Electrophysiological evaluation of the effect of peptide toxins on voltage-gated ion channels: a scoping review on theoretical and methodological aspects with focus on the Central and South American experience.

The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: "electrophysiology", "patch-clamp techniques", "Ca2+ channels", "K+ channels", "cnidarian venoms", "cone snail venoms", "scorpion venoms", "spider venoms", "snake venoms", "cardiac myocytes", "dorsal root ganglia", and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel's structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins' source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering.

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

Electrophysiological evaluation of the effect of peptide toxins on voltage-gated ion channels: a scoping review on theoretical and methodological aspects with focus on the Central and South American experience

Abstract The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: "electrophysiology", "patch-clamp techniques", "Ca2+ channels", "K+ channels", "cnidarian venoms", "cone snail venoms", "scorpion venoms", "spider venoms", "snake venoms", "cardiac myocytes", "dorsal root ganglia", and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel's structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins' source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering.

Bioaccessibility and uptake/epithelial transport of vitamin E: Discoveries and challenges of in vitro and ex vivo assays.

Vitamin E comprises compounds consisting of a chromanol ring and an isoprenoid side-chain, and is an essential lipid-soluble nutrient with several physiological functions. Vitamin E intake has been reported as inadequate for some populations. Only a fraction of dietary vitamin E is effectively released from the food matrix (bioaccessible fraction), absorbed (enterocyte uptake/epithelial transport) and transported in lipoproteins to reach the target tissues (bioavailable fraction), depending on the food structure, composition, and processing. Therefore, research concerning the fate of vitamin E through the gastrointestinal tract is of paramount importance for developing healthy foods and guiding effective public policies. The combination of simulated in vitro gastrointestinal digestion followed by intestinal epithelial transport and/or enterocyte uptake assays using ex vivo cell models has been successfully used to mimic the physiological conditions and predict the bioaccessibility and epithelial transport of compounds. The objective of this review was to summarize the current knowledge and challenges for predicting the bioaccessibility and uptake/epithelial transport of vitamin E by in vitro and ex vivo assays. Here, we revisited the metabolism of vitamin E and introduced in vitro and ex vivo methods for estimating the bioaccessibility and intestinal absorption of vitamin E. This review compiles data on vitamin E bioaccessibility in vitro and uptake/epithelial transport ex vivo for different food matrices, and discusses the factors that can affect their measurement. Additionally, co-culture approaches using hepatic lineages to assess vitamin E bioavailability are further presented.

Long Non-Coding RNAs Might Regulate Phenotypic Switch of Vascular Smooth Muscle Cells Acting as ceRNA: Implications for In-Stent Restenosis.

Coronary in-stent restenosis is a late complication of angioplasty. It is a multifactorial process that involves vascular smooth muscle cells (VSMCs), endothelial cells, and inflammatory and genetic factors. In this study, the transcriptomic landscape of VSMCs' phenotypic switch process was assessed under stimuli resembling stent injury. Co-cultured contractile VSMCs and endothelial cells were exposed to a bare metal stent and platelet-derived growth factor (PDGF-BB) 20 ng/mL. Migratory capacity (wound healing assay), proliferative capacity, and cell cycle analysis of the VSMCs were performed. RNAseq analysis of contractile vs. proliferative VSMCs was performed. Gene differential expression (DE), identification of new long non-coding RNA candidates (lncRNAs), gene ontology (GO), and pathway enrichment (KEGG) were analyzed. A competing endogenous RNA network was constructed, and significant lncRNA-miRNA-mRNA axes were selected. VSMCs exposed to "stent injury" conditions showed morphologic changes, with proliferative and migratory capacities progressing from G0-G1 cell cycle phase to S and G2-M. RNAseq analysis showed DE of 1099, 509 and 64 differentially expressed mRNAs, lncRNAs, and miRNAs, respectively. GO analysis of DE genes showed significant enrichment in collagen and extracellular matrix organization, regulation of smooth muscle cell proliferation, and collagen biosynthetic process. The main upregulated nodes in the lncRNA-mediated ceRNA network were PVT1 and HIF1-AS2, with downregulation of ACTA2-AS1 and MIR663AHG. The PVT1 ceRNA axis appears to be an attractive target for in-stent restenosis diagnosis and treatment.

A human preadipocyte cell strain with multipotent differentiation capability as an in vitro model for adipogenesis.

Murine 3T3 cell lines constitute a standard model system for in vitro study of mammalian adipogenesis although they do not faithfully reflect the biology of the human adipose cells. Several human adipose cell lines and strains have been used to recapitulate human adipogenesis in vitro, but to date there is no generally accepted in vitro model for human adipogenesis. We obtained a clonal strain of human subcutaneous adipose stromal cells, IPI-SA3-C4, and characterized its utility as an in vitro model for human subcutaneous adipogenesis. IPI-SA3-C4 cells showed a high proliferative potential for at least 30 serial passages, reached 70 cumulative population doublings and exhibited a population doubling time of 47 h and colony forming efficiency of 12% at the 57th cumulative population doublings. IPI-SA3-C4 cells remained diploid (46XY) even at the 56th cumulative population doublings and expressed the pluripotency markers POU5F1, NANOG, KLF4, and MYC even at 50th cumulative population doublings. Under specific culture conditions, IPI-SA3-C4 cells displayed cellular hallmarks and molecular markers of adipogenic, osteogenic, and chondrogenic lineages and showed adipogenic capacity even at the 66th cumulative population doublings. These characteristics show IPI-SA3-C4 cells as a promising potential model for human subcutaneous adipogenesis in vitro.

Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Molecular Cloning and Transient Expression of Recombinant Human PPARγ in HEK293T Cells Under an Inducible Tet-on System.

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.

La agregación de TDP-43 como posible blanco terapéutico contra la Esclerosis Lateral Amiotrófica / TDP-43 aggregation as a possible therapeutic target for Amyotrophic Lateral Sclerosis / Agregação de TDP-43 como um possível alvo terapêutico para a Esclerose Lateral Amiotrófica

Los agregados de TDP-43 representan una de las característica histopatológicas más importantes de varias enfermedades neurodegenerativas, entre las que se incluye la Esclerosis Lateral Amiotrófica (ELA). TDP-43 está localizada principalmente en el núcleo. Sin embargo, los pacientes afectados por ELA presentan agregados de TDP-43 en el citoplasma de las neuronas comprometidas, con lo que se despoja al núcleo de TDP-43 funcional. Aún se desconoce si la degeneración causada por la agregación de TDP-43 es debida a una toxicidad intrínseca de los agregados o a la pérdida de función de TDP-43 como consecuencia del vaciamiento del núcleo. Varias investigaciones, incluidas las de estos autores, indican que la pérdida de función es el factor fundamental responsable de la neurodegeneración observada en presencia de inclusiones de TDP-43. Por otro lado, aún no existen tratamientos efectivos para la ELA. Por lo tanto, es de crucial importancia conocer las bases moleculares que conllevan al desarrollo de la enfermedad, con el objetivo de encontrar posibles estrategias terapéuticas. Para ello, estos autores han desarrollado un modelo celular capaz de imitar la agregación de TDP-43 y sus consecuencias. Finalmente, se ha utilizado este modelo para analizar el efecto de diferentes compuestos capaces de degradar los agregados de TDP-43 y se ha demostrado que esta podría ser una estrategia terapéutica válida para la ELA.

TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). TDP-43 is mainly a nuclear protein, but it shuffles from the nucleus to the cytoplasm. In patients’ brains, TDP-43 is retained in the cytoplasm of the affected motorneurons to form insoluble aggregates, which results in TDP-43 nuclear clearance. There is still no consensus whether TDP-43-mediated neurodegeneration results from a gain or loss of function of the protein or a combination of both. The work from several laboratories, including this, points towards a strong loss of function component. On the other hand, there is no effective treatment or cure for ALS. Thus, there is obviously a need to find new therapeutic strategies for ALS. In order to gain new insights into the molecular mechanism of the disease, and with the aim of looking for new methodologies that can revert it, a cellular model of TDP-43 aggregation that can mimic the phenotypic consequences found in ALS patients has been developed. Finally, this model was used to search for compounds that can dissolve these aggregates, and it was shown that the clearance of TDP-43 aggregates could be a therapeutic strategy for ALS.

Os agregados proteicos TDP-43 são características histopatológicas importantes de muitas doenças neurodegenerativas, incluindo a Esclerose Lateral Amiotrófica (ALS). A proteína TDP-43 se localiza principalmente no núcleo, porém nos cérebros de indivíduos afetados, a proteína TDP-43 fica retida no citoplasma dos neurônios motores, o que leva a formação de agregados insolúveis, resultando em deposição nuclear. Ainda não existe um consenso se a neurodegeneração mediada por TDP43 é causada por ganho ou perda da função da proteína ou uma combinação de ambos. O trabalho de muitos laboratórios, bem como este trabalho, apontam para uma forte perda da função da proteína. Por outro lado, não existe um tratamento efetivo ou cura para a ALS. Portanto, existe uma grande necessidade de identificar novos tratamentos para a ALS. Para entender o mecanismo molecular da doença, e com o objetivo de identificar novas metodologias para reverter a doença, desenvolvemos o modelo celular de agregados de TDP-43, o qual mimetiza as consequências fenotípicas encontradas em pacientes com ALS. Por fim, utilizamos esse modelo para identificar compostos que podem dissolver os agregados, e demonstramos que a liberação de inclusões de TDP-43 poderiam ser usados como tratamentos para a ALS.

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.