nteraction between FAK/αB-crystalline is important for viability of the glioblastoma cells

Abstract Glioblastoma multiforme is a tumor of the central nervous system. Focal Adhesion Kinase (FAK) and αB-crystalline are two proteins involved in glioblastoma development. In this study, we investigated whether the FAK/αB-crystalline interaction is important for glioblastoma cells, we aimed to investigate the interaction of these two proteins in the glioblastoma multiforme cell line U87-MG. Two peptides named FP01 peptide (derived from αB-crystalline) and FP02 peptide (derived from FAK) were synthesized for this study. Treatment of U87-MG with the peptides FP01 and FP02 in the concentration at 50 µM reduced the viability cellular to around 41% and 51%, respectively. Morphological alterations in the cells treated with the peptides when compared to the control were observed. This study suggests that the interaction between FAK and αB-crystalline is important for the viability of glioblastoma cells

Human venous blood derivatives as fetal bovine serum substitute for fibroblast culture cells in a fibrin construct

Aim: Venous blood derivatives (VBDs) have been suggested as substitutes for Fetal Bovine Serum (FBS) to improve the clinical transition of cell-based therapies. The literature is not clear about which is the best VBDs substitute. The present study aimed to evaluate the influence of VBDs on cell viability and describe a new method to seed these cells in a 3D Platelet-Rich Fibrin (PRF). Methods: Blood was processed to obtain Platelet-Poor Plasma from PRF (P-PRF), Human Serum (HS), Platelet-Poor Plasma from PRP (P-PRP), activated-PRP (a-PRP), and Platelet lysate (PL). Cells were supplemented with each VBD at 10% and FBS at 10% was the control. Cell viability (fibroblast 3T3/NIH) test was evaluated with MTT assay in two ways: i) cell-seeded and expanded with VBD; ii) cell-seed with FBS and expanded with VBD. To seed the Fibrin construct, cells were suspended in PBS and dropped into the blood sample before performing Choukroun's protocol for PRF. Constructs were cultured for 7 days in VBD supplements and FBS. Histological and Immunohistochemical analysis with vimentin was performed. Cell viability was analyzed by one-way ANOVA. Results: VBD's production time was very heterogeneous. Cells expanded in HS and a-PRP has grown faster. VBD-supplemented culture media provided cell culture highly sensible to trypsin/EDTA 0.25%. Cells seeded and expanded with VBD presented viability comparable to FBS in HS, a-PRP, and P-PRP (p>0.05) and lower in P-PRF and PL groups (p<0.05). The viability of cell seed with FBS and expanded with VBD was similar between P-PRF, a-PRP, PL, and FBS (p>0.05) and lower in HS and P-PRP (p<0.005). PRF-seeded cells showed a positive expression of vimentin and were able to maintain all cells supplemented with VBD. Conclusion: VBD supplements were able to maintain fibroblast cells in 2D and 3D cultures. The new method of the fibrin-cell construct was efficient to insert the cells into the fibrin network

The biological effects of different LED wavelengths in the health field. A review / Os efeitos biológicos de diferentes comprimentos de onda de LED na área da saúde. Uma revisão

Introduction: the use of light emitting diodes (LED) in domestic and public vias have increased in the last 20 years. In addition, the LED light has been used as a light source for medical applications. Objective: since humans are increasingly exposed to LEDs, there is an urgency to investigate the possible biological effects on tissues caused by this exposure. So, researchers have been focused their investigations in the application of this light in the health field. Material and method: in this review, a search in important databases was performed on the biological effects caused after application of different LED light protocols in in vitro and in vivo studies. Result: although most published papers have shown positive results, some of them reported negative biological effects of light LEDs technology on humans' cells/tissues. Conclusion: therefore, the comprehension of the biological effects caused by light LEDs will provide a better assessment of the risks involved using this technology.

Introdução: o uso de diodos emissores de luz ("LED") em vias domésticas e públicas tem aumentado nos últimos 20 anos. Além disso, a luz LED tem sido usada para aplicações médicas. Objetivo: pelo fato de seres humanos estarem cada vez mais expostos aos LEDs, há urgência em investigar os possíveis efeitos biológicos nos tecidos causados por esta exposição. Assim, pesquisadores têm focado suas investigações no uso desta luz na área da saúde. Material e método: nesta revisão foi realizada uma pesquisa em bancos de dados conceituados sobre os efeitos biológicos causados após aplicação de diferentes protocolos de luz LED em estudos in vitro e in vivo. Resultado: embora a maioria dos artigos publicados tenham mostrado resultados positivos, alguns deles relataram efeitos biológicos negativos da tecnologia de LEDs nas células/tecidos humanos. Conclusão: portanto, a compreensão dos efeitos biológicos causados pela luz LED proporcionará uma melhor avaliação dos riscos envolvidos no uso desta tecnologia.

Narrative review of generalities of the genus Leptospira and its virulence factors associated with renal pathophysiology

Worldwide, leptospirosis is the most highly prevalent zoonosis. Although the wide range of clinical manifestations of leptospirosis in humans is well-documented, knowledge of the mechanisms through which this pathogen causes kidney disease remains limited. This narrative review of the scientific literature presents experimental studies of pathophysiology and kidney disease in leptospirosis, both in humans and animals, and the results show that virulence factors are involved in kidney damage by inducing interstitial tubular nephritis, which is the most frequent pathological manifestation, additionally, to the acute non-oliguric renal lesion with hypokalemia, and loss of magnesium and sodium. Finally, it is concluded that in leptospirosis, the initial lesion in the kidney is caused by damage to the cell membrane of the proximal tubular region cells by pathogenic Leptospira virulence factors, thus exacerbating the immune response

Caracterización genotípica y biofilms de E. coli shigatoxigénica aisladas de casos clínicos humanos / Genotypic characterization and biofilms of shigatoxigenic E. coli isolated from human clinical cases

Resumen Escherichia coli shigatoxigénica (STEC) está involucrada en el desarrollo del síndrome urémico hemolítico, entre otras enfermedades que son de gran importancia para la salud pública e inocuidad alimentaria a nivel mundial. La capacidad de STEC de formar biofilms en los alimentos y en diferentes superficies podría conducir a la contaminación cruzada por el desprendimiento de las células bacterianas. El objetivo del presente trabajo fue detectar la presencia de genes que codifican factores de adherencia mediante la técnica de PCR y determinar la capacidad de formación de biofilms por medio de cultivo en microplacas de poliestireno de 96 pocillos y la técnica de cristal violeta, en cepas de STEC aisladas de muestras clínicas humanas en la ciudad de Mar del Plata, Argentina. El perfil de genes de adherencia más frecuente fue efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas las cepas de STEC formaron biofilms con valores de densidad óptica entre 0,209 y 3,251 y el 54,4% (31/57) de las mismas fueron clasificadas como fuertes formadoras de biofilms. La capacidad de formación de biofilms de STEC constituye un riesgo evidente en la transmisión de este patógeno al ser humano a tener en cuenta para su vigilancia y control.

Abstract Shigatoxigenic Escherichia coli (STEC) is involved in the development of hemolytic uremic syndrome, among other diseases that are relevant to public health and food safety worldwide. The ability of STEC to form biofilms in food and on different surfaces could lead to cross-contamination by shedding bacterial cells. The aim of this work was to detect the presence of genes encoding adherence factors by the PCR technique and to determine the biofilm formation ability by culture in 96-well polystyrene microplates and the crystal violet technique, in STEC strains isolated from human clinical samples in Mar del Plata city, Argentina. The most frequent adherence gene profile was efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43.9%). All STEC strains formed biofilms with optical density values between 0.209 and 3.251. Also, the 54.4% (31/57) of STEC strains were classified as strong biofilm formers. The ability of STEC to form biofilms constitutes an evident risk in the transmission of this pathogen to humans, which must be taken into account for its surveillance and control.

Resumo A Escherichia coli shigatoxigênica (STEC) está envolvida no desenvolvimento da síndrome hemolítica urêmica, entre outras doenças relevantes para a saúde pública e segurança alimentar em todo o mundo. A capacidade do STEC de formar biofilmes nos alimentos e em diferentes superfícies poderia levar à contaminação cruzada através do desprendimento de células bacterianas. O objetivo do presente trabalho foi detectar a presença de genes que codificam fatores de aderência através da técnica PCR e determinar a capacidade de formação de biofilme por cultura em microplacas de poliestireno de 96 poços e da técnica de cristal violeta, em cepas STEC isoladas de amostras clínicas humanas na cidade de Mar del Plata, Argentina. O perfil de genes de aderência mais frequente foi efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas as cepas de STEC formaram biofilmes com valores de densidade ótica entre 0,209 e 3,251. Também, os 54,4% (31/57) das estirpes STEC foram classificados como fortes formadores de biofilmes. A habilidade de formação de biofilmes de STEC constitui um risco evidente na transmissão deste patógeno ao humano, que deve ser levado em consideração para sua vigilância e controle.

César Milstein / César Milstein

Resumen El presente comentario, escrito en virtud del decreto del gobierno nacional que designó a 2021 como año de homenaje al Dr. César Milstein, es una visión personal sobre el significado del desarrollo de los anticuerpos monoclonales, del aporte que brindó y brinda a la ciencia y tecnología actual y del contexto del correspondiente Premio Nobel en el ámbito de la Inmunología y las Ciencias Médicas. El objetivo de este artículo es transmitir, a modo de homenaje, algunas de mis experiencias en la especialidad y la relación profesional y personal con el creador de la producción de anticuerpos monoclonales por fusión de células somáticas, tecnología que continúa realizando grandes aportes al conocimiento.

Abstract The present commentary, which was written by virtue of the national government decree that designated 2021 as the year of the tribute to Dr. César Milstein, is a personal vision of the meaning of the development of monoclonal antibodies, of the contribution that he has always made to the current science and technology, and of the context of the corresponding Nobel Prize within the sphere of Immunology and the Medical Sciences. The aim of this article is to share, as a tribute, some of my experiences in the specialty and my professional and personal relationship with the creator of the immense breakthrough still being delivered by his development.

Resumo O presente comentário, escrito em virtude do decreto do governo nacional que nomeou o ano 2021 como ano de homenagem ao Dr. César Milstein, é uma visão pessoal sobre o significado do desenvolvimento dos anticorpos monoclonais, da contribuição que ofereceu e oferece à ciência e tecnologia atual e do contexto do correspondente Prêmio Nobel no âmbito da Imunologia e das Ciências Médicas. O objetivo deste artigo é transmitir, à maneira de homenagem, algumas das minhas experiências na especialidade e na relação profissional e pessoal com o criador do imenso avanço que continua nos oferecendo seu descobrimento.

Perspectivas atuais da engenharia de tecidos da articulação temporomandibular / Perspectivas Actuales de la Ingeniería de Tejidos para la Articulación Temporomandibular / Current Perspectives of Tissue Engineering for the Temporomandibular Joint

Introdução: As limitações das terapias atuais para doenças degenerativas da articulação temporomandibular (ATM) levaram ao aumento do interesse em estratégias regenerativas. A engenharia de tecidos (ET), combinando células-tronco, arcabouços e fatores de crescimento, pode fornecer uma substituição biológica funcional e permanente das estruturas da ATM, além de prevenir o avanço de doenças degenerativas. Objetivo: Este artigo descreve as perspectivas atuais da ET das estruturas da ATM em modelos animais. Metodologia: As abordagens da ET foram categorizadas de acordo com as estruturas primárias da ATM: 1) o disco articular, 2) o côndilo mandibular e 3) a fossa glenóide e eminência articular. Resultados: As áreas com a maior quantidade de estudos são o côndilo mandibular e disco articular, em estudos que abordam o uso de arcabouços tridimensionais, de origem sintética e/ou natural, podendo ou não estar associados a células tronco (diferenciadas ou não) e a fatores de crescimento. Conclusão: A ET da ATM ainda é uma área relativamente nova, em desenvolvimento e em constante avanço. Os avanços tecnológicos desenvolvidos nessa área têm o potencial de auxiliar no desenvolvimento de terapias mais eficientes e menos invasivos... (AU)

Introducción: Las limitaciones de las terapias actuales para las enfermedades degenerativas de la articulación temporomandibular (ATM) han llevado a un mayor interés en las estrategias regenerativas. La ingeniería de tejidos, que combina células, andamios y factores de crecimiento, puede proporcionar un reemplazo biológico funcional y permanente de las estructuras de la ATM, además de prevenir el avance de enfermedades degenerativas. Objetivo: Este artículo describe las perspectivas actuales de la ingeniería de tecidos de las estructuras de la ATM en modelos animales. Metodología: Los enfoques de ingeniería de tejidos se clasificaron según las estructuras primarias de la ATM: 1) el disco articular, 2) el cóndilo mandibular y 3) la fosa glenoidea y la eminencia articular. Resultados: Las áreas con mayor número de estudios son el cóndilo mandibular y el disco articular, en estudios que abordan el uso de estructuras tridimensionales, de origen sintético y/o natural, que pueden o no estar asociadas a células (diferenciadas o no) y con factores de crecimiento. Conclusión: La ingeniería de tejidos de la ATM es todavía un área relativamente nueva, en desarrollo y en constante avance. Los avances tecnológicos desarrollados en esta área tienen el potencial de ayudar en el desarrollo de terapias más eficientes y menos invasivas... (AU)

Introduction: The limitations of current therapies for degenerative diseases of the temporomandibular joint (TMJ) have led to increased interest in regenerative strategies. Tissue engineering (TE), combining stem cells, scaffolds, and growth factors, can provide a functional and permanent biological replacement of TMJ structures, in addition to preventing the advancement of degenerative diseases. Aim: This article describes current TE perspectives of TMJ structures in animal models. Methods: TE approaches were categorized according to the primary TMJ structures: 1) the articular disc, 2) the mandibular condyle, and 3) the glenoid fossa and articular eminence. Results: The areas with the greatest number of studies are the mandibular condyle and articular disc, in studies that address the use of three-dimensional scaffolds, of synthetic and/ or natural origin, which may or may not be associated with stem cells (differentiated or not) and with growth factors. Conclusion: TE of the TMJ is still a relatively new, developing, and constantly advancing area. The technological advances developed in this area have the potential to assist in the development of more efficient and less invasive therapies... (AU)

Lymphomatous Polyposis: Case Report and Literature Review / Poliposis linfomatosa: reporte de caso y revisión de la literatura

Abstract Lymphomatous polyposis (LP) is the endoscopic feature of primary gastrointestinal mantle cell lymphoma (MCL), a rare type of B-cell non-Hodgkin's lymphoma (NHL) and a typical but rare endoscopic pattern of gastrointestinal tract involvement (GIT) by nodal MCL. We present the case of a 62-year-old man with nodal MCL, with LP as a manifestation of GIT, and review the literature.

Resumen La poliposis linfomatosa (PL) es la característica endoscópica del linfoma de células del manto (LCM) gastrointestinal primario, un tipo infrecuente de linfoma no Hodgkin (LNH) de células B, así como un patrón endoscópico típico, pero infrecuente, del compromiso del tracto gastrointestinal (TGI) por LCM nodal. Presentamos el caso de un hombre de 62 años con LCM nodal, con PL como manifestación del compromiso gastrointestinal, y realizamos una revisión de la literatura.

Prospecção de novos candidatos a fármacos antituberculose: uma abordagem in vitro / Prospecting new candidates for antituberculosis drugs: an in vitro approach

A tuberculose (TB) é uma doença infecciosa causada pelo Mycobacterium tuberculosis que apresenta alta morbidade e mortalidade. Atualmente, a tuberculose afeta 9,9 milhões de pessoas em todo o mundo e é responsável por cerca de 1,3 milhões de mortes, sendo um sério problema de Saúde Pública global. O tratamento da TB é composto por uma associação de fármacos e dura seis meses. A frequência de doentes infectados com isolados resistentes aos fármacos de primeira linha, principalmente os isolados multirresistentes, é uma ameaça mundial que caracteriza um importante problema de saúde pública no controle da TB em vários países. Existem poucos medicamentos para o tratamento da TB muti-resistente e com base nesta problemática o presente estudo visou a avaliação do potencial farmacológico in vitro de fármacos aprovados, utilizando a abordagem de reposicionamento de fármacos. A partir de uma triagem in vitro de 89 fármacos, foram selecionados os que apresentaram atividade antituberculose para a avaliação da concentração inibitória mínima, os fármacos foram avaliados em exposição a 23 isolados do complexo Mycobacterium tuberculosis e a cepa ATCC H37 RV 27294 in vitro utilizando o ensaio de resazurina. Além disso, foram realizados ensaios de citotoxicidade contra células de mamíferos NCTC L929 em cultura, determinando-se os Índices de Seletividade in vitro. Dentre os 89 fármacos testados, quatro (amiodarona, flunarizina, ivermectina e pentamidina) apresentaram atividade antituberculose a 10 µM. A Concentração Inibitória Mínima (CIM) 90% da amiodarona resultou em 5-10 µM, flunarizina 10 µM, ivermectina 0,15 ­ 10 µM e pentamidina 1,25-10 µM. Os índices de seletividade observados para a amiodarona foram de 4,21 ­ 8,43, flunarizina >20, ivermectina 1,17 ­ 78,47 e pentamidina 14,40 ­ 115,2. Com base na atividade e citotoxicidade da amiodarona, foi realizado um ensaio fluorimétrico utilizando Sytox Green para avaliar a permeabilidade de membrana plasmática do complexo M. tuberculosis na presença deste fármaco. Foi observado que a amiodarona não alterou a permeabilidade da membrana plasmática do Complexo M. tuberculosis, tendo seu mecanismo de ação letal por outra via.

Cytotoxic evaluation and LC-MS/MS analysis of aerial parts of Eryngium kotschyi Boiss. grown in Turkey

Abstract Increasing biological activity and phytochemical investigations on Eryngium species showed its potential as pharmaceutical approach. Eryngium kotschyi Boiss. is one of the species of Eryngium genus and is endemic to Turkey. It is known that this plant is traditionally used in the South-western part of Turkey for the treatment of various diseases. This study focuses on cytotoxic activities of methanol extract and ethyl acetate, n-butanol and water sub-extracts from E. kotschyi in A549, COLO 205 and MDA-MB-231 cell lines by Sulforhodamin B assay and qualitative and quantitative determination of phytochemical constituents in active extract by LC-MS/MS. From the result of the study, it was seen that E. kotschyi ethyl acetate (EKE) sub-extract showed the strongest cytotoxic effect with the low IC50 values (50.00; 31.96 and 22.26 µg/mL in A549; COLO 205 and MDA-MB-231 cells at 48 h, respectively). Preliminary examination of the mass spectrums revealed the presence of 15 phytochemical compounds in active sub-extract and 7 of them was quantified. According to quantitative analyses the main compounds of EKE sub-extract were rosmarinic acid (485.603 µg/mgextract), chlorogenic acid (62.355 µg/mgextract) and caffeic acid (59.266 µg/mgextract). Moreover, this preliminary study on inhibitory activity of EKE sub-extract suggests further toxicologic investigations and detailed investigation on cytotoxic effect of various combinations of determined compounds

A influência de macrófagos M1, M2 e TAM, e do receptor P2X7 na invasividade e resistência de neuroblastoma / The influence of different macrophages' phenotypes, and of the P2X7 receptor, on the invasiveness and chemoresistance of neuroblastoma

O neuroblastoma é um tumor sólido muito comum em crianças. O estágio mais avançado da doença é altamente agressivo e invasivo, além de pouco responsivo à terapia, que é limitada por mecanismos de resistência e reincidência relacionados à metástase. Muitos estudos tem sido feitos para identificar mecanismos de invasão e quimioresistência de células tumorais, afim de aumentar a sobrevida dos pacientes com câncer. Nesse trabalho, nós estudamos o efeito dos macrófagos, as células imunes mais abundantes no microambiente tumoral, os TAMs (do inglês tumor-associated macrophage) e do receptor P2X7, um purinoreceptor acionado por ATP, nesses processos. Os TAMs respondem e atuam de acordo com a miríade de fatores que encontram, podendo gerar populações heterogêneas e com funções distintas, tanto antitumorais, como pró-tumorais. Altos níveis de ATP extracelular são encontrados no microambiente tumoral, podendo então ativar o receptor P2X7. Este receptor tem sido relacionado tanto a funções inflamatórias como funções na resolução da inflamação de macrófagos. Além disso, o receptor P2X7 está envolvido em uma variedade de eventos celulares, incluindo a secreção de mediadores pró-inflamatórios, a proliferação celular e a apoptose de células tumorais. Primeiramente, foi avaliado o papel do receptor P2X7 na polarização de macrófagos da derivados medula óssea de camundongos wild-type e nocaute para o P2X7 na presença e ausência de fatores secretados por células de neuroblastoma, e então foi estudada a influência desses diferentes macrófagos polarizados em eventos celulares de grande relevância clínica para o neuroblastoma: a invasividade e quimiorresistência. Os resultados demonstraram que, apesar do reconhecido envolvimento do receptor P2X7 na inflamação, a ausência deste receptor não atenua a expressão de marcadores característicos do fenótipo inflamatório, M1. O aumento da expressão do receptor P2Y2, também envolvido na inflamação, nessas células, sugere um mecanismo genético de compensação para não atenuação da inflamação em macrófagos que não expressam o receptor P2X7. Contudo, a ausência do receptor P2X7 levou a alterações no fenótipo alternativo, M2, de modo que a expressão de Tnf, marcador de M2, não foi reprimido. TAMs noucates para P2X7 tiveram a expressão de arg1, marcador de M2, suprimida, reforçando a importância do receptor P2X7 no estabelecimento de fenótipos ativados alternativamente. Nossos dados também sugerem que ausência do receptor P2X7 em TAMs permite a aquisição de um fenótipo capaz de tornar as células de neuroblastoma que expressam P2X7 mais invasivas e mais quimioresistentes à vincristina. Por outro lado, TAMs, independentemente da presença ou ausência do receptor P2X7, induziram a proliferação e quimioresistência das células de neuroblastoma silenciadas para o receptor P2X7, o que nos leva a concluir que o receptor P2X7 em TAMs é desfavorável à progressão de tumores expressando P2X7

Neuroblastoma is a highly common childhood solid tumor. The most advanced stage of the disease is highly aggressive and invasive, besides from being poorly responsive to therapies, which are limited by resistance and recurrence mechanisms related to metastasis. Several studies attempt to identify invasion and resistance mechanisms of the tumor cells in order to increase overall survival of the patients. On the present work, we investigated the effect of macrophages, the most abundant immune cells on the tumor microenvironment, called TAMs (tumor-associated macrophages), and of the P2X7 receptor, an ATP-gated purinoceptor, on these processes. TAMs and cancer cells crosstalk, and behave accordingly to a miriad of factors present at the TME, generating heterogeneous populations with distinct functionalities, either pro- or antitumor. High extracellular levels of ATP are found in the TME, being able to activate the P2X7 receptor. This receptor mediates both pro- and anti-inflammatory functions in macrophages. In addition, it is involved in several cellular events, including the secretion of pro-inflammatory mediators, cell proliferation and tumor cell apoptosis. At first, we evaluated the role of the P2X7 receptor on the polarization of bone marrow-derived macrophages (BMDM), either wild-type or knockout for the P2X7 receptor, in presence or absence or factors secreted by neuroblastoma cells. Next, we investigated the influence of the polarized macrophages in highly relevant cellular events for neuroblastoma, such as invasiveness and chemoresistance. Our results showed that, despite the known involvement of P2X7 receptor on inflammation, its absence did not decrease the expression if inflammatory markers of M1 macrophage populations. An increase in the expression of the P2Y2 receptor, also involved in inflammation, on these cells suggest a genetic compensation mechanism for preventing attenuation of inflammation when P2X7 is lacking. However, P2X7 receptor absence did compromise the M2 phenotype, driving the expression of Tnf. TAMs knockout for the P2X7 receptor were not able to express arg1, also an M2 marker, reinforcing a role of the P2X7 receptor on establishing alternative macrophage phenotypes. Our data also suggest that TAMs lacking the P2X7 receptor acquire a phenotype capable of turning P2X7R-expressing neuroblastoma cells more invasive and chemoresistant to vincristine. On the other hand, TAMs, independently on the presence of the P2X7 receptor, induced proliferation and resistance of neuroblastoma cells silenced for P2X7 receptor expression, leading us to the conclusion that the P2X7 receptor in TAMs is unfavorable for the progression of P2X7R-expressing tumors

Análise proteômica de células BEAS-2B expostas a benzo[α]pireno e nicotinamida ribosídeo / Proteomic Analysis of BEAS-2B cells exposed to benzo[α]pyrene and nicotinamide riboside

Responsável por milhões de óbitos anuais e um grande custo para a saúde pública, o câncer é a segunda maior causa de mortes no mundo. Dentre seus diversos tipos, o câncer de pulmão, além da alta incidência, é um dos mais letais. A exposição a substâncias tóxicas provenientes da combustão de matéria orgânica, assim como o consumo de cigarro, são os principais responsáveis pela alta incidência de câncer de pulmão. Dentre estas substâncias, está o benzo[α]pireno (B[α]P), um carcinógeno completo, ou seja, capaz de iniciar e promover o processo de carcinogênese. Resultados anteriores obtidos pelo grupo demonstraram que células BEAS-2B expostas a 1 µM de B[α]P apresentaram alterações das concentrações de metabólitos intracelulares, indução de estresse redox e hipermetilação do DNA. A exposição a 1 µM de nicotinamida ribosídeo (NR), um dos precursores de NAD+, foi capaz de proteger as células BEAS-2B contra a transformação induzida por B[α]P, além de impedir totalmente que células não expostas a B[α]P formassem colônias em soft-agar. A utilização da proteômica neste trabalho permitiu verificar a abundância das proteínas nos quatro diferentes grupos de exposição: Controle, B[α]P, B[α]P + NR e NR. Após 120 h de exposição as células foram coletadas, as proteínas extraídas e preparadas para análise. Foram descobertas 3024 proteínas posteriormente analisadas com o objetivo de elucidar vias possivelmente envolvidas na proteção contra o processo de transfomação maligna. Os grupos NR e Controle demonstram ser mais parecidos em relação ao seu conteúdo, enquanto os grupos B[α]P e B[α]P + NR foram mais semelhantes entre si. A análise de proteínas exclusivas revelou menos processos relacionados ao reparo de DNA no grupo tratado apenas com B[α]P quando comparado com B[α]P + NR. A análise estatística do total de proteínas utilizando o teste ANOVA (p < 0,05, N = 5) revelou 564 proteínas diferencialmente expressas entre os grupos. A clusterização nos permitiu observar a diferença na abundância de proteínas entre os quatro tratamentos. As proteínas estão envolvidas em funções como a regulação do metabolismo, resposta a estresse, transdução de sinal, regulação de expressão gênica e morte celular. Um dos clusters (cluster 1), contendo 59 proteínas, revelou poucos processos na análise de enriquecimento, mas as proteínas contidas nele apresentam funções como controle da divisão celular, apoptose e proteção ao estresse redox. Nele podemos observar que, no geral, o tratamento com B[α]P aumentou a abundância de algumas proteínas, o que foi revertido no grupo B[α]P + NR. O tratamento apenas com NR diminuiu a abundância das proteínas contidas nesse cluster. Outro cluster (cluster 4) apresentou 51 proteínas de abundância diminuída durante a exposição ao B[α]P, o que se reverteu no grupo B[α]P + NR. As proteínas desse cluster estão envolvidas em etapas importantes da via glicolítica, de crescimento, adesão, migração e invasão celular. Apesar de ser descrito que a exposição a NR pode aumentar a eficiência do reparo de DNA, os resultados apresentados nesse trabalho indicam que o efeito protetor pode estar relacionado com a modulação do ciclo celular ou alterações na adesão celular

Responsible for millions of annual deaths and a great health expense, cancer is the second leading cause of death in the world. Among its many types, lung cancer, besides its high incidence, is also one of the most lethal. Exposure to toxic substances resulting from the combustion of organic matter, as well as cigarette consumption, are the mainly responsible for the high incidence of lung cancer. One of these substances is benzo[α]pyrene (B[α]P), a complete carcinogen, able to initiate and promote the carcinogenesis process. Results obtained previously demonstrated that BEAS-2B cells exposed to 1 µM BaP presented alterations in the levels of intracellular metabolites, induction of oxidative stress, and hypermethylation of DNA. The exposure to 1 µM nicotinamide riboside (NR), one of the precursors of NAD+, was able to protect BEAS-2B cells against the transformation induced by B[α]P, moreover, it also totally prevented the colonies formation on soft agar in cells not exposed to B[α]P. The use of proteomics allowed us to verify the abundance of proteins in the four different exposure groups: Control, B[α]P, B[α]P + NR e NR. After 120h of exposure, the cells were collected followed by the extraction of the proteins. A total of 3024 proteins were identified and analyzed aiming to elucidate possible pathways involved in the protective effect against the malignant transformation induced by B[α]P. The NR and Control groups showed to be more similar, while B[α]P and B[α]P + NR were more similar. The analysis of exclusive proteins revealed fewer processes related to DNA repair in B[α]P when compared with B[α]P + NR. The statistical analysis of the total proteins using the ANOVA test (p <0.5, N = 5) revealed 564 proteins differentially expressed between the groups. The heatmap showed the difference in protein abundance between the four treatments. Proteins are involved in functionssuch asthe regulation of metabolism, stress response, signal transduction, regulation of gene expression, and cell death. One of the clusters (cluster 1), containing 59 proteins, revealed a few processes in the enrichment analysis, but the proteins contained in it have functions such as control of cell division, apoptosis, and protection from redox stress. It is possible to observe, in general, treatment with B[α]P increased the abundance of some proteins, which was partially reversed in group B[α]P + NR. On the other hand, the NR treatment decreased the abundance of proteins contained in this cluster. Another cluster (cluster 4) showed 51 proteins of decreased abundance during exposure to B [α] P, which was partially reversed in group B[α]P + NR. The proteins in this cluster are involved in important stages of the glycolytic pathway, also in growth, adhesion, migration, and cell invasion. Although it has been described that exposure to NR can increase the efficiency of DNA repair, the results presented in this work indicate that the protective effect may be related to the modulation of the cell cycle or cell adehsion modifications

In vitro evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics

Abstract The consumption of cosmetics has been increasing every year and is expected to reach $675 billion by 2020 at an estimated growth rate of 6.4% per year. Exposure to skin irritants is the major cause of non-immunological inflammation of the skin. Therefore, the safety evaluation of cosmetic preservatives should be increased. Thus, the present work aimed to evaluate the cytotoxicity as the viability endpoint and the eye irritation potential of preservatives used in cosmetics. Cytotoxicity assays were performed using MTT and NRU in human keratinocytes (HaCaT), human dermal fibroblasts, adult (HDFa), and human hepatoma cells (HepG2). The eye irritation potential was evaluated using the Hen's Egg Test-chorioallantoic membrane (HET-CAM). The evaluated preservatives were methylparaben (MP), propylparaben (PP), phenoxyethanol (PE), and a mixture of methylchloroisothiazolinone and methylisothiazolinone (CMI/MI). All preservatives showed cytotoxic potential within the permitted concentrations for use in cosmetic products. In the HET-CAM test, PE and CMI/MI, MP, and PP were classified as severe, moderate, and poor irritants, respectively. Our results indicate that proper safety evaluations are required to ensure the beneficial properties of preservatives on cosmetic products without exceeding exposure levels that would result in adverse health effects for consumers.

Selective Cytotoxic effect of Probiotic, Paraprobiotic and Postbiotics of L. casei strains against Colorectal Cancer Cells: Invitro studies

Abstract This study highlights the cytotoxic effect of three L. casei strains on colorectal cell lines in invitro conditions. Different concentrations of live, heat killed (HK) and cell free supernatant (CFS) of three L.casei strains were subjected to CaCo2 and MRC5 cell lines. The viability of the treated and untreated cells was determined after 72 hrs by MTT assay, and IC50 estimated. Apoptosis was evaluated by Annexin V-propidium iodide method using flow cytometry. The live, HK and CFS of the L. casei strains showed cytotoxic effects on colorectal cell lines with significant differences. The cytotoxicity effects of live cells on CaCo2 cells were significantly higher (p˂0.01) than the HK cells. A dose dependent response was observed, as higher concentrations resulted in enhanced cytotoxicity effects. Live L.casei 1296-2cells inhibited 91% of CaCo2 cell growth, with IC50 of less than 108 cfu/ml. MRS medium and concentrations of CFS at above 20% v/v, were cytotoxic to the normal cell lines. Flow cytometry analyses of L. casei 1296-2 indicated that cytotoxicity effects on CaCo2 cells is related to apoptotic induction. Invitro studies indicate that Live and CFS of L. casei 1296-2 might be promising candidate for the control of colorectal cancers

Bioprocess optimization of interferon ß-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

Caracterização funcional do gene codificador de proteína CD14 e do gene não codificador de proteína LINC01133 em linhagens celulares humanas de câncer de mama triplo negativo / Functional characterization of the CD14 protein-coding gene and the non-protein-coding LINC01133 gene in triple negative human breast cancer cell lines

Dentre os subtipos de câncer de mama, o triplo negativo (TNBC) é o que apresenta as maiores taxas de mortalidade, sendo, portanto, considerado um enorme desafio para a clínica. O uso de moléculas como marcadores tumorais vem auxiliando o clínico no diagnóstico, no prognóstico e, até mesmo, no tratamento do TNBC, sendo essenciais na redução de suas altas taxa de mortalidade. No entanto, um pequeno grupo de marcadores tumorais são validados na prática clínica, estimulando à busca por novos alvos, e sua caracterização funcional, como forma de se entender a Biologia desta doença. Assim, o objetivo deste trabalho é caracterizar funcionalmente o gene codificador de proteína CD14 e o gene não codificador de proteína LINC01133 em linhagens celulares humanas de TNBC, no intuito de descobrir o papel destas moléculas na progressão tumoral. Na primeira parte deste trabalho, analisou-se a expressão do CD14 frente à um painel de linhagens celulares que representam os diferentes subtipos dos tumores mamários. O CD14 exibiu elevados níveis de expressão nas linhagens nãotumorigênicas MCF10A e MCF12A e baixos níveis na linhagem triplo negativa Hs578T. A partir destes resultados, o CD14 foi superexpresso na linhagem Hs578T. Ensaios de caracterização funcional mostraram que a superexpressão do CD14 reduziu a capacidade migratória e invasiva das células, efeito que foi hipoteticamente relacionado ao aumento da expressão da E-caderina. No entanto, observou-se aumento no potencial tumorigênico, levando-nos a sugerir seu envolvimento num possível mecanismo utilizado pelas células para compensar a significativa redução do potencial migratório e invasivo. Os resultados obtidos indicam que o nível basal de expressão do CD14 observado na linhagem Hs578T é importante, podendo contribuir para a desenvolvimento primário do tumor, atuando como um oncogene. Na segunda parte deste trabalho, analisou-se a expressão de 10 RNAs longos não codificadores (lncRNAs), frente ao mesmo painel de linhagens descritoanteriormente. Dentre estes, o lncRNA LINC01133 exibiu baixos níveis de expressão nas linhagens não-tumorigênicas MCF10A e MCF12A e elevados níveis na linhagem triplo negativa Hs578T, sendo, então, escolhido como alvo de estudo. A partir destes resultados, decidimos superexpressar, de forma indutível, o LINC01133 na linhagem MCF10A e nocautear este gene, via sistema CRISPR/Cas9, na linhagem Hs578T. Ensaios de caracterização funcional mostraram que a superexpressão do LINC01133 na linhagem MCF10A reduziu a proliferação celular e inibiu o crescimento de colônias dependente de ancoragem, mas, em contrapartida, aumentou o crescimento de colônias independente de ancoragem e a capacidade migratória e invasiva destas células. No entanto, sugerimos que isto não seja suficiente para tornar estas células tumorigênicas e metastáticas. Por outro lado, o nocauteamento do LINC01133 na linhagem triplo negativa Hs578T aumentou de forma considerável todos os parâmetros de malignidade analisados. Baseado nos dados obtidos, sugerimos que o elevado nível de expressão do LINC01133 na linhagem Hs578T é importante na regulação negativa de processos relacionados com a progressão tumoral, atuando com um supressor tumoral. Os dados obtidos em nosso estudo contribuem para o enriquecimento de informações relacionadas à Biologia do TNBC, auxiliando, desta forma, no desenvolvimento de potenciais protocolos clínicos e terapêuticos utilizandos estes biomarcadores

Among the breast cancer subtypes, the triple negative (TNBC) displays the highest mortality rates, being, therefore, considered a major challenge for the clinic. The use of molecules as tumor markers has helped clinicians in the diagnosis, prognosis and even in treatment of TNBC, being essential in reducing its high mortality rate. However, a small group of tumor markers is validated in clinical practice, stimulating the search for new targets, and their functional characterization, as a way to understand the biology of this disease. Thus, the aim of this work is to functionally characterize the CD14 protein-coding gene and the non-protein-coding LINC01133 gene in human TNBC cell lines, in order to probe into the role of these molecules in tumor progression. In the first part of this work, the expression of CD14 was analyzed in a panel of cell lines that represent the different subtypes of breast tumors. High expression levels of CD14 were observed in the non-tumorigenic MCF10A and MCF12A lineages and low levels in the triple negative Hs578T lineage. Based on these results, CD14 was overexpressed in the Hs578T lineage. Functional characterization assays showed that CD14 overexpression reduced the migratory and invasive capacity of cells, an effect that was hypothetically related to increased E-cadherin expression. However, increased in the tumorigenic potential was observed, leading us to suggest its involvement in a possible mechanism used by cells to compensate for the significant reduction in the migratory and invasive potential. The results obtained indicate that CD14 expression basal level observed in the Hs578T lineage may be important to contribute to the primary development of tumor, thus acting as an oncogene. In the second part of this work, the expression of 10 long non-coding RNAs (lncRNAs) was analyzed against the same lineage panel described above. Among these, the LINC01133 lncRNA exhibited low expression levels in the non-tumorigenic MCF10A and MCF12A lineages and high levels in the triple negative Hs578T lineage, being, then, chosen as a target for this study. Based on these results, we decided toinducibly overexpress LINC01133 in the MCF10A lineage and knockout this gene, via the CRISPR/Cas9 system, in the Hs578T lineage. Functional characterization assays showed that overexpression of LINC01133 in the MCF10A lineage reduced cell proliferation and inhibited anchorage-dependent colony growth, but, on the other hand, increased anchorage-independent colony growth and the migratory and invasive capacity of these cells. However, we suggest that this is not sufficient to render these cells tumorigenic and metastatic. On the other hand, the knockout of LINC01133 in the triple negative Hs578T lineage considerably increased all the analyzed malignancy parameters. Based on the results obtained, we suggest that the high expression level of LINC01133 in the Hs578T lineage is important for down-regulation of processes related to tumor progression, acting as a tumor suppressor. The data obtained in our study contribute to the enrichment of information related to TNBC Biology, thus assisting in the development of potential clinical and therapeutic protocols using these biomarkers

Expressão e localização celular da diguanilato ciclase DgcP em Pseudomonas aeruginosa / Expression and cell localization of the diguanylate cyclase DgcP in Pseudomonas aeruginosa

A bactéria Gram-negativa Pseudomonas aeruginosa é um patógeno oportunista frequentemente associado a vítimas de queimaduras graves ou indivíduos com fibrose cística, sendo os isolados resistentes a carbepenêmicos dessa espécie considerados pela OMS como uma das maiores ameaças ao controle de infecções. O estabelecimento da infecção por esse patógeno é dependente de uma série de fatores de virulência, entre eles o pilus tipo IV (T4P), que possui papel importante na adesão a superfícies e motilidade do tipo twitching, essenciais para a colonização do hospedeiro. Uma das moléculas importantes na diferenciação entre as formas séssil e planctônica de P. aeruginosa é o segundo mensageiro bis-(3,5)-di-guanosina monofosfato cíclico (c-di-GMP), cuja síntese é feita enzimaticamente por diguanilato ciclases (DGCs). DgcP é uma DGC localizada nos polos da célula, que tem sua atividade de síntese de c-di-GMP aumentada na presença da proteína FimV, essencial para a montagem do T4P em P. aeruginosa. Neste trabalho, ensaios de microscopia de fluorescência, organização e expressão gênica foram realizados com o objetivo de aumentar a compreensão sobre o papel de DgcP em relação a sua expressão e aos fatores que regulam o T4P de P. aeruginosa. A proteína DgcP em fusão com mNeonGreen no C-terminal, expressa a partir do locus cromossômico, se localiza de maneira predominantemente bipolar tanto na linhagem selvagem quanto nos mutantes ΔpilA, ΔpilR e ΔchpA, evidenciando que seu padrão de localização não depende dos sistemas de regulação Pil-Chp e PilS-PilR. Ensaios de RT-PCRmostraram que dgcP se encontra em operon com PA14_72430 e dsbA1, indicando um papel celular conjunto entre esses genes, até o momento, desconhecido. Por fim, ensaios de qRT-PCR revelaram que os níveis de mRNA de dgcP são invariáveis nas linhagens WT, ΔpilA, ΔpilR, ΔchpA e ΔfimV, cultivadas em meio líquido ou meio sólido. Os resultados aqui mostrados, combinados com trabalhos prévios do nosso e de outros grupos, sugerem que DgcP é uma diguanilato ciclase responsável por geração constante de c-di-GMP nos polos da célula, possivelmente, atuando na sinalização local dependente do dinucleotídeo cíclico, cuja localização e atividade não são dependentes dos sistemas de regulação que atuam sobre o T4P

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe burn victims or individuals with cystic fibrosis, which carbapenem-resistant isolates were classified by th World Health Organization classified one of the greatest threats to infection control. The establishment of infection by this pathogen is dependent on a series of virulence factors, including the type IV pilus (T4P), which plays an important role in adhesion to surfaces and twitching motility, essential features for host colonization. Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that involved in processes of biofilm formation, motility, and virulence. The diguanylate cyclase DgcP synthetizes cdi-GMP and it is located at the cell poles, and its activity depends on the scaffold protein FimV, essential for T4P assembly in P. aeruginosa. By increasing c-di-GMP levels, DgcP decreases flagellum-dependent motility and increases biofilm formation. In this work, fluorescence microscopy, gene organization and expression assays were performed to understand the whether DgcP localization and expression are under the control of T4P regulatory proteins. Fluorescence microscopy analysis showed that DgcP localizes predominantly at both cell poles in ΔpilA, ΔpilR, and ΔchpA mutants, showing that its localization pattern does not depend on the Pil-Chp and PilS-PilR systems. Furthermore, RT-PCR assays showed that dgcP is found in an operon with PA14_72430 and dsbA1, indicating an unknown putative related cellular role for these genes. Finally, qRT-PCR assays indicated that DgcP expression is invariant in ΔpilA, ΔpilR, ΔchpA, and ΔfimV mutants, either in liquid or solid medium. The results shownhere, combined with previous work by ours and other groups, suggest that DgcP is a diguanylate cyclase responsible for constant generation of c-di-GMP at the cell poles, possibly acting in local signaling dependent on the cyclic dinucleotide, but that is not under the control of the known T4P regulatory systems

Producción de astaxantina bajo factores de estrés utilizando un biorreactor a escala de laboratorio de 5 L / Astaxanthin production under stress factors using a 5L laboratory scale bioreactor / Astaxanthin production under stress factors using a 5L laboratory scale bioreactor

Resumen Introducción. Haematococcus pluvialis es una microalga que produce astaxantina, un beta-caroteno y antioxidante muy usado en la industria. Para obtener una mayor producción de astaxantina se planteó como objetivo utilizar diferentes factores de estrés, en un biorreactor a escala de laboratorio de 5 litros. Metodología. Se cultivó la microalga en el medio RM, pH 6,8, temperatura 20±2°C, aire filtrado, iluminación con lámparas blancas 20h luz/4h oscuridad, irradianza 70 μE m-2s-1, diferentes concentraciones de acetato de sodio y cloruro de sodio. Se determinó crecimiento celular, cambios morfológicos y cuantificación de astaxantina y clorofila por espectrofotometría. Se realizó un análisis estadístico utilizando ANOVA (95%). Resultados. Utilizando 0,299 mg/L de acetato de sodio se obtuvo un crecimiento celular de 2,0 x 104 Cel/mL y una concentración de astaxantina de 2,530 μg/mL, mientras que con 1,6 mg/L de acetato de sodio el crecimiento celular fue de 3,5 x 104 Cel/mL y una concentración de astaxantina de 1,9 μg/ml. El tratamiento al cual se le adicionó 1,6 g/L de acetato de sodio y 6,4 g/L de cloruro de sodio presentó la mayor producción astaxantina 7,3 μg/ml. El tratamiento con acetato de sodio 0,320 g/L + cloruro de sodio 1,28 g/L presentó el mayor crecimiento celular con 1,64x105 células/ml. Conclusión. Esta investigación destaca la importancia de cultivar inicialmente la microalga utilizando el biorreactor Tecferm de 5 litros y después de su fase exponencial someterla a factores de estrés con acetato de sodio y cloruro de sodio lográndose así la mayor producción de astaxantina 7,325 μg/ml.

Abstract Introduction. Haematococcuspluvialis is a microalgae that produces astaxanthin, a beta-carotene and antioxidant widely used in industry. In order to obtain a higher production of astaxanthin, the objective was to use different stress factors, in a 5-liter laboratory-scale bioreactor. Methodology. The microalgae was cultivated in the RM medium, pH 6.8, temperature 20 ± 2°C, filtered air, illumination with white lamps 20h light/4h darkness, irradiance 70 μE m-2s-1, different concentrations of sodium acetate and chloride of sodium. Cell growth, morphological changes and quantification of astaxanthin and chlorophyll were determined by spectrophotometry. Statistical analysis was performed using ANOVA (95%). Results. Using 0.299 mg/L of sodium acetate a cell growth of 2.0 x 104 Cel/mL and an astaxanthin concentration of 2.530 μg/mL were obtained, while with 1.6 mg/L of sodium acetate the cell growth It was 3.5 x 104 Cel/mL and an astaxanthin concentration of 1.9 μg/mL. The treatment to which 1.6 g L of sodium acetate and 6.4 g/L of sodium chloride were added showed the highest astaxanthin production, 7.3 μg/ml. Treatment with 0.320 g/L sodium acetate + 1.28 g/L sodium chloride showed the highest cell growth with 1.64x105 cells/ml. Conclusion. This research highlights the importance of initially cultivating the microalgae using the 5-liter Tecferm bioreactor and, after its exponential phase, subjecting it to stress factors with sodium acetate and sodium chloride, thus achieving the highest production of 7.325 μg/ml astaxanthin.