Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad.

Malaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.

The impact of a story on learning ketone body metabolism.

Biochemistry is a core subject in the cross-disciplinary training on Biotechnology engineering courses. Metabolic pathways teaching has traditionally integrated hands-on laboratory experiences and traditional lectures, which detail a large number of reactions at a molecular level, their enzymes and regulation. The current scenario of Covid-19 outbreak have motivated the development of complementary tools that expand the horizon of metabolism teaching. In this study, we employed a story-based methodology to strengthen the metabolic pathways learning and to measure students' perception. Specifically, a peer-reviewed tale describing the ketone body metabolism was used during five semesters as a didactic strategy to teach this biochemical process. A questionnaire assessed the students' understanding and acceptance of the methodology (n = 83). Our findings showed that a high proportion of students (83.13%) were able to relate the story to the topics studied in the classroom (ketogenesis and ketolysis). On the other hand, they were satisfied and suggested that such methodology is effective and fun. In summary, most of the survey responses related to acceptance of story-based strategy ranged from 72% to 97%. Collectively, these results indicated that the story is appropriate to decomplex pathways, becoming a simple tool for driving motivation, learning and engagement of students. The narrative represents a bridge to connect the intriguing series of chemical reactions involved in the anabolism and degradation of 3-hydroxybutyrate (3-OHB), acetoacetate, and acetone with previously learned knowledge, emotions, and key concepts. In conclusion, the tale was useful to decode ketone body-related pathways and making metabolism learning more interesting and easier.

Picturins and Pictuseptins, two novel antimicrobial peptide families from the skin secretions of the Chachi treefrog, Boana picturata.

The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named "Picturins" (PTR), "Pictuseptins" (PTS), and "Boanins" (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, -2 and - 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, -2 and - 4 have 22 amino acid residues in length, while PTS -3, -5 and - 6 are composed of 26 residues. Whereas BNS are four 28-37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to -3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 µM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 µM) followed by PTS-3 (11.7 µM) and PTS-2 (14.24 µM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. BIOLOGICAL SIGNIFICANCE: Nature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance.

An experimental protocol for molecular biology lab at an Amazonian University.

Laboratory-based practical classes are an essential component in teaching molecular biology for undergraduate students. Universidad Regional Amazonica Ikiam is a higher education institution located in the Ecuadorian Amazon rainforest, a high biodiversity place, including amphibians. Based on this, we have established a practical molecular biology program with eight sessions that contextualize the biodiverse surroundings of the University. This program stimulates synchronization of information between theory and practice and improves research skills. During these sessions, students are motivated to identify and characterize antimicrobial peptides from Ecuadorian frog skin secretions, using molecular biology techniques and biochemistry and microbiology knowledge. This practical course was held twice with a total of 56 students from the fifth semester of the biotechnology engineering. The evaluation of the practical program was carried out through a questionnaire applied to students using the Likert scale. Overall, this form of teaching had high receptivity and presented benefits for student learning. Interestingly, 80% of respondents strongly agreed that this course provided tools and knowledge for the development of their undergraduate dissertation. Therefore, practical courses tailored to the student's context can stimulate student learning and interest. Additionally, this experimental methodology is interdisciplinary and can be applied to other research fields and subjects.

Unravelling the Skin Secretion Peptides of the Gliding Leaf Frog, Agalychnis spurrelli (Hylidae).

Frog skin secretions contain medically-valuable molecules, which are useful for the discovery of new biopharmaceuticals. The peptide profile of the skin secretion of Agalychnis spurrelli has not been investigated; therefore, the structural and biological characterization of its compounds signify an inestimable opportunity to acquire new biologically-active chemical scaffolds. In this work, skin secretion from this amphibian was analysed by molecular cloning and tandem mass spectrometry. Although the extent of this work was not exhaustive, eleven skin secretion peptides belonging to five peptide families were identified. Among these, we report the occurrence of two phyllokinins, and one medusin-SP which were previously reported in other related species. In addition, eight novel peptides were identified, including four dermaseptins, DRS-SP2 to DRS-SP5, one phylloseptin-SP1, and three orphan peptides. Phylloseptin-SP1 and dermaseptins-SP2 were identified in HPLC fractions based on their molecular masses determined by MALDI-TOF MS. Among the antimicrobial peptides, dermaseptin-SP2 was the most potent, inhibiting Escherichia coli, Staphylococcus aureus, and ORSA with a minimum inhibitory concentration (MIC) of 2.68 µM, and Candida albicans with an MIC of 10.71 µM, without haemolytic effects. The peptides described in this study represent but a superficial glance at the considerable structural diversity of bioactive peptides produced in the skin secretion of A. spurrelli.

Comprehensive two-dimensional gas chromatography-mass spectrometry combined with multivariate data analysis for pattern recognition in Ecuadorian spirits.

The current methodology used in quality control of Ecuadorian beverages such as Pájaro azúl, Puro and Pata de vaca is carried out by using conventional gas chromatography; however, it does not allow the fingerprinting of these Ecuadorian spirit beverages and their possible cases of adulteration. In order to overcome this drawback, comprehensive two-dimensional gas chromatography-mass spectrometry (GC × GC-MS) was combined with multivariate data analysis, revealing that compounds like citronellal, citronellol, geraniol, methyl anthranilate, (-)-trans-α-bergamotene, (-)-cis-α-bergamotene and D-limonene can be considered key elements for pattern recognition of these traditional beverages and product adulteration cases. Thus, the two-dimensional chromatographic fingerprints obtained by GC × GC-MS coupled with chemometric analysis, using Principal Component Analysis and Fisher-ratio can be considered as a potential strategy for adulteration recognition, and it may used as a quality assurance system for Ecuadorian traditional spirits.

New Advances in Toxicological Forensic Analysis Using Mass Spectrometry Techniques.

This article reviews mass spectrometry methods in forensic toxicology for the identification and quantification of drugs of abuse in biological fluids, tissues, and synthetic samples, focusing on the methodologies most commonly used; it also discusses new methodologies in screening and target forensic analyses, as well as the evolution of instrumentation in mass spectrometry.

Genetic and phenotypic diversity of Rhizobium isolates from Southern Ecuador / Diversidade genética e fenotípica de isolados de Rhizobium do sul do Equador

ABSTRACT Rhizobium-legume symbioses play relevant roles in agriculture but have not been well studied in Ecuador. The aim of this study was to characterize the genetic and phenotypic diversity of Rhizobium isolates associated with Phaseolus vulgaris from southern Ecuador. Morpho-cultural characterization, biochemical tests and physiological analyses were conducted to authenticate and determine the diversity of bacteria Rhizobium-like isolates. The genetic diversity of the isolates was determined by molecular techniques, which consisted of bacteria DNA extraction and amplification and sequencing of the 16S rRNA gene. The nodulation parameters and nitrogen fixation for P. vulgaris under greenhouse conditions were also assessed to determine the phenotypic diversity among isolates. Furthermore, bacteria indole-acetic-acid production was evaluated by the colorimetric method. Morpho-cultural and biochemical characteristic assessments demonstrated that Rhizobium-like bacteria was associated with the P. vulgaris nodules. The diversity among the isolates, as determined by physiological analyses, revealed the potential of several isolates to grow at different pH values, salinity conditions and temperatures. Partial sequencing of the 16S rRNA gene identified the Rhizobium genus in every sampling site. From a total of 20 aligned sequences, nine species of Rhizobium were identified. Nodule formation and biomass, as well as nitrogen fixation, showed an increase in plant phenotypic parameters, which could be influenced by IAA production, especially for the strains R. mesoamericanum NAM1 and R. leguminosarum bv. viciae COL6. These results demonstrated the efficiency of native symbiotic diazotrophic strains inoculants for legume production. This work can serve as the basis for additional studies of native Rhizobium strains and to help spread the use of biofertilizers in Ecuadorian fields.

RESUMO A simbiose Rhizobium-leguminosa desempenha um relevante papel na agricultura, entretanto não tem recebido suficiente atenção de estudos científicos no Equador. O objetivo deste artigo foi caracterizar a diversidade genética e fenotípica de isolados de Rhizobium associados com Phaseolus vulgaris do sul do Equador. A caracterização morfo-cultural, testes bioquímicos e análises fisiológicas foram realizados para autenticar e determinar a diversidade de isolados de bactérias Rhizobium. A diversidade genética foi determinada por por técnicas moleculares consistindo na extração de DNA genômico bacteriano, amplificação e sequenciamento parcial do gene 16SrRNA; e parâmetros de nodulação e fixação de nitrogênio de P. vulgaris sobre condições de estufa foram testados para determinar a diversidade fenotípica entre os isolados. Além disso, a produção de ácido indolacético foi avaliada por um método colorimétrico. A análise fisiológica da diversidade entre os isolados revelou o potencial de crescimento de diversos isolados em diferentes níveis de pH, salinidade e temperatura. O sequenciamento parcial do gene 16S rRNA mostrou o gênero Rhizobium em todas os locais de amostragem. De um total de 20 sequências alinhadas, 9 espécies de Rhizobium foram identificadas. A formação de nódulos e biomassa, bem como a fixação de nitrogênio mostraram um aumento nos parâmetros fenotípicos das plantas, os quais devem ser influenciados pela produção de IAA, especialmente pelas cepas R. mesoamericanum NAM1 e R. leguminosarum bv. viciae COL6. Estes resultados demonstram a eficiência de cepas diazotróficas simbióticas nativas para produção de inoculantes para leguminosas e fornece informações valiosas e uteis para a agricultura sustentável equatoriana. Neste sentido, este trabalho deve ser um elemento essencial para a realização de futuras pesquisas aplicadas relacionadas a cepas Rhizobium nativas e espalhar o uso de biofertizantes em campos equatorianos.