effects of percutaneous transluminal angioplasty on the vasa vasorum in dogs with experimentally created abdominal aortic stenoses., Materials and Methods: Two stenoses were created in the abdominal aorta in each of 21 dogs. After 6 weeks, the more cephalic stenosis was dilated; the other stenosis served as an untreated control. Groups of three dogs were killed at 24 hours, 3 and 6 weeks, and 4, 8, 12, and 18 months after angioplasty. The aortae were studied by means of histologic examination, microangiography, scanning electron microscopy, and the Spalteholz technique., Results: At nondilated stenoses, the vasa vasorum was interrupted in the outer adventitia and unchanged in the outer media. At dilated stenoses, the number of precapillary arterioles in the outer media progressively increased up to 8 months; thereafter, the number of precapillary arterioles began to decrease. At 18 months, the number was normal., Conclusion: Angioplasty brings about changes in the number of precapillary arterioles in the outer media of the aorta in our canine model of focal abdominal aortic stenosis.

Purpose: Experimental stenoses were created in canine aortae to compare the effects of stent placement and balloon angioplasty on the vasa vasorum., Materials and Methods: A balloon-expandable Palmaz stent was placed in the proximal stenosis, and angioplasty was performed in the distal stenosis in each of eight dogs. Two dogs were killed at 4, 8, 12, and 18 months, respectively. Specimens were studied by means of microangiography, histology, scanning electronic microscopy, and Spalteholz technique., Results: At up to 12 months, there was proliferation of the vasa vasorum and a rich plexus formed in each of the animals at each treated site. More vasa vasorum were seen after stent placement than after angioplasty. The new vessels were mainly venules located in the media. At 18 months, there was regression of the venules and the vasa vasorum distribution returned to baseline., Conclusion: The authors conclude that the effects of stents on the vasa vasorum are temporary.