Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs

<p>Abstract</p> <p>Background</p> <p>Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications.</p> <p>Results</p> <p>Thirty-one SVMP cDNAs were full length cloned from a single specimen of <it>Bothrops neuwiedi </it>snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described.</p> <p>Conclusions</p> <p>In <it>B. neuwiedi </it>venom gland, class P-II SVMPs were represented by three different types of transcripts that may have arisen by interclass recombination with P-I and P-III sequences after the divergence of the different classes of SVMPs. Our observations indicate that exon shuffling or post-transcriptional mechanisms may be driving these recombinations generating new functional possibilities for this complex group of snake toxins.</p

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

The Primary Duct of Bothrops jararaca Glandular Apparatus Secretes Toxins

Submitted by Sandra Infurna ([email protected]) on 2018-09-27T15:39:15Z No. of bitstreams: 1 joseantonio_portesjr_etal_IOC_2018.pdf: 6165008 bytes, checksum: b14c5e58fb2db4ccaae9c736e6652f8f (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-09-27T15:47:59Z (GMT) No. of bitstreams: 1 joseantonio_portesjr_etal_IOC_2018.pdf: 6165008 bytes, checksum: b14c5e58fb2db4ccaae9c736e6652f8f (MD5)Made available in DSpace on 2018-09-27T15:47:59Z (GMT). No. of bitstreams: 1 joseantonio_portesjr_etal_IOC_2018.pdf: 6165008 bytes, checksum: b14c5e58fb2db4ccaae9c736e6652f8f (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil.Instituto Butantan. Laboratório de Farmacologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Farmacologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Biologia Celular. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil.Instituto Butantan. Laboratório de Farmacologia. São Paulo, SP, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil.Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland

Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

Made available in DSpace on 2015-09-21T17:25:46Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) carolina_nicolau_etal_IOC_2013.pdf: 1668584 bytes, checksum: 6ae4f3b359524876188b347a8f43cd15 (MD5) Previous issue date: 2013Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo CruzI. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.Universidade São Paulo. Instituto de Matemática e Estatística. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Instituto Butantan. Laboratório de Fisiopatologia. São Paulo, SP, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Universidade Federal do Oeste do Pará. Santarém, Pará, Brasil.Instituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Instituto Butantan. Laboratório de Fisiopatologia. São Paulo, SP, Brasil.Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil / Faculdades Integradas do Tapajó. Santarém, Pará, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo CruzI. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.nstituto Butantan. Laboratório de Imunopatologia. São Paulo, SP, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq). Brasil.In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotro´pico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted

Principal Component Analysis relative to toxin composition.

<p>Loading (top) and score (bottom) plots of the principal components 1 and 2 of the venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) according to their protein composition including as variables the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A), or the normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The Principal Component Analysis was based on the covariance matrix and all calculations were carried out in the software Minitab 16.</p

Comparison of the elution profiles of venoms from snakes classified in different genera.

<p>Samples containing 5 mg of crude lyophilized venom from <i>Bothrops atrox</i>, <i>Bothrops jararacussu</i>, <i>Bothropoides jararaca</i>, <i>Bothropoides neuwiedi</i>, <i>Rhinocerophis alternatus</i> and <i>Rhinocerophis cotiara</i>, species maintained at Instituto Butantan herpetarium, were applied to a Vydac C-18 column (4.6×250 mm, 10-µm particle size) coupled to an Agilent 1100 HPLC system. The fractions were eluted at 1 mL/min, with a gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, 45–70% B over 20 min and 70–100% B over 10 min). The separations were monitored at 214 nm.</p

Venom clustering according to toxin composition.

<p>The venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) were classified according to their protein composition by hierarchical clustering of the observations, including as a variable the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A) or normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The procedure used an agglomerative hierarchical method linked by the minimum Euclidean distance between an item in one cluster and an item in another cluster (nearest neighbor) using the Minitab 16 software.</p

Comparison of electrophoretic profile (A) and <i>Bothrops</i> antivenom antigenic reactivity (B) of venoms from snakes classified in different genera.

<p>Samples containing 10 µg <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i>(JSU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) venoms were fractionated by SDS-PAGE (12.5% acrylamide gels) under non-reducing conditions and were either stained with Coomassie blue (<b>A</b>) or transferred to nitrocellulose membranes, which were then incubated with SAB (1∶1,000) as the primary antibody and peroxidase-labeled goat anti-horse IgG (1∶1,000). The reactive bands were detected by incubation with 4-chloro-α-naphthol and H₂O₂ (<b>B</b>). The numbers at the left indicate the mobility of the molecular mass markers in kDa. These results represent three independent runs.</p

The relationship between clinics and the venom of the causative Amazon pit viper (Bothrops atrox).

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

ELISA reactivity with <i>Bothrops</i> antivenom and MAJar-3 monoclonal antibody of fractions collected from chromatograms of venom from snakes classified in different genera.

<p>Samples containing 100 µL 1-µg/mL fractions collected at the elution times represented in the chromatograms were used to coat maxisorb microplates (Nunc), which were incubated with SAB (1∶1,000) or a monoclonal antibody against jararhagin (class P-III SVMP) MAJar-3 (1∶50), followed by incubation with anti-horse IgG (1∶2,000) or anti-mouse IgG (1∶1,000) labeled with peroxidase. The reactions were developed with ortho-phenylenediamine/H₂O₂ as the enzyme substrate, and the products were detected at 490 nm. The ELISA reactivity was calculated as % reactivity, taking as 100% the maximal OD value obtained in each of three independent experiments performed in duplicate.</p