Caracterização eletrofisiológica dos peptídeos To4 E To5, duas β-toxinas presentes na peçonha do escorpião Tityus obscurus

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2017.Toxinas de escorpiões têm sido alvo de pesquisa dada suas propriedades farmacológicas. Estas toxinas agem sobre quatro tipos de canais iônicos (Na+ , K+ , Ca++ e Cl- ). As que agem sobre os canais iônicos para sódio (NaScTx) são divididas em dois grupos α- ou β-toxinas segundo seu alvo molecular dentro desses canais iônicos. As primeiras interferem na cinética de inativação e as do segundo grupo afetam a cinética de ativação do canal iônico alvo. Muitas destas toxinas já foram identificadas e testadas sobre diferentes alvos farmacológicos. A maioria é proveniente de escorpiões da família Buthidae, conhecidos como os escorpiões de relevância médica devido à capacidade toxica de sua peçonha. No entanto, ainda falta elucidar sobre toxinas das peçonhas de espécies potencialmente perigosas à saúde humana. A pesquisa de toxinas está sendo promissora para diversos estudos e desenvolvimento biotecnológico. Dado que a maioria age sobre canais iônicos, elas se tornam ferramentas para estudar interações ou mecanismos moleculares de ação desses alvos. Portanto, as toxinas podem favorecer direta ou indiretamente no desenvolvimento de estratégias para tratar, por exemplo, doenças como as canalopatias. Tityusobscurus é um escorpião que ocorre no norte do Brasil, onde acontecem acidentes por sua picada. Os componentes de sua peçonha estão sendo pesquisados e os alvos farmacológicos das NaScTxs descritas ainda não foram identificados. Neste trabalho, pela primeira vez, duas NaScTxs (To4 e To5) foram purificadas da peçonha de T. obscurus por cromatografia RP-HPLC, sequênciadas por in-sourcedecay ISD e por meio da biblioteca de cDNA previamente descrita. A validação do alvo molecular especifico destas toxinas foi realizada testando sete subtipos de canais iônicos humanos hNav1.1 – hNav1.7 usando a técnica eletrofisiológica de patch clamp. Adicionalmente, tentou-se expressar a toxina To5 heterólogamente em Escherichia coli, visando comparar a atividade eletrofisiológica desta com a nativa. Nossos resultados mostraram que tanto a To4, quanto a To5 são β-toxinas que afetam a ativação e correntes de todos os canais iônicos testados. Estas toxinas não mostraram algum efeito tipo α, assim, descarta-se a possibilidade de estas agirem sobre outro sitio destes canais testados. Futuros testes eletrofisiológicos poderão ser feitos sobre canais iônicos próprios de inseto, já que estas duas toxinas potencialmente agem nos dois grupos taxonômicos. Dada sua capacidade promiscua sobre os hNavs testados, as toxinas To4 e To5 mostram ser componentes virtualmente tóxicos da peçonha deste escorpião. Estes e outros componentes dessa peçonha tornam do T. obscurus, um escorpião potencialmente perigoso à saúde humana.Scorpion toxins have been research targets due its pharmacological properties. They can target four ion-channels types (Na+ , K+ , Ca++ e Cl- ). Such toxins active on sodium channels (NaScTx) have two interaction points on them, thus being classified as α- or β-toxins. The first group interferes with the kinetic inactivation, and the second group affects the kinetic activation from sodium channels (Navs). Several toxins were identified and tested on pharmacological targets. Most of these are from Buthidae scorpion family, a taxonomic group considered as of medical relevance. It is necessary to elucidate toxins from potentially dangerous scorpion venom species yet not described. The toxins study is being promissory to diverse research and biotechnological development. Due that the most of them target on ion channels, they would become tools to ion channels interaction studies or action molecular mechanisms analysis of them. Thus, toxins could promote direct or indirectly the strategies development to treat, for example, diseases as channelopathies. Tityus obscurus scorpion from north of Brazil is cause of accidents for sting. Its venom components are being investigated, among them, the described NaScTxs do not have the target Navdefined. By first time, in this work, two T. obscurusNaScTxs (To4 e To5) were purified from venom by RP-HPLC chromatography, sequenced by in-source decay ISD and by cDNA library previously described. The specific molecular target validation was testing the human Navs (hNav1.1 – hNav1.7) by electrophysiological technique patch clamp. Additionally, To5 toxin was expressed in Escherichia coli to perform futures electrophysiological tests. Our results showed than To4 and To5 are β-toxins affecting the both activation and ionic currents from all hNavs isoforms tested. These toxins did not show α- activity discarding other possible target site on these hNavs. Electrophysiological tests on insect Navs are recommended because these toxins are potentially active on them. The promiscuous capacity of the To4 and To5 toxins on hNavs make of them compounds potentially toxic to humans. The T. obscurus venom would be potentially dangerous to human health

Caracterização de novos peptídeos bloqueadores de canais para K+ isolados da peçonha do escorpião Tityus sp.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2013.As peçonhas escorpiônicas têm sido alvo de pesquisas por possuírem componentes com diversas atividades biológicas, as quais são responsáveis pelas alterações fisiológicas observadas no escorpionismo. Dentre esses componentes, estão as toxinas que agem em canais para potássio (KTxs), as quais têm sido caracterizadas e divididas em subfamílias segundo a sua estrutura primária. O objetivo desta pesquisa foi procurar novas KTxs na peçonha do escorpião colombiano Tityus sp. utilizando duas estratégias: a transcritômica e a proteômica. Uma biblioteca de cDNA foi construída, a partir da glândula de peçonha do escorpião e da sua peçonha foram isoladas e caracterizadas KTxs por cromatografia líquida de alta eficiência (HPLC), espectrometria de massa do tipo MALDI-TOF/TOF e tipo ESI. Os peptídeos isolados foram testados por meio da técnica eletrofisiológica de patch clamp (whole cell). A partir da biblioteca de cDNA, foram caracterizadas cinco sequências precursoras que codificam prováveis KTx: três delas são pertencentes à subfamília α-KTx15, uma à subfamília α-KTx12 e uma à subfamília α-KTx18. Da peçonha, foram sequenciadas outras quatro KTxs com massas moleculares de 2430, 3590, 3640 e 4171 Da. Estas KTxs foram + capazes de diminuir a corrente de K em células do gânglio dorsal de ratos. A caracterização destas novas KTxs reflete o amplo espectro de expressão deste tipo de toxinas e as variadas estratégias moleculares utilizadas pelos escorpiões para produção de componentes neuroativos. ______________________________________________________________________________ ABSTRACTThe venom scorpions have been object of investigations by its different activities biological from components, which are responsible by the physiologic alterations observed in the scorpionism. Within these components, the toxins that target potassium channels (KTxs) are present, which have been characterized and divided according primary structure. The aim was search new KTxs in the Colombian scorpion Tityus sp. venom using two strategies: the transcriptomic and proteomic. The scorpion venom gland a cDNA library was constructed, and from venom, KTxs were characterized using high performance liquid chromatographic (HPLC), mass spectrometry MALDI-TOF/TOF and ESI type technical. The isolated peptides were tested with patch clamp (whole cell) electrophysiological technique. Four precursors likely KTxs were characterized: three α-KTx15, one α-KTx12 and one α-KTx18 like subfamilies. Additionally, four KTxs-like were sequenced from scorpion venom with molecular masses 2430, 3590, 3640 and 4171 Da. These slightly decreased levels potassium current on dorsal root ganglion. The characterization these new KTxs reveals a wide spectrum of KTxs-type toxins and one varied molecular strategy for neuroactive compounds production also

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

Structural and Functional Analyses of Cone Snail Toxins

Cone snails are marine gastropod mollusks with one of the most powerful venoms in nature. The toxins, named conotoxins, must act quickly on the cone snails´ prey due to the fact that snails are extremely slow, reducing their hunting capability. Therefore, the characteristics of conotoxins have become the object of investigation, and as a result medicines have been developed or are in the trialing process. Conotoxins interact with transmembrane proteins, showing specificity and potency. They target ion channels and ionotropic receptors with greater regularity, and when interaction occurs, there is immediate physiological decompensation. In this review we aimed to evaluate the structural features of conotoxins and the relationship with their target types