Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines

From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg2+ and Ca2+ enhanced its enzymatic activity, whereas Zn2+ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM–cancer-cell interactions.</p

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Comparison of four energy-based vascular sealing and cutting instruments: a porcine model. Surg Endosc

Abstract Aim To compare the safety and efficacy of four energybased vascular sealing and cutting instruments. Methods Blood vessels of various types and diameters were harvested from four pigs using four instruments: Harmonic ACE TM (Ethicon Endo-Surgery, Cincinnati, OH), LigaSure TM V and LigaSure Atlas TM (Valleylab, Inc., Boulder, CO; a division of Tyco Healthcare), and EnSeal TM vessel fusion system (SurgRx, Inc. Redwood City, CA). The diameters of the vessels, speed and adequacy of the cutting and sealing process, and bursting pressures were compared. An additional set of specimens was sealed and left in situ for up to 4 h after which the vessels were harvested and histopathologically analyzed for the degree of thermal injury. Results The bursting pressures were significantly higher with EnSeal TM compared to all other instruments (p \ 0.0001). The sealing process was significantly shorter with Harmonic ACE TM and significantly longer with LigaSure Atlas TM (p \0.0001). The mean seal width was larger with the LigaSure Atlas TM compared to the other instruments, and it was smaller with EnSeal TM and Harmonic ACE TM . Less radial adventitial collagen denaturation was present with EnSeal TM and LigaSure TM V than with the other two instruments; there were no significant differences in collagen denaturation although proximal thermal injury to the smooth muscle in the media of the vessel wall was less common with LigaSure Atlas TM than with the other instruments; however, the numbers were too small for statistical analysis. Conclusions The bursting pressures with EnSeal TM were significantly higher than with all the other instruments. Harmonic ACE TM was the fastest sealing instrument and LigaSure Atlas TM was slowest. EnSeal TM created less radial thermal damage to the adventitial collagen of the vessels and LigaSure Atlas TM created less thermal damage to the media of the vessels. The clinical significance of these findings is unknown. Keywords Vascular sealing Á Energy-based Á Bursting pressure Á Thermal injury Á Porcine model Recent advances in surgical technology include the use of various energy sources for sealing, coagulating, and cutting blood vessels as opposed to performing these procedures mechanically by tying, suturing, and even clipping or stapling them. The use of energy-based instruments has become even more popular in laparoscopic surgery because the traditional techniques of surgical hemostasis (pressure, tying, suturing) are not as easily laparoscopically applied. The efficacy and reliability of various energy-based vascular sealing instruments have been reported to be equivalent to the results with metallic clips and silk tie

findMySequence: a neural-network-based approach for identification of unknown proteins in X-ray crystallography and cryo-EM

Although experimental protein-structure determination usually targets known proteins, chains of unknown sequence are often encountered. They can be purified from natural sources, appear as an unexpected fragment of a well characterized protein or appear as a contaminant. Regardless of the source of the problem, the unknown protein always requires characterization. Here, an automated pipeline is presented for the identification of protein sequences from cryo-EM reconstructions and crystallographic data. The method's application to characterize the crystal structure of an unknown protein purified from a snake venom is presented. It is also shown that the approach can be successfully applied to the identification of protein sequences and validation of sequence assignments in cryo-EM protein structures.</jats:p

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergon) Induces Glycoprotein VI Shedding and Impairs Platelet Function

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the alpha-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and alpha 2 beta 1 integrin. Neither the alpha 2 beta 1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble similar to 55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox

Fibrinogen-clotting enzyme, pictobin, fromBothrops pictus snake venom. Structural and functional characterization

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friableiporous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor alpha 2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondria! network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold beta/beta hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.National Council for Scientific and Technological Development (CNPq) 490269/2013-3 Minas Gerais State Research Foundation (FAPEMIG) APQ-01858-15 AUC00022-16 Programa de Pos Graduacao in Toxinology, Instituto Butantan, SP Programa Nacional de Innovación para la Competitividad y Productividad - Innovate Peru 131-FINCyT-2013 Vicerrectorado de Investigación y Posgrado -Universidad Nacional Mayor de San Marcos, Peru B19101621 B17101271 FONDECYT-Chile postdoctoral fellowship 3170813 CONICYT-Chile PCI-Biotechnology Redbio0027 Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) CONICYT FONDECYT 1181823 ICM P09-015-F EQM140038 EQM140156 German Research Foundation (DFG) Eb177/13-