Microscopic Image Segmentation to Quantification of Leishmania Infection in Macrophages

The determination of infection rate parameter from in vitro macrophages infected by Leishmania amastigotes is fundamental in the study of vaccine candidates and new drugs for the treatment of leishmaniasis. The conventional method that consists in the amastigotes count inside macrophages, normally is done by a trained microscope technician, which is liable to misinterpretation and sampling. The objective of this work is to develop a method for the segmentation of images to enable the automatic calculation of the infection rate by amastigotes. Segmentation is based on mathematical morphology in the context of a computer vision system. The results obtained by computer vision system presents a 95% accuracy in comparison to the conventional method. Therefore, the proposed method can contribute to the speed and accuracy of analysis of infection rate, minimizing errors from the traditional methods, especially in situations where exhaustive repetitions of the procedure are required from the technician.A determinação de parâmetros como taxa de infecção em monocultura de macrófagos cultivados in vitro com Leishmania é fundamental no estudo de candidatos vacinais e novos fármacos para o tratamento de leishmanioses. O método convencional que consiste na contagem de amastigotas no interior de macrófagos, normalmente é realizada por um especialista treinado em microscopia óptica, o que está sujeito a erros de interpretação e amostragem. O objetivo do trabalho é desenvolver um método para a segmentação de imagens como etapa preliminar para o cálculo automático da taxa de infecção por amastigotas. A segmentação é baseada em morfologia matemática no contexto de um sistema de visão computacional. Os resultados obtidos pelo método computacional demonstraram acerto de 95% quando comparados ao método convencional. Conclui-se que a metodologia computacional baseada na segmentação de imagem como pré-requisito para o cálculo de taxa de infecção, pode contribuir para a rapidez e a precisão na obtenção dos resultados e na minimização de erros cometidos no método tradicional, especialmente em situações em que exaustivas repetições do procedimento são exigidas ao observador

Lycopene-rich extract from red guava ( Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M]+• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC50]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC50=1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC50≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway.The authors acknowledge the computational time provided by CENAPAD/SP on the project proj697. Alexandra Plácido is gratefully to FCT by her grant SFRH/BD/97995/2013, financed by POPH–QREN–Tipologia 4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. The work at REQUIMTE/LAQV received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT) through project UID/QUI/50006/2013. Adriany das G. N. Amorim is grateful to CAPES by for the doctoral fellowship process no. 99999.004236/2014-09 in Federal University of Piauí (UFPI). Eder A. Barbosa is grateful to PNPD/CAPES for its post-doctoral fellowship.info:eu-repo/semantics/publishedVersio

Neuroprotective effects on microglia and insights into the structure–activity relationship of an antioxidant peptide isolated from Pelophylax perezi

© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly citedTryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia in the framework of the project POCI-01-0145-FEDER-031158 – PTDC/BII-BIO/31158/2017. The authors would like to thank the participation and scientific support of the Unit projects UIDB/50006/2020 | UIDP/50006/2020, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universal Faixa ‘B’ (grant number 32103/2018-0). A.P. is a recipient of a post-doctoral grant from the project PTDC/BII-BIO/31158/2017. The authors would like to thank the researcher Roberto Resendes (CiBio, University of the Azores, Ponta Delgada, São Miguel, Azores, Portugal) for the logistical support in the collection of samples. C.P.A acknowledges FCT-MCTES fellowship PD/BD/136860/2018. A.B.-N. and F.C.D.A.L. acknowledge CNPq (grants 420449/2018-3 and 428211/2018-6) for financial support.info:eu-repo/semantics/publishedVersio

Novel ocellatin peptides mitigate LPS-induced ROS formation and NF-kB activation in microglia and hippocampal neurons

© The Author(s) 2020. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per-mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.Alexandra Plácido is a recipient of a post-doctoral grant from the project FCT (PTDC/BII-BIO/31158/2017). Renato Socodato and Camila Cabral Portugal hold postdoctoral fellowships from FCT (Refs: SFRH/BPD/91833/2012 and FRH/BPD/91962/2012, respectively). This work was funded through project UID/QUI/50006/2013-POCI/01/0145/FEDER/007265 (LAQV/REQUIMTE) with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT 2020info:eu-repo/semantics/publishedVersio

Quantitative and qualitative diagnostic of urban trees squares in Aracaju, SE, Brazil

Aracaju is a city that was planned with geometric precision and simplicity. Since its settlement, it has gone through constant changes in its urban environment. Due to these changes, fewer green areas remain in the urban environment. Well-planned urban afforestation is very important, once trees provide benefits to the city in ecological (climate and pollution), biological (physical health of human beings) and psychological (mental health of population) processes. However, the absence of afforestation planning and the use of inappropriate species are the main obstacles to achieving these benefits. This study was carried out to evaluate the floristic composition of twenty-two main squares in Aracaju-SE, as a way to know the current status and allow some interventions to improve the technical adequacy of urban green space. The floristic survey was conducted by means of a Census, from September 2006 until July 2007, in each square making the recognition and identification of all trees present using traditional methods and herborization.Aracaju foi uma cidade planejada com simplicidade e rigor geométrico. Desde a sua fundação, ela vem passando por constantes alterações, havendo cada vez menos áreas verdes no ambiente urbano. A arborização urbana bem planejada é muito importante, pois as árvores trazem benefícios à cidade nos processos de ordens ecológica (clima e poluição), biológica (saúde física do homem) e psicológica (saúde mental do homem). Porém, a falta de planejamento dessa arborização e o uso inadequado de algumas espécies são empecilhos para se atingirem esses benefícios. Dessa forma, este trabalho foi realizado com o objetivo de avaliar a composição florística de 22 praças de Aracaju, SE, como forma de conhecer a atual situação de sua arborização e possibilitar intervenções para melhorar a adequação técnica do espaço verde urbano. O levantamento florístico foi realizado através de censo, no período de setembro de 2006 a julho de 2007, em cada praça, com o reconhecimento e identificação de todos os indivíduos arbóreos, utilizando-se as técnicas tradicionais de coleta e herborização. Realizou-se a identificação das espécies com o auxílio de literatura específica. Foram identificados 1.290 indivíduos, distribuídos em 20 famílias botânicas, 46 gêneros e 64 espécies, sendo nove espécies não identificadas. Das espécies identificadas, 58% são exóticas no território brasileiro e 42%, espécies nativas. As praças apresentam similaridade entre si, com coeficiente de Jaccard variando de 0,00 a 0,66, e a Praça Almirante Tamandaré e a Praça Graccho Cardoso foram as de maior similaridade (66%).Viçosa, M

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H]+ = 1543.69 Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide.info:eu-repo/semantics/publishedVersio

ASPECTOS CLINICOS DE UMA REABILITAÇÃO COM PRÓTESES FIXAS SOBRE IMPLANTES OSSEOINTEGRADOS: RELATO DE CASO

Os implantes fazem parte do planejamento dos tratamentos reabilitadores na Odontologia, e quando empregados com critério, tem-se a possibilidade de restituir ao paciente as funções imprescindíveis do sistema estomatognático. A realização de estudos no âmbito dessa temática tem comprovado que os pacientes inválidos orais tem usufruído dos aspectos benéficos das próteses sobre implante, principalmente quando comparado com as próteses totalmente mucosuportadas. Em face destas considerações o presente relato de caso clínico, possui como Objetivo discutir os aspectos de interesse protético na reabilitação de paciente desdentado total com implantes osseointegráveis, descrevendo a execução do tratamento reabilitador protético maxilomandibular através de próteses  implantosuportadas  e  parafusadas.  Descrição  do  Caso:  Para  isso, realizou-se tratamento seqüencial de uma prótese total implanto suportada e parafusada, composto por duas etapas, sendo uma cirúrgica e uma protética, efetivadas em um intervalo de tempo de quatro meses. Consideração Final: ao final do tratamento observou-se a reabilitação maxilomandibular com próteses totais fixas sobre implantes e a melhora na auto-estima relatada pelo paciente