Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

Isolation and characterization of microsatellite markers in two species of the neotropical Epicharis (Hymenoptera, Apidae, Centridini) genus and cross-amplification in related species.

Genetics can provide invaluable tools for management and conservation of bee populations, which are declining worldwide. Among these tools, microsatellite are very useful molecular markers for population analyses. The aim of this study was to isolate and characterize microsatellites for Epicharis (Anepicharis) dejeanii and Epicharis (Epicharis) nigrita, two Neotropical species of solitary bees, both exhibiting the habit of nesting in aggregations. Microsatellite loci were identified from two enriched genomic libraries. The characterization and analysis of loci were carried out using 35 females of E. dejeanii and 34 of E. nigrita. In total, we report the development of 12 microsatellite loci for E. dejeanii and 13 for E. nigrita. For E. dejeanii, all loci were polymorphic, the number of alleles per locus ranged from 2 to 12, averaging 8.7 and, observed and expected heterozygosity were 0.485 (range 0.229-0.857) and 0.633 (range 0.288-0.843), respectively. For E. nigrita, only nine out of 13 loci amplified were polymorphic, the number of alleles per locus ranged from 2 to 12, averaging 5.5. For this species, the observed and expected heterozygosity were 0.440 (range 0.118-0.676) and 0.545 (range 0.167-0.814), respectively. Cross-amplification of primers was successful in other Centridini species. The two sets of loci described for E. dejeanii and E. nigrita species are polymorphic and informative and show promising applicability for both population genetic approaches and relatedness on these and other Centridini species.

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems.

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1-3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Seroprevalence of Leptospira spp. in Colorado equids and association with clinical disease.

Detection of Leptospira interrogans is difficult as a result of intermittent leptospiruria and brief leptospiremia. Hence, diagnosis relies heavily on serologic testing, the reference method of which is the microscopic agglutination test (MAT). In horses, clinical leptospirosis has been associated with abortion, recurrent uveitis, and sporadic cases of hepatic and renal disease. Little information exists on the seroprevalence of antibodies to L. interrogans in equids in the United States; past nationwide studies suggest that the seroprevalence in some areas is as high as 77% (reciprocal titer ≥ 100). We tested sera from 124 apparently healthy horses previously submitted for equine infectious anemia (EIA) serology using MAT for 6 serovars-Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. When using a reciprocal MAT titer cutoff of ≥ 100, 102 of 124 (82%) of the samples were positive for at least one serovar. Seropositivity was significantly associated with increasing age. Query of specimens from clinical cases submitted to the Colorado State University Veterinary Diagnostic Laboratory for MAT since 2010 indicated significantly greater seroprevalence (p = 0.015) of pathogenic serovar Pomona in clinical cases compared to sera submitted from healthy equids for routine EIA testing. Information from our diagnostic laboratory submission forms also suggests a correlation between uveitis or other ophthalmic problems and serovar Pomona.

Synthesis of thiazolidin-4-ones and thiazinan-4-ones from 1-(2-aminoethyl)pyrrolidine as acetylcholinesterase inhibitors.

The present study describes the synthesis of a novel series of thiazolidin-4-one and thiazinan-4-one using 1-(2-aminoethyl)pyrrolidine as amine precursor. All compounds were synthesised by one-pot three component cyclocondensation reaction from the amine, a substituted benzaldehyde and a mercaptocarboxylic acid. The compounds were obtained in moderate to good yields and were identified and characterised by 1H, 13 C, 2 D NMR and GC/MS techniques. The compounds also were screened for their in vitro acetylcholinesterase (AChE) activity in hippocampus and cerebral cortex on Wistar rats. The six most potent compounds have been investigated for their cytotoxicity by cell viability assay of astrocyte primary culture, an important cell of central nervous system. We highlighted two compounds (6a and 6k) that had the lowest IC50 in hippocampus (5.20 and 4.46 µM) and cerebral cortex (7.40 and 6.83 µM). These preliminary and important results could be considered a starting point for the development of new AChE inhibitory agents.

Oral glucose tolerance test: unnecessary requests and suitable conditions for the test / Teste oral de tolerância à glicose: solicitações desnecessárias e condições adequadas a realização do teste

ABSTRACT Introduction: The oral glucose tolerance test (OGTT) is an important test for diagnosis of diabetes mellitus (DM) that needs criteria to be requested and analytical performance evaluation. Objectives: Determine the prevalence of unnecessary OGTT requests and analyze patients' suitability criteria for glucose load. Method: Cross-sectional, descriptive and analytical study conducted with 554 patients who had OGTT requested from January to April 2018. Data from laboratory tests were collected through Complab Advanced version 6.9.6 system, organized into a Microsoft® Excel table and analyzed using Epi InfoTM, version 7.2.1.0. The accuracy of the glucometer paired analysis was performed by determinations of Student's t test, using SPSS version of IBM 21TM. Patients with a previous diagnosis of DM and/or who showed OGTT request as a screening test along with blood glucose or glycated hemoglobin were classified as unnecessary requests. Results: Among the studied patients, 17% (94) had unnecessary OGTT requests, 53 (53.4%) patients had blood glucose ≥ 140 mg/dl and previous DM and/or OGTT used as screening; 41 (43.6 %) patients with capillary blood glucose < 140 mg/dl, but with a diagnosis of DM. The glucometer proved to be accurate with a high correlation (r 2 = 0.97, p < 0.0001) with serum glucose. Approaches during screening and capillary blood glucose prevented unnecessary exposure to glucose overload in 67% (63) of the patients. Conclusion: The high prevalence of unnecessary OGTT requests underscores the need of criteria for OGTT requesting and the standardization of procedures for screening in the exam.

RESUMEN Introducción: La prueba de tolerancia a la glucosa oral (PTGO) es un importante examen para diagnóstico de la diabetes mellitus (DM) que requiere criterios de solicitud y estandarización en su consecución. Objetivos: Establecer la prevalencia de solicitudes innecesarias de PTGO y analizar los criterios de aptitud del paciente para una sobrecarga de glucosa. Método: Estudio de corte transversal, descriptivo y analítico, llevado a cabo con 554 pacientes que tuvieron PTGO solicitado entre enero y abril de 2018. Datos de las pruebas de laboratorio fueron recolectados con el sistema Complab Advanced versión 6.9.6, tabulados en Microsoft® Excel y analizados en Epi InfoTM, versión 7.2.1.0. La precisión del glucómetro fue medida por análisis pareado de las determinaciones en la prueba t de Student, mediante el IBM SPSS® versión 21TM. Pacientes con diagnóstico previo de DM y/o que presentaron solicitud de PTGO como prueba de cribado junto con glucemia o hemoglobina glucosilada fueron clasificados como peticiones innecesarias. Resultados: Entre los pacientes investigados, el 17% (94) tuvieron peticiones innecesarias de PTGO: 53 (53,4%) con glucemia capilar ≥ 140 mg/dl y DM previo y/o PTGO usado como cribado; 41 (43,6%) con glucemia capilar < 140 mg/dl, pero con diagnóstico de DM. El glucómetro se mostró preciso, con alta correlación (r2 = 0,97, p < 0,0001) con la glucemia sérica. Los enfoques durante el cribado y la glucemia capilar evitaron la exposición innecesaria a la sobrecarga de glucosa en el 67% (63) de los pacientes. Conclusión: El alta prevalencia de peticiones innecesarias de PTGO destaca la necesidad de criterios para la solicitud de la PTGO, así como la estandarización de procedimientos para cribado en la consecución de esa prueba.

RESUMO Introdução: O teste oral de tolerância à glicose (TOTG) é um importante exame para diagnóstico do diabetes mellitus (DM) que necessita de critérios para solicitação e padronização em sua realização. Objetivos: Determinar a prevalência de solicitações desnecessárias de TOTG e analisar os critérios de aptidão do paciente para sobrecarga glicêmica. Método: Estudo de corte transversal, descritivo e analítico, realizado com 554 pacientes que tiveram TOTG solicitado entre janeiro e abril de 2018. Dados dos exames laboratoriais foram coletados utilizando o sistema Complab Advanced versão 6.9.6, tabulados no Microsoft® Excel e analisados no Epi InfoTM, versão 7.2.1.0. A acurácia do glicosímetro foi medida por análise pareada das determinações em teste t de Student, por meio do IBM SPSS® versão 21TM. Pacientes com diagnóstico prévio de DM e/ou que apresentaram solicitação de TOTG como teste de triagem junto com glicemia ou hemoglobina glicada foram classificados como solicitações desnecessárias. Resultados: Dos pacientes estudados, 17% (94) tiveram solicitações desnecessárias de TOTG: 53 (53,4%) com glicemia capilar ≥ 140 mg/dl e DM prévio e/ou TOTG usado como triagem; 41 (43,6%) com glicemia capilar < 140 mg/dl, mas com diagnóstico de DM. O glicosímetro mostrou-se preciso, com elevada correlação (r 2 = 0,97, p < 0,0001) com a glicemia sérica. As abordagens durante a triagem e a glicemia capilar evitaram a exposição desnecessária à sobrecarga de glicose em 67% (63) dos pacientes. Conclusão: A alta prevalência de solicitações desnecessárias de TOTG ressalta a necessidade de critérios para solicitação do TOTG, bem como a padronização de procedimentos para triagem na realização desse exame.

Synthesis and biological evaluation of benzothiazin-4-ones: a possible new class of acetylcholinesterase inhibitors.

A series of nineteen benzothiazin-4-ones from N-(3-aminopropyl) piperidine, 4-(2-aminoethyl)morpholine or 1-(2-aminoethyl)piperidine, aliphatic or aromatic aldehyde and thiosalicylic acid, were synthesized in good yields by multicomponent one-pot reactions. The solvent was toluene and this efficient procedure afforded the desired heterocycles in 5 h. Identification and characterization were achieved by NMR and GC-MS techniques. In vitro AChE activities of all compounds were evaluated in cerebral cortex and hippocampus of rats and in general, the results in cortex were more promising than hippocampus. The benzothiazinone 5Bd showed the best AChE inhibition activity IC50 8.48 µM (cortex) and IC50 39.80 µM (hippocampus). The cytotoxicity of seven compounds in MCR-5 human fibroblast cell by SRB test in 24 h were evaluated and 5Bd suggest preliminary safety, showing no cytotoxicity at 100 µM. Finally, these important findings could be a starting point for the development of new AChE inhibitors agents and will provide the basis for new studies.

Molecular networking prospection and characterization of terpenoids and C15-acetogenins in Brazilian seaweed extracts.

Molecular networking (MN) can efficiently dereplicate extracts and pure compounds. Red algae of the genus Laurencia are rich in halogenated secondary metabolites, mainly sesquiterpenes and C15-acetogenins. Brown algae of the genus Dictyopteris produce mainly C11-hydrocarbons, sesquiterpenes and sulfur-containing compounds, while Dictyota and Canistrocarpus are reported to contain mainly diterpenes. This study performs an exploratory MN analysis of 14 extracts from algae collected in Brazil (including the oceanic islands) and characterizes the secondary metabolites from the analyzed species. The extracts and some isolated metabolites were analyzed by LC-MS using the FastDDA algorithm, and the MS/MS spectra were submitted to GNPS and displayed in Cytoscape 3.5.1. The GNPS platform generated 68 individual nodes and nine family networks. The MN exploratory analysis indicated chemical differences among species, and also in sampling sites for the same species. For some extracts, it was possible to identify mass values that could correspond to terpenoids and C15-acetogenins that have already been isolated from those or related species. An interesting chemodiversity was highlighted between Laurencia catarinensis from two nearby islands, and this was revealed and was also suggested by the family networks. Many nodes in the MN could not be characterized, and these metabolites can be used as targets for isolation in future works.

Mitochondrial DNA diversity of orchid bee Euglossa fimbriata (Hymenoptera: Apidae) populations assessed by PCR-RFLP.

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (phi(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (phi(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

Eufriesea violacea (Blanchard) (Hymenoptera: Apidae): an orchid bee apparently sensitive to size reduction in forest patches.

Eufriesea violacea (Blanchard) is a very seasonal euglossine species, more frequently found in the southern and southeastern regions of Brazil. A number of studies have revealed large variations in the abundance of males of this species present in Atlantic Forest remnants throughout both regions. In this paper, we report variations in the abundance of E. violacea males sampled in several forest patches of different sizes (ranging from 10 to 580 ha), and we propose that this species is possibly sensitive to the reduction in size of forest remnants. Surveys were carried out in nine forest remnants of Atlantic rainforest located in northern Paraná State, southern Brazil. Male euglossine bees were collected with an entomological net when visiting scent-baits, between 10:00 am and 1:00 pm, from October to December of 2001 and 2006. A total of 360 E. violacea males were captured in the nine forest fragments studied. The number of bees attracted to scent baits in each forest patch varied from zero to 261. A very high association (r = 0.993) was detected between the forest patch size and the visitation rate of E. violacea males at different sites, with the highest mean number of males visiting baits/sampling (43.5) being observed for bees from the largest forest remnant. Although alternative hypothesis should not be discharged for the decline in the abundance or absence of E. violacea in small forest patches, our results indicate that populations of this euglossine species need larger forest areas for existing.

Eufriesea violacea (Blanchard) (Hymenoptera: Apidae): an orchid Bee apparently sensitive to size reduction in forest patches / Eufriesea violacea (Blanchard) (Hymenoptera: Apidae): uma abelha das orquídeas aparentemente sensível à redução no tamanho dos fragmentos florestais

Eufriesea violacea (Blanchard) is a very seasonal euglossine species, more frequently found in the southern and southeastern regions of Brazil. A number of studies have revealed large variations in the abundance of males of this species present in Atlantic Forest remnants throughout both regions. In this paper, we report variations in the abundance of E. violacea males sampled in several forest patches of different sizes (ranging from 10 to 580 ha), and we propose that this species is possibly sensitive to the reduction in size of forest remnants. Surveys were carried out in nine forest remnants of Atlantic rainforest located in northern Paraná State, southern Brazil. Male euglossine bees were collected with an entomological net when visiting scent-baits, between 10:00 am and 1:00 pm, from October to December of 2001 and 2006. A total of 360 E. violacea males were captured in the nine forest fragments studied. The number of bees attracted to scent baits in each forest patch varied from zero to 261. A very high association (r = 0.993) was detected between the forest patch size and the visitation rate of E. violacea males at different sites, with the highest mean number of males visiting baits/sampling (43.5) being observed for bees from the largest forest remnant. Although alternative hypothesis should not be discharged for the decline in the abundance or absence of E. violacea in small forest patches, our results indicate that populations of this euglossine species need larger forest areas for existing.

Eufriesea violacea (Blanchard) é uma espécie de Euglossini bastante sazonal, de ocorrência mais frequente no Sul e Sudeste do Brasil. Vários estudos revelaram grandes variações na abundância de machos de E. violacea em remanescentes de Mata Atlântica dessas duas regiões brasileiras. Neste artigo, relatamos variações na abundância de machos de E. violacea de vários fragmentos florestais de tamanhos distintos (variando de 10 a 580 ha), e sugerimos que a espécie seja provavelmente sensível à redução no tamanho da área do fragmento florestal. As amostragens foram realizadas em nove remanescentes florestais de Mata Atlântica, localizados no Norte do Paraná, Sul do Brasil. Machos euglossíneos foram coletados com rede entomológica durante suas visitas às iscas-odores, entre 10:00h e 13:00h, de outubro a dezembro de 2001 e 2006. O total de 360 machos de E. violacea foi coletado nos fragmentos estudados. O número de abelhas atraídas às iscas em cada área variou de zero a 261 indivíduos. O tamanho do fragmento foi proporcionalmente relacionado (r = 0,993) à taxa de visitação de machos de E. violacea nas diferentes áreas, sendo que o maior número médio de machos (43,5) por amostragem foi observado para abelhas do fragmento florestal maior. Embora hipóteses alternativas para o declínio na abundância ou ausência de E. violacea em fragmentos florestais menores não devam ser descartadas, esses resultados indicam que populações de E. violacea necessitam de áreas florestais maiores para sua sobrevivência.