Molecular approaches for structural characterization of a new potassium channel blocker from Tityus stigmurus venom: cDNA cloning, homology modeling, dynamic simulations and docking

AbstractPotassium channels are involved in the maintenance of resting membrane potential, control of cardiac and neuronal excitability, neurotransmitters release, muscle contractility and hormone secretion. The Tityus stigmurus scorpion is widely distributed in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hypoesthesia, edema, erythema, paresthesia, headaches and vomiting. Most potassium channel blocking peptides that have been purified from scorpion venoms contain 30–40 amino acids with three or four disulfide bridges. These peptides belong to α-KTx subfamily. On the other hand, the β-KTx subfamily is poorly characterized, though it is very representative in some scorpion venoms. A transcriptomic approach of T. stigmurus scorpions developed by our group revealed the repertoire of possible molecules present in the venom, including many toxins of the β-KTx subfamily. One of the ESTs found, named TSTI0003C has a cDNA sequence of 538bp codifying a mature protein with 47 amino acid residues, corresponding to 5299Da. This β-KTx peptide is a new member of the BmTXKβ-related toxins, and was here named TstKMK. The three-dimensional structure of this potassium channel toxin of the T. stigmurus scorpion was obtained by computational modeling and refined by molecular dynamic simulations. Furthermore, we have made docking simulations using a Shaker kV-1.2 potassium channel from rats as receptor model and proposed which amino acid residues and interactions could be involved in its blockade

Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin

AbstractA new antimicrobial peptide, herein named Stigmurin, was selected based on a transcriptomic analysis of the Brazilian yellow scorpion Tityus stigmurus venom gland, an underexplored source for toxic peptides with possible biotechnological applications. Stigmurin was investigated in silico, by circular dichroism (CD) spectroscopy, and in vitro. The CD spectra suggested that this peptide interacts with membranes, changing its conformation in the presence of an amphipathic environment, with predominance of random coil and beta-sheet structures. Stigmurin exhibited antibacterial and antifungal activity, with minimal inhibitory concentrations ranging from 8.7 to 69.5μM. It was also showed that Stigmurin is toxic against SiHa and Vero E6 cell lines. The results suggest that Stigmurin can be considered a potential anti-infective drug

Insights of antiparasitic activity of sodium diethyldithiocarbamate against different strains of Trypanosoma cruzi

Funding Information: This research was funded by Global Health and Tropical Medicine: Grant number IHMT-UID/multi/04413/2013 and Grant number PTDC/CVT-CVT/28908/2017, FCT-Portugal. Funding Information: We would like to thank Prof. João Aristeu da Rosa and Dr. Aline Rimodi Rimeiro at UNESP (Universidade Estadual de São Paulo) Araraquara (Brazil) for offering four different strains of T. cruzi. JWFO, CJGM, and BAC thanks to the financial support (PhD and Post-doctoral fellowships) provided by Capes/Brazil; MSS and HAOR thanks to CNPq/Brazil for the Research Grant (Bolsa de Produtividade). We also would like to thank the Department of Materials Engineering at UFRN for allowing the use of their scanning electron microscope, and the Department of Biochemistry at UFRN for allowing the use of their Flow Cytometer. We are also grateful to Paulo Fanado for editing this manuscript. Publisher Copyright: © 2021, The Author(s).Chagas disease is caused by Trypanosoma cruzi and affects thousands of people. Drugs currently used in therapy are toxic and have therapeutic limitations. In addition, the genetic diversity of T. cruzi represents an important variable and challenge in treatment. Sodium diethyldithiocarbamate (DETC) is a compound with pharmacological versatility acting as metal chelators and ROS generation. Thus, the objective was to characterize the antiparasitic action of DETC against different strains and forms of T. cruzi and their mechanism. The different strains of T. cruzi were grown in LIT medium. To evaluate the antiparasitic activity of DETC, epimastigote and trypomastigote forms of T. cruzi were used by resazurin reduction methods and by counting. Different response patterns were obtained between the strains and an IC50 of DETC ranging from 9.44 ± 3,181 to 60.49 ± 7.62 µM. Cell cytotoxicity against 3T3 and RAW cell lines and evaluated by MTT, demonstrated that DETC in high concentration (2222.00 µM) presents low toxicity. Yet, DETC causes mitochondrial damage in T. cruzi, as well as disruption in parasite membrane. DETC has antiparasitic activity against different genotypes and forms of T. cruzi, therefore, representing a promising molecule as a drug for the treatment of Chagas disease.publishersversionpublishe

Erythrina velutina Willd. alkaloids : piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets formodulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. The scarce studies regarding non-model plants impair advances in this field. Erythrina spp. are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids, which can act on several pathology-related biological targets. Objective: Herein the purpose is to employ combined transcriptome and metabolome analyses (seeds and leaves) of a non-model medicinal Fabaceae species grown in its unique arid natural habitat. The study tries to propose a putative biosynthetic pathway for the bioactive alkaloids by using an omic integrated approach. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a Illumina NextSeq 500 platform. Regarding the targeted metabolite profiling, Nuclear Magnetic Resonance and the High-Performance Liquid Chromatography coupled to a micrOTOF-QII, High Resolution Mass Spectrometer, were used. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids by metabolome tools. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: These results contribute by indicating potential biotechnological targets Erythrina alkaloids biosynthesis as well as to improve molecular databases with omic data from a non-model medicinal plant. Furthermore, they reveal an interesting chemical diversity in Erythrina velutina harvested in Caatinga. Last, but not least, this data may also contribute to tap Brazilian biodiversity in a rational and sustainable fashion, promoting adequate public policies for preservation and protection of sensitive areas within the Caatinga

Anthelmintic effect of Cassia fistula and Combretum leprosum protein fractions against goat gastrointestinal nematodes

<div><p>Abstract In this study, we evaluated the ovicidal and larvicidal activity of protein preparations obtained from Cassia fistula L. and Combretum leprosum Mart. leaves on the gastrointestinal parasites of goats. Protein preparations were obtained after the extraction of C. fistula L. and C. leprosum Mart. leaves, followed by protein fractionation (with ammonium sulfate saturation percentages of 30%, 30%-60%, and 60%-90%) and dialysis, which resulted in protein fractions (called F1, F2, and F3, respectively). The fractions were evaluated by egg hatching (the eggs were recovered in stool samples from naturally infected goats) and larval development tests. The results reveled that the inhibition of hatching of eggs caused by the protein fractions of C. fistula (38%) were similar to that of the control drug, thiabendazole. In addition, the fractions of C. fistula caused significant inhibition (61-69%) of larval development also. However, C. leprosum did not reveal significant inhibition of egg hatching and larval development. We conclude that C. fistula L. showed better ovicidal and larvicidal activity against endoparasites.</p></div