Optimization of parameters for obtaining callus, suspension, and root cultures of meadowsweet (filipendula ulmaria) to isolate the largest number of biologically active substances with geroprotective properties / Otimização de parâmetros para obtenção de culturas de calos, suspensão e raiz de Ulmária (Filipendula ulmaria) para isolar o maior número de substâncias biologicamente ativas com propriedades geroprotetoras

The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.

O estudo de substâncias biologicamente ativas - metabólitos secundários de plantas que apresentam propriedades geroprotetoras - é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.

Optimization of parameters for obtaining callus, suspension, and root cultures of meadowsweet (filipendula ulmaria) to isolate the largest number of biologically active substances with geroprotective properties

Abstract The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.

Resumo O estudo de substâncias biologicamente ativas metabólitos secundários de plantas que apresentam propriedades geroprotetoras é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.

Potential Clinical Application of Organs-on-a-Chip in Periodontal Diseases: A Systematic Review of In Vitro Studies.

The periodontium is a unique organ from the standpoint of building an organ-on-a-chip (OoC) since it is a system that is continually threatened by microorganisms, their noxious compounds, and antigenic components. At the same time, periodontal health depends on a balanced connection between the host and the bacteria in the oral cavity, which is a complex micro-ecological environment. The objective of this systematic review of in vitro studies is to revise the potential clinical application of OoC in periodontal diseases. PRISMA was used to guide this analysis. The review framework made use of several databases, including SCOPUS, PubMed/MEDLINE, SCIELO, and LILACS as well as the gray literature. This systematic review comprised seven studies. The clinical efficacy of OoC in periodontal diseases was observed in models of the gingival crevice for the research of periodontitis, periodontal medication analysis, the interaction of multiple microbial species, pH measurements in in situ-grown biofilm, testing antimicrobial reagents, evaluation of mucosal interactions with microorganisms, and a device for quantitative exploration of microorganisms. OoC has the potential to advance our understanding of periodontal diseases by providing a more accurate representation of the oral microenvironment and enabling the development of new treatments.

Reprogramming of the Genome-Wide DNA Methylation Landscape in Three-Dimensional Cancer Cell Cultures.

During the last century, 2D cell cultures have been the tool most widely used to study cancer biology, drug discovery, genomics, and the regulation of gene expression at genetic/epigenetic levels. However, this experimental approach has limitations in faithfully recreating the microenvironment and cellular processes occurring in tumors. For these reasons, 3D cell cultures have recently been implemented to optimize the conditions that better recreate the biological and molecular features of tumors, including cell-cell and cell-extracellular matrix (ECM) interactions, growth kinetics, metabolic activities, and the development of gradients in the cellular microenvironment affecting the availability of oxygen and nutrients. In this sense, tumor cells receive stimuli from the local environment, resulting in significant changes in their signaling pathways, gene expression, and transcriptional and epigenetic patterns. In this review, we discuss how different types of 3D cell culture models can be applied to characterize the epigenetic footprints of cancer cell lines, emphasizing that DNA methylation patterns play an essential role in the emergence and development of cancer. However, how 3D cancer cell cultures remodel the epigenetic programs is poorly understood, with very few studies in this emerging topic. Here, we have summarized the studies on the reprogramming of the epigenetic landscape of DNA methylation during tumorigenesis and discuss how it may be affected by microenvironmental factors, specifically in 3D cell cultures.

Obtaining 2,3-Dihydrobenzofuran and 3-Epilupeol from Ageratina pichinchensis (Kunth) R.King & Ho.Rob. Cell Cultures Grown in Shake Flasks under Photoperiod and Darkness, and Its Scale-Up to an Airlift Bioreactor for Enhanced Production.

Ageratina pichinchensis (Kunth) R.King & Ho.Rob. is a plant used in traditional Mexican medicine, and some biotechnological studies have shown that its calluses and cell suspension cultures can produce important anti-inflammatory compounds. In this study, we established a cell culture of A. pichinchensis in a 2 L airlift bioreactor and evaluated the production of the anti-inflammatory compounds 2,3-dihydrobenzofuran (1) and 3-epilupeol (2). The maximum biomass production (11.90 ± 2.48 g/L) was reached at 11 days of culture and cell viability was between 80% and 90%. Among kinetic parameters, the specific growth rate (µ) was 0.2216 days-1 and doubling time (td) was 3.13 days. Gas chromatography coupled with mass spectrometry (GC-MS) analysis of extracts showed the maximum production of compound 1 (903.02 ± 41.06 µg/g extract) and compound 2 (561.63 ± 10.63 µg/g extract) at 7 and 14 days, respectively. This study stands out for the significant production of 2,3-dihydrobenzofuran and 3-epilupeol and by the significant reduction in production time compared to callus and cell suspension cultures, previously reported. To date, these compounds have not been found in the wild plant, i.e., its production has only been reported in cell cultures of A. pichinchensis. Therefore, plant cell cultured in an airlift reactor can be an alternative for the improved production of these anti-inflammatory compounds.

Anti-hyperglycemic activity of aqueous extracts of cells produced in Ibervillea sonorae (S. Watson) Green cell culture on murine model / Actividad anti-hiperglucémica del extracto acuoso de células producidas en cultivo celular de Ibervillea sonorae (S. Watson) Green en un modelo murino

Ibervillea sonorae (S. Watson) Greene, is a plant native to Mexico, where its roots have been used traditionally for treating Diabetes Mellitus. The aim of this work was to establishment of cell cultures of stem explants of I. sonorae and evaluation of the anti-hyperglycemic activity of cell aqueous extract on a murine model of streptozotocin-induced diabetic rats. Cell extracts had 2.29 mg palmitic acid/g extracted, and other compounds with pharmacological activities like palmitoyl ethanolamide and palmitoyl tryptamine were also identified. Diabetic rats treated with aqueous cell extract decreased glucose levels from 350 mg/dL to 145 mg/dL, AST and ALT from 164 U/L to 49 U/L and 99 U/L to 53 U/L, respectively. Additionally, there were no changes in the cellular morphology of the pancreas, liver, kidneys, and spleen. These results revealed that the cell aqueous extract from stem explants has anti-hyperglycemic activity.

Ibervillea sonorae (S. Watson) Greene, es una planta originaria de México, donde sus raíces se han utilizado tradicionalmente para el tratamiento de la Diabetes Mellitus. El objetivo de este trabajo fue el establecimiento de cultivos celulares de explantes de tallo de I. sonorae y la evaluación de la actividad anti-hiperglucémica del extracto acuoso celular en un modelo de ratas diabéticas inducidas con estreptozotocina. El extracto celular contiene 2.29 mg de ácido palmítico/g extracto y se identificaron otros compuestos como palmitoil etanolamida y palmitoil triptamina. Las ratas diabéticas tratadas con extracto celular disminuyeron los niveles de glucosa de 350 mg/dL a 145 mg/dL, AST y ALT de 164 U/L a 49 U/L y 99 U/L a 53 U/L, respectivamente. Además, no hubo cambios en la morfología celular del páncreas, hígado, riñones y bazo. Estos resultados indican que el extracto de células de explantes de tallo de I. sonorae tiene actividad anti-hiperglucémica.

A Protocol to Produce Genetically Edited Primary Oral Keratinocytes Using the CRISPR-Cas9 System.

The Nobel Prize awarded gene editing system, CRISPR-Cas9, is probably one of the greatest achievements of the last decades. CRISPR-Cas9 can introduce irreversible genomic changes in its target DNA by simple specifying a 20-nucleotide sequence within its RNA guide. Due to its simplicity, efficacy, and relative low cost in comparison with other genome editing systems, it has become the most common gene editing system used in research laboratories. Here we describe a step-by-step protocol to produce genetically edited primary oral keratinocytes using the CRISPR-Cas9 system.

Green Synthesis of Silver Nanoparticles Using Randia aculeata L. Cell Culture Extracts, Characterization, and Evaluation of Antibacterial and Antiproliferative Activity.

The demand for metallic nanoparticles synthesized using green methods has increased due to their various therapeutic and clinical applications, and plant biotechnology may be a potential resource facilitating sustainable methods of AgNPs synthesis. In this study, we evaluate the capacity of extracts from Randia aculeata cell suspension culture (CSC) in the synthesis of AgNPs at different pH values, and their activity against pathogenic bacteria and cancer cells was evaluated. Using aqueous CSC extracts, AgNPs were synthesized with 10% (w/v) of fresh biomass and AgNO3 (1 mM) at a ratio of 1:1 for 24 h of incubation and constant agitation. UV-vis analysis showed a high concentration of AgNPs as the pH increased, and TEM analysis showed polydisperse nanoparticles with sizes from 10 to 90 nm. Moreover, CSC extracts produce reducing agents such as phenolic compounds (162.2 ± 27.9 mg gallic acid equivalent/100 g biomass) and flavonoids (122.07 ± 8.2 mg quercetin equivalent/100 g biomass). Notably, AgNPs had strong activity against E. coli, S. pyogenes, P. aeruginosa, S. aureus, and S. typhimurium, mainly with AgNPs at pH 6 (MIC: 1.6 to 3.9 µg/mL). AgNPs at pH 6 and 10 had a high antiproliferative effect on cancer cells (IC50 < 5.7 µg/mL). Therefore, the use of cell suspension cultures may be a sustainable option for the green synthesis of AgNPs.

Cell culture: a promising environment for research on cardiology / Cultivos celulares: un entorno promisorio para la investigación en cardiología

Abstract At present, tissue engineering is transforming the area of cardiovascular regenerative medicine, which combines the principles and methods of materials engineering and biological sciences, interacting with biochemical and physicochemical factors, for the understanding of their structure-function relationship. Thus, the course of diseases is reoriented by implementing methods and procedures involved in the regeneration of organs and tissues by means of the interaction with biocompatible matrices, pre-treated organs or stem cell management, among others, thus recovering the functionality in the system affected by acquired pathologies, alterations or congenital defects. Consequently, these procedures are increasingly becoming one the most promising treatment alternative for patients who suffer from any type of functional deficit. Known that all these possibilities make cell cultures a promising study environment to be used in biomedical applications, especially in tissue engineering and regenerative medicine, this manuscript presents a general reviews of established cell lines or primary tissue lines and how cell cultures serve as a model before experimental work on laboratory animals and human subjects which makes it a valuable tool for broad models of study in the research on cardiology.

Resumen En la actualidad, la ingeniería de tejidos está transformando el área de la medicina regenerativa cardiovascular, combinando los principios y métodos de la ingeniería de materiales y las ciencias biológicas, interactuando entre factores bioquímicos y fisicoquímicos, para la comprensión de su relación estructura-función. Así, el curso de las enfermedades se viene a reorientar mediante la implementación de métodos y procedimientos implicados en la regeneración de órganos y tejidos a través de la interacción con matrices biocompatibles, órganos pretratados o manejo de células madre, entre otros, recuperando así la funcionalidad en el sistema afectado por enfermedades adquiridas y alteraciones o defectos congénitos. En consecuencia, estos procedimientos se están convirtiendo en una de las alternativas de tratamiento cada vez más prometedoras para los pacientes que sufren de algún tipo de alteración funcional. Considerando que todas estas posibilidades hacen de los cultivos celulares un entorno de estudio prometedor para ser utilizado en aplicaciones biomédicas, especialmente en ingeniería de tejidos y medicina regenerativa, este manuscrito presenta una revisión general de las líneas celulares establecidas o líneas de tejido primario y cómo los cultivos celulares sirven como modelo de evaluación antes del trabajo experimental en animales de laboratorio y sujetos humanos, lo cual los convierte en una herramienta valiosa para amplios modelos de estudio en la investigación en cardiología.

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells.

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal-glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10-P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-ßIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and ßIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and ßIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal-glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.

Systematic review of preclinical studies on the neutrophil-mediated immune response to air pollutants, 1980-2020.

Preclinical evidence about the neutrophil-mediated response in exposure to air pollutants is scattered and heterogeneous. This has prevented the consolidation of this research field around relevant models that could advance towards clinical research. The purpose of this study was to systematic review the studies of the neutrophils response to air pollutants, following the recommendations of the Cochrane Collaboration and the PRISMA guide, through 54 search strategies in nine databases. We include 234 studies (in vitro, and in vivo), being more frequent using primary neutrophils, Balb/C and C57BL6/J mice, and Sprague-Dawley and Wistar rats. The most frequent readouts were cell counts, cytokines and histopathology. The temporal analysis showed that in the last decade, the use of mice with histopathological and cytokine measurement have predominated. This systematic review has shown that study of the neutrophils response to air pollutants started 40 years ago, and composed of 100 different preclinical models, 10 pollutants, and 11 immunological outcomes. Mechanisms of neutrophils-mediated immunopathology include cellular activation, ROS production, and proinflammatory effects, leading to cell-death, oxidative stress, and inflammatory infiltrates in lungs. This research will allow consolidating the research efforts in this field, optimizing the study of causal processes, and facilitating the advance to clinical studies.

Membrane translocation and activation of GnRH receptor sensitize prostate cancer cells to radiation.

BACKGROUND: GnRH analogs are widely used as neoadjuvant agents for radiotherapy in prostate cancer (PCa) patients, with well-documented effects in reducing tumor bulk and increasing progression-free survival. GnRH analogs act locally in the prostate by triggering apoptosis of PCa cells via activation of the GnRH receptor (GnRHR). During PCa progression, the distribution of GnRHR within the cell is altered, with reduced expression in the cell membrane and remaining sequestered in the endoplasmic reticulum. Pharmacoperone IN3 is able to relocalize GnRHR to the cell membrane. The aim of this study was to evaluate the effect of radiation on PCa cells pretreated with leuprolide, alone or in combination with IN3, as radiosensitizers. MATERIAL AND METHODS: PC3 and human PCa primary cell cultures were treated with IN3 for 24 h, followed by different doses of leuprolide for 48 h and, finally, single doses of radiation (3, 6, and 9 Gy). After radiation, cell survival, apoptosis, cell cycle distribution, and colony growth were evaluated. RESULTS: Radiation reduced cell survival and increased apoptosis in a dose-dependent manner. This effect was also directly related to leuprolide concentration. Pretreatment with IN3 enhanced apoptosis and decreased cell survival, also observing a higher proportion of cells arrested in G2. CONCLUSION: Neoadjuvant leuprolide increases radiation-mediated apoptosis of PCa cells. This effect was enhanced by pretreatment with pharmacoperone IN3. Clinical use of IN3 as a radiosensitizer combined with androgen deprivation therapy to improve survival of patients with PCa remains to be evaluated.

Differential regulatory effect of progesterone on the proliferation and apoptosis of uterine leiomyoma tissue explants and primary leiomyoma cell cultures.

OBJECTIVE: The growth of uterine leiomyomas is regulated by progesterone, although the underlying molecular mechanisms are not fully understood. METHODS: Primary leiomyoma cells were isolated by standard method from 16 samples of uterine leiomyoma tissue. Uterine leiomyoma explants and primary leiomyoma cell cultures were exposed to progesterone in concentrations of 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml for 24 h. Cell apoptosis was assessed with Annexin V assays performed in cell cultures by flow cytometry. The expression of PR-A, PR-B, Ki67, Akt, ERK, PTEN and PPARγ mRNAs was estimated in cultured leiomyoma cells and tissue explants by real time RT-PCR. RESULTS: Treatment with progesterone promoted viability and proliferation of cultured leiomyoma cells in a dose-dependent manner. Low and high doses of progesterone decreased early apoptosis of leiomyoma cells. High concentrations of progesterone increased the number of living cells in Annexin V assays. High doses of progesterone increased the expression of Ki67 mRNA, while low doses increased the expression of PR-A mRNA in cultured leiomyoma cells and tissue explants. In cell cultures, low doses of progesterone increased the expression of PR-B mRNA and the expression of PTEN and PPARγ mRNAs in a dose-dependent manner. Exposure of leiomyoma tissue explants to progesterone led to increased expression of PR-B and ERK mRNAs in a dose-dependent manner. CONCLUSIONS: The effects of progesterone on the apoptosis and proliferation of leiomyoma cells was dose-dependent and different in cell cultures and leiomyoma explants, possibly as a result of impacts derived from the tumor microenvironment.

Mini Review: Virus Interference: History, Types and Occurrence in Crustaceans.

Virus interference is a phenomenon in which two viruses interact within a host, affecting the outcome of infection of at least one of such viruses. The effect of this event was first observed in the XVIII century and it was first recorded even before virology was recognized as a distinct science from microbiology. Studies on virus interference were mostly done in the decades between 1930 and 1960 in viruses infecting bacteria and different vertebrates. The systems included in vivo experiments and later, more refined assays were done using tissue and cell cultures. Many viruses involved in interference are pathogenic to humans or to economically important animals. Thus the phenomenon may be relevant to medicine and to animal production due to the possibility to use it as alternative to chemical therapies against virus infections to reduce the severity of disease/mortality caused by a superinfecting virus. Virus interference is defined as the host resistance to a superinfection caused by a pathogenic virus causing obvious signs of disease and/or mortality due to the action of an interfering virus abrogating the replication of the former virus. Different degrees of inhibition of the superinfecting virus can occur. Due to the emergence of novel pathogenic viruses in recent years, virus interference has recently been revisited using different pathogens and hosts, including commercially important farmed aquatic species. Here, some highly pathogenic viruses affecting farmed crustaceans can be affected by interference with other viruses. This review presents data on the history of virus interference in hosts including bacteria and animals, with emphasis on the known cases of virus interference in crustacean hosts. Life Science Identifiers (LSIDs) Escherichia coli [(Migula 1895) Castellani & Chalmers 1919] Aedes albopictus (Skuse 1894) Liocarcinus depurator (Linnaeus 1758): urn:lsid:marinespecies.org:taxname:107387 Penaeus duorarum (Burkenroad 1939): urn:lsid:marinespecies.org:taxname:158334 Carcinus maenas (Linnaeus 1758): urn:lsid:marinespecies.org:taxname:107381 Macrobrachium rosenbergii (De Man 1879): urn:lsid:marinespecies.org:taxname:220137 Penaeus vannamei (Boone 1931): urn:lsid:zoobank.org:pub:C30A0A50-E309-4E24-851D-01CF94D97F23 Penaeus monodon (Fabricius 1798): urn:lsid:zoobank.org:act:3DD50D8B-01C2-48A7-B80D-9D9DD2E6F7AD Penaeus stylirostris (Stimpson 1874): urn:lsid:marinespecies.org:taxname:584982.

Optimization of Flavonoid Production in Plant Cell Culture of Thevetia peruviana Elicited with Methyl Jasmonate and Salicylic Acid

Abstract Thevetia peruviana is a medicinal plant that has valuable secondary terpenoid-type metabolites and phenolic compounds. Some flavonoid compounds of pharmaceutical interest stand out in the latter group. The concentration of these bioactive compounds in natural conditions is limited by environmental; therefore, it has been considered necessary to make in vitro plant cell suspension cultures that admit the use of elicitors to increase the content of active principles. Accordingly, in this study, for the optimization of flavonoid production in cell suspension culture of T. peruviana, different parameters related to elicitation with methyl jasmonate (MeJa), and salicylic acid (SA) were evaluated, at stirred flask scale. Firstly, 3 μM MeJa and 300 μM SA were added separately in cell cultures of T. peruviana, to assess their potential effects. Secondly, several experimental conditions were evaluated, for optimization purpose. In the first part, MeJa and SA increased the total flavonoid content, in 1.07 and 1.3 times, respectively, compared to the control culture; in the second part, total flavonoid content produced in MeJa mediated cell suspension cultures were 4.14 mg QE/g DW (milligrams of quercetin equivalent per gram of dry biomass) with: concentration 0.3 μM, addition time day 5 and harvest time 90 h. On the other hand, total flavonoid content produced in SA mediated cell suspension cultures were 3.75 mg QE/g DW with: concentration 100 μM, addition time day 0 and harvest time 96 h. Elicitation of cell suspension cultures of T. peruviana with MeJa and SA under their ideal parameter values increased flavonoid content.

Importancia del mapeo óptico de cultivos de cardiomiocitos HL-1 en estudios electrofisiológicos / Importance of the optical mapping of HL-1 cardiomyocyte cultures in electrophysiological studies

Introducción: El desarrollo de herramientas para investigar la actividad electrofisiológica cardiaca ha permitido profundizar en el conocimiento sobre los mecanismos subyacentes a las arritmias cardiacas. Los sistemas de mapeo óptico constituyen una tecnología que responde a la necesidad de superar varios obstáculos en la experimentación. Objetivo: Proporcionar una visión general de la importancia del mapeo óptico en cultivos celulares HL-1, en las investigaciones en electrofisiología cardiaca. Métodos: Se realizó una revisión sobre los estudios electrofisiológicos que involucran la línea celular HL-1 utilizando la técnica de mapeo óptico. Conclusiones: Los trabajos se caracterizan por la implementación de la técnica respecto a la tecnología de los equipos de mapeo, a la utilización de diferentes colorantes y al objetivo de la investigación. Están enfocados en el estudio de mecanismos arritmogénicos, procesos de estiramiento mecánico o remodelación del tejido y en el análisis de nuevos biomateriales. Lo anterior, sustenta la relevancia del mapeo óptico en la investigación cardiaca(AU)

Introduction: The development of tools to study cardiac electrophysiological activity has made it possible to broaden knowledge about the mechanisms underlying cardiac arrhythmias. Optical mapping systems constitute a technology that responds to the need to overcome several hurdles in experimentation. Objective: Provide an overview of the importance of optical mapping in HL-1 cell cultures in cardiac electrophysiology research. Methods: A review was conducted of electrophysiological studies involving the HL-1 cell line using the optical mapping technique. Conclusions: The studies are characterized by implementation of the technique with respect to the technology of mapping equipment, the use of different colorants and the purpose of the research. They focus on the study of arrhythmogenic mechanisms, mechanical stretch processes or tissue remodeling as well as the analysis of new biomaterials. The above substantiates the relevance of optical mapping in cardiac research(AU)

Amyloid-ß oligomers in cellular models of Alzheimer's disease.

Amyloid-ß (Aß) dysmetabolism is tightly associated with pathological processes in Alzheimer's disease (AD). Currently, it is thought that, in addition to Aß fibrils that give rise to plaque formation, Aß aggregates into non-fibrillar soluble oligomers (AßOs). Soluble AßOs have been extensively studied for their synaptotoxic and neurotoxic properties. In this review, we discuss physicochemical properties of AßOs and their impact on different brain cell types in AD. Additionally, we summarize three decades of studies with AßOs, providing a compelling bulk of evidence regarding cell-specific mechanisms of toxicity. Cellular models may lead us to a deeper understanding of the detrimental effects of AßOs in neurons and glial cells, putatively shedding light on the development of innovative therapies for AD.

Do alterations in gene expressions influence tumorigenesis in the transmissible venereal tumor in dogs? / Alterações nas expressões gênicas influenciam na tumorigênese no tumor venéreo transmissível em cães?

Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.(AU)

O tumor venéreo transmissível canino (TVTC) se trata de uma neoplasia transmissível, que se propaga naturalmente entre os cães pela transferência halogênica de células tumorais, principalmente, durante o coito. É a mais antiga linhagem tumoral conhecida na natureza e relatos sobre mutações gênicas vêm sendo ampliadas. Além disso, este tumor compartilha uma série de mutações genéticas com alguns cânceres em seres humanos, dentre eles, carcinomas de pulmão, melanoma, próstata, mama, entre outros tipos de câncer. Assim, quantificou-se a expressão de genes supressores de tumores, como TP53, P21 e genes relacionados à apoptose, como BAX, BCL-2 e BCL-xL, tanto in vivo quanto in vitro (cultura celular primária). No presente estudo, na comparação das expressões gênicas, o gene TP53 se mostrou elevado na maioria dos casos em tecidos (65%) e em cultura celular primária (100%), enquanto BCL-2, BCL-xL e BAX apresentaram-se variáveis entre os animais analisados. Ademais, nessas situações os resultados sugerem que não ocorreu regulação apoptótica desses genes pelo TP53. O gene P21 mostrou-se, em sua maioria, normal (70%), embora a ausência (6%) e subexpressões (24%) também tenham sido observadas. A análise estatística do gene BCL-xL demonstrou diferenças significativas entre os tecidos dos animais, quando comparadas às culturas celulares, entretanto, para os demais genes, não foi observada diferença estatística entre os grupos. Preliminarmente, os resultados sugerem a presença de alterações nas expressões gênicas dos genes TP53, P21, BAX, BCL-2 e BCL-xL, levando a perda de função desses genes, os quais afetam a tumorigênese do CTVT.(AU)

Phytochemical Screening of Callus and Cell Suspensions Cultures of Thevetia peruviana

Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.

Do alterations in gene expressions influence tumorigenesis in the transmissible venereal tumor in dogs? / Alterações nas expressões gênicas influenciam na tumorigênese no tumor venéreo transmissível em cães?

ABSTRACT: Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.

RESUMO: O tumor venéreo transmissível canino (TVTC) se trata de uma neoplasia transmissível, que se propaga naturalmente entre os cães pela transferência halogênica de células tumorais, principalmente, durante o coito. É a mais antiga linhagem tumoral conhecida na natureza e relatos sobre mutações gênicas vêm sendo ampliadas. Além disso, este tumor compartilha uma série de mutações genéticas com alguns cânceres em seres humanos, dentre eles, carcinomas de pulmão, melanoma, próstata, mama, entre outros tipos de câncer. Assim, quantificou-se a expressão de genes supressores de tumores, como TP53, P21 e genes relacionados à apoptose, como BAX, BCL-2 e BCL-xL, tanto in vivo quanto in vitro (cultura celular primária). No presente estudo, na comparação das expressões gênicas, o gene TP53 se mostrou elevado na maioria dos casos em tecidos (65%) e em cultura celular primária (100%), enquanto BCL-2, BCL-xL e BAX apresentaram-se variáveis entre os animais analisados. Ademais, nessas situações os resultados sugerem que não ocorreu regulação apoptótica desses genes pelo TP53. O gene P21 mostrou-se, em sua maioria, normal (70%), embora a ausência (6%) e subexpressões (24%) também tenham sido observadas. A análise estatística do gene BCL-xL demonstrou diferenças significativas entre os tecidos dos animais, quando comparadas às culturas celulares, entretanto, para os demais genes, não foi observada diferença estatística entre os grupos. Preliminarmente, os resultados sugerem a presença de alterações nas expressões gênicas dos genes TP53, P21, BAX, BCL-2 e BCL-xL, levando a perda de função desses genes, os quais afetam a tumorigênese do CTVT.