Total de Trabalhos: 00020 XXXIII Reunião Anual da FeSBE 12.001 EFFECTS OF CPX ON TRANSFECTION EFFICIENCY AND OVER DIFFERENTIATION IN C2C12

Title: EFFECTS OF CPX ON TRANSFECTION EFFICIENCY AND OVER DIFFERENTIATION IN C2C12. 1Severino, M., 1Esteca, M. V., 1,2Tamborlin, L., 1,2Luchessi A. D., 1Baptista I. L., 1Faculdade de Ciências Aplicadas, Unicamp, Brasil, 2Instituto de Biociências, Unesp, Brasil. Introdution: The differentiation process in skeletal muscle cells is highly organizated and essential to the regeneration. C2C12 culture cells emerge as reliable model to understand the differentiation process, since those cells have the ability to differentiate in multinucleate myotubes from single myoblasts. These in vitro models are powerful tools for allow genetic manipulation, for example, through the use of plasmids and several transfection reagents. On this study, we used a newly described reagent, ciclopirox (CPX), used to improve transfection efficiency on other cell types, in polyethylenimine (PEI) transfection system. Objective: The aim of this study was to investigate the effects of ciclopirox over the transfection efficiency and the differentiation process of C2C12 cells. Methodology: C2C12 cells in consolidated culture, was transfected with PEI and a GFP-flag plasmid; other wells also received ciclopirox. To analyze the effect of ciclopirox during the the differentiation,we have used horse serum 2% supplementation for 2 and 4 days. The morphometric parameters were quantified by counting cells, through images made by optical microscopy and fluorescence; we also used western blot to quantify where there was higher GFP-flag production, Cyclin (proliferation marker) and Myogenin (differentiation marker). Fluorescence data was analyzed using T-test (p<0,05). Results: The percentage of transfected cell was higher in PEI transfection compared to PEI+CPX (PEI 35,5% vs PEI+CPX 21% per mm2 n=3). We also analyzed the transfection efficiency using Western Blot; we observed that CPX reduced the GFP production (~30% of reduction), suggesting that ciclipirox have a stressing effect over C2C12 cells. We compared the number of cells post transfection, and before differentiation. We observed 102 cells/mm2 in PEI procedure vs 75 cells/mm2 in PEI+CPX, evidencing a reduction of 35%, even starting from 1x105 (n=4). Those data indicate that CPX treatment induced less proliferation compared to PEI transfected cells. Using Western blot, we detected Flag (GFP), Cyclin and Myogenin proteins during differentiation. We observed that when we applied ciclopirox the Myogenin levels was strong reduced in PEI+CPX (86% vs Control) than PEI transfection (60% vs Control) and control group; the levels of Flag decreased during differentiation (reduction of 40% when compared 2 to 4 days), however in wells that receiving CPX that decrease was slightly higher (30%); the Cyclin levels in PEI+CPX was also reduced compared to PEI (50% of reduction). Our data suggest that in CPX transfection an early differentiation could occur, but without a full progress, as observed in myogenin expression. Conclusion: Our results showed that in PEI transfection, the protein production was higher compared to PEI+CPX. Moreover, when CPX was applied, it reduced C2C12 proliferation with higher compromised to differentiation, and that could make those cells becoming resistant to the incorporation of the plasmid.


Resumo
Introduction: Metacaspases (MCAs) are cysteine-peptidases found in lower eukaryotes, such as plants, fungi and protozoa, these enzymes are similar to caspases and are closely correlated with cell death in these organisms. The fungi Paracoccidioides brasiliensis (Pb) is the causal agent of paracoccidioidomycosis, a biochemical characterization of the PbMCA activity is important to design new substrates or inhibitor for this protein being an important therapeutic target for the paracoccidioidomycosis.
Objective: Clone the PbMCA in heterologous expression in E. coli, expressing and purifying this enzyme.
Methodology: pET28a(+) vector (Invitrogen) was chosen to cloning PbMCA. From the synthetic cDNA, amplification was performed with specific primers for PbMCA gene with the HotStar HiFidelity DNA Polymerase (Qiagen). pET28a(+) plasmid and the PCR product were digested with NheI and EcoRI restriction enzymes (Sinapse), these material was binding by T4 ligase (Sinapse). After ligation process PbMCA clone was transformed by thermal shock in E. coli DH5-alpha strain, positive colonies was select by PCR and this material was sequenced by Sanger´s method. Positive colony was chosen and a alkaline lysis was performed to and this material was transformed in an E. coli BL21(DE3) strain and a PCR was make to choose a positive clone. PbMCA was expressed using 0.25 mM of IPTG for 16 hours in 20 o C, the material obtained was purified with nickel-sepharose column (GE Lifescience) in different imidazole concentrations (0 -500 mM).
Results: PbMCA gene amplification was analyzed by 1% agarose electrophoresis and shows a 1427 bp band corresponding at PbMCA. Digestion of the PbMCA and vector (pET28a (+)) was analyzed also with a 1% agarose electrophoresis, and presenting bands around 1400 bp and 5369 bp, respectively. These material was ligated with T4 ligase, the recombinant DNA was introduced into the E.coli DH5alpha strain and confirmed by colony PCR with T7 primers, agarose gel analysis showed the presence of 5 positive colonies, the plasmid was extracted from these colonies and with this material sequencing was performed , through this we realized that cloning was performed without the presence of mutations. Positive colonies were then transfected into the competent bacterium BL21 (DE3) -strain capable of inducing gene expression -and also confirmed by colony PCR, verifying that all colonies were positive. Large-scale expression of PbMCA was analyzed on SDS-PAGE gel, where a band of about 52 kDa was visualized, indicating that the protein was successfully expressed, after expression the PbMCA was purified by column chromatography with nickel, purification was also analyzed by SDS-PAGE where it was found that the protein was left pure with 300 mM imidazole.

Conclusion:
PbMCA was cloned, expressed and purified successful, our next objectives was testing the activity of this enzyme and procedure some biochemical studies of PbMCA. Introduction: Hyperandrogenism is defined as the state of excessive production and secretion of androgens. The growth, regeneration, maintenance, and adaptation of skeletal muscle tissues are dependent on a number of factors, including serum testosterone (T). Skeletal muscle is one of the most metabolically active tissues and is therefore highly susceptible to hormonal variations, especially in relation to testosterone. The C2C12 cell line is a sub-clone of the murine myoblast cell line; these cells are able to differentiate rapidly, forming contractile myotubes and producing characteristic muscle proteins. Because of their high myogenic potential, C2C12 cells provide an excellent tool for the understanding of specific processes, both in vitro and in vivo.
Objective: To evaluate the effects of different testosterone concentrations on cells counts and viability in C2C12 cell cultures.
Methodology: C2C12 cells were cultured (in triplicates) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fungizone, streptomycin, penicillin, and 2% of normal equine serum, in 48-well plates (5000 cells / 200 μL medium -day zero). The cultures were kept at 37 °C under a humid atmosphere of 5% CO2 and 95% atmospheric air for up to 10 days, in the absence (control, C) and presence of T (at 10 -10 M and 10 -5 M). The testosterone concentrations used mimicked the normal condition and hyperandrogenemia. The culture medium was exchanged every 48 h. Cell quantification was performed with a Bio-Rad TC20  cell counter. Cell viability was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction method, using absorbance at a wavelength of 570 nm. The data were analyzed using ANOVA and Fisher's test for multiple comparisons. The results are reported as the means  SD, with p-values<0.05.
Conclusion: Testosterone stimulated cell multiplication and viability in a dose-dependent manner after 5 days of culture. After 10 days of culture, cell multiplication and viability were restricted by a T concentration of 10 -10 M, while a T concentration of 10 -5 M avoided these inhibitory effects. Thus, the culture of C2C12 cells remained viable for up to 10 days, hence providing a useful in vitro model for studies concerning the effects of T on skeletal muscle cells.

Autores
Anderson Martelli 1 , Viviane Theodoro 1 , Gabriela Maiara Pastre 1 , Karina Marques de Melo 1 , Fernanda Oliveira de Gaspari de Gaspi 1 , Maria Esméria Corezola do Amaral 1 , Rodrigo Augusto Dalia 1 , Andrea Aparecida de Aro 1 , Marcelo Augusto Marretto Esquisatto 1 , Thiago Antônio Moretti Andrade 1 , Glaucia Maria Tech dos Santos 1 Instituição 1 FHO|Uniararas -Centro Universitário Herminio Ometto Resumo Introduction: The complications associated with diabetes are several, mainly regarding the difficulty of repairing burns. Therefore, the study of Casearia sylvestris, an alternative in the repair of lesions associated with diabetes, becomes of great relevance. Objective: Investigate the action of the hydroalcoholic extract from the leaves of C. sylvestris in the repair of second-degree burns in diabetic and non-diabetic rats. Methodology: Study approved in CEUA-UNIARARAS (nº 023/2015). A total of forty non-diabetic and forty diabetes-induced rats (150 mg/kg of intravenous aloxane) were used and those with glycemia above 200 mg/dL, 30 days after induction, were included in the study. The second-degree burns on the skin of the dorsum of the animals were performed with a metal plate (2.0 cm in diameter/120ºC/20 seconds), after anesthesia (ketamine: 1.0 mL/kg and xylazine: 0.2 mL/kg). The experimental groups were: (C) non-diabetic animals treated topically with carbopol gel; (G) non-diabetic treated with an extract of C. sylvestris in carbopol; (DM-C) diabetics treated with carbopol; (DM-G) diabetics treated with C. sylvestris in carbopol. Lesion samples were collected on the 3rd, 7th, 14th and 21st days after the lesion for biochemical analysis: Dosages (µg/g of dry tissue) of hydroxyproline (HO-Pro) and glycosaminoglycans (GAG) for MEC remodeling evaluation and protein expression of TGF-β1, Collagen types I and III by the Western blotting technique (arbitrary units). The quantitative values were analyzed using ANOVA and Tukey's post-test (p<0.05) and expressed as mean±standard error. Results: On the 14th experimental day, it was observed that C. sylvestris treatment favored increased hydroxyproline quantification in the DM-G group (33.40±3.44) in relation to DM-C (18.47±5.71). In relation to glycosaminoglycans quantification, an increase in DM-G (3d:5.03±2.34, 7d:4.07±0.95) was observed in relation to DM-C (3.67±0.28) and G (7d:0.87±0.1). In the Western blotting analysis for TGF-β1 protein expression, in the day 3 was observed an increase in G (11,478.05±3,022.05) in relation to the other groups. On the 7th day, DM-G (23,544.32±2,489.94) was superior to the other groups with a reduction in the later periods. In the determination of collagen type III, on the 3rd and 14th days group G (3d:21,081.86±3,535.98, 14d:20,800.87±1,360.41) was superior to the others and the group DM-G (3d:13,599±2,299) higher than the DM-C group. On day 7, group G (7d:16.589±705) was superior to group C (7d:7,205±52.42). On the 14th and 21st days, there was an increase in group C (14d:12,273 ± 2,001, 21d:10, 719±5,191) in relation to the DM-G group. Group G at day 21 (15,127±1,126) was superior to DM-C and DM-G groups. In the analysis of Collagen type I, a gradual increase was observed in groups G and DM-G during the whole experimental period, being higher in DM-G on the 14th day (17,143±566) in relation to C and G. On the 21st day G (14,001±1,481) was superior to C and DM-C and DM-G (18,766 ± 1,343) superior to the other groups. Conclusion: C. sylvestris can be considered an alternative for the healing process of second-degree burns in diabetics, as it favored repair by increasing inflammation in the initial periods with subsequent reduction, also providing reorganization of the extracellular matrix and collagenesis. Funding: FHO.

HISTOLOGICAL CHARACTERIZATION OF PERIPROSTATIC ADIPOSE FROM OBESE MICE AND ITS ROLE ON PROSTATE TISSUE.
Autores Gabriela R. Passos Gabriela Passos 1 , Fabiola zakia Mónica Fabiola zakia Mónica 1 Instituição 1 UNICAMP -Universidade Estadual de Campinas Resumo Introduction: The lower urinary tract symptoms (LUTS) has a high prevalence worldwide and affect both genders, however, obese men are more likely to develop these symptoms. Obesity promotes an increase of adipose tissue, which is an important source of proinflammatory, pro-contractile and pro-proliferative substances. The adipocytes are divided into white and brown adipose tissue. The white adipose tissue (WAT) is composed of large diameter solid lipid droplet, cytoplasm and nucleus. While the brown adipose tissue (BAT) is composed of smaller cells containing small lipid droplets. In a recent study published by our group the prostate weight and epididimal fat of obese mice are significantly greater when compared to lean mice tissues and these animals presented prostate hipercontractility.
Objective: Therefore, the aim of this study is to characterize the periprostatic adipose tissue from obese mice.
Methodology: All experimental protocols were approved by Ethics Committee for the use of experimental animals (CEUA 4720-1/2-2017). C57BL/6 male mice with 4 weeks old were fed with standard chow (control group, N=5) or high-fat diet (obese group, N=5) for 12 weeks. At week 12, prostate and periprostatic adipose tissue were collect for histopathology and immunohistochemistry to evaluate proliferative (ki-67). Student's t-test analysis was used. P<0.05 was accepted as significant.

Results:
The histological analysis showed that the adipocyte area from periprostatic adipose is significantly greater in obese than in lean mice. With respect to the histological analysis of prostate sections, in the control group a simple columnar epithelium and a thin layer of smooth muscle was observed. On the other hand, the prostate from obese mice showed epithelial hyperplasia. These results were confirmed with the proliferative index (Ki-67) test, which showed greater values (an increase of 93%; P Conclusion: Since the area from periprostatic adipose tissue is significantly greater in obese than in lean mice, our results suggest that the adipose tissue could release proliferative substances thus contributing to the development of prostatic hyperplasia. Campomanesia adamantium O. Berg., popularly known as "guavira", is a plant native to Brazil, occurring in the Cerrado, including Mato Grosso do Sul State, and it is used for several purposes. Leaves, fruits and roots of this plant have medicinal properties already described as effect to decrease body mass, hypoglycemic, hypolipidemic and antileukemic.
Objectives: To evaluate the antioxidant activity, cytotoxicity and anti-adipogenic effect of Campomanesia adamantium leaves.
Methods: Aqueous extract of the leaves of C. adamantium (EAFCa) was prepared by solvent accelerated extraction (DIONEX35). The antioxidant potential of EAFCa was investigated by the DPPH capture method, its cytotoxicity in 3T3F442a pre-adipocytes and its effects on the differentiation of adipocytes by accumulation of lipids in cell culture of 3T3F442a pre-adipocytes with oil red staining. The data were submitted to one-way analysis of variance (ANOVA) followed by Student Newman Keuls test using the Prism 5 GraphPad Software. The results were considered significant when p<0.05.

Results:
The concentration required to inhibit 50% free radicals (IC50) in the DPPH assay of the EAFCA was 11.6 ± 1.5 μg/mL, compared to BHT 28.1 ± 6.3 μg/mL and ascorbic acid 2.1 ± 0.6 μg/mL, synthetic and natural control antioxidants, respectively. EAFCa did not induce death of 3T3F442a pre-adipocytes incubated with different concentrations of the extract (3.1 to 100 μg/mL). The differentiation of these cells with adipogenic cocktail led to a 35.3 ± 7.4% increase in lipid content compared to undifferentiated cells. The EAFCa at concentrations of 25, 50 and 100 μg/mL prevented this accumulation, similarly to the action observed for the quercetin control, at a concentration of 50 μg / mL, which showed similar lipid content to undifferentiated cells. Conclusion: In summary, the preliminary results indicate the antioxidant and anti-adipogenic potential of EAFCa without presenting effects of cytotoxicity.

Introduction: Yellow fever (YF) is an infectious, non-contagious disease caused by an RNA virus of the
Flaviviridaefamily, which is transmitted to human by the bite of hematophagous mosquitoes. It causes severe disease involving especially the liver, with lesions characterized by midzonal apoptosis. The mechanism by which liver cells undergo apoptosis in response to YF infection remains unclear.
Objective: To provide further information on the mechanism of apoptosis in YF infected hepatic cells we investigated whether the virus causes endoplasmic reticulum (ER) stress and intracellular Ca 2+ signaling disturbs.
Methodology: Explants and biopsies from patients diagnosed with hepatitis (Certificate of Presentation for Ethical Consideration: 77877417.9.0000.5125) caused by YF infection were analyzed by hematoxylin and eosin staining and immunohistochemistry. HepG2 cells were infected with the attenuated YF strain (17DD) and isolated YF (RB) to evaluate the viral cytotoxicity by violet crystal and by rezasurin metabolism. Proteins involved in Ca 2+ signaling and ER stress were assessed by immunofluorescence and immunoblot. Mitochondrial Ca 2+ signaling, with Rhod-2/AM, was monitored in individual cells by time lapse confocal microscopy after adenosine triphosphate (ATP) or tumor necrosis factor (TNF-a) stimulation. The results were expressed as means and standard errors of the mean, and tested by one-way analysis of variance followed by Tukey posttests, where p Results: Histological analysis of liver samples from YFpatientsshowed a range from mild to severe hepatic damage, where Councilman bodies, macro and microvesicular steatosis, and lytic necrosis were found within the midzonal region of the lobule in mild cases, or in the whole organ in the fulminant hepatitis cases. Immunohistochemistry staining showed that several hepatocytes were positive to flavivirus antigen and to apoptosis (ssDNA). Moreover, the 1,4,5-trisphosphate receptor subtype 3 (InsP3R-3), an isoform that is absent under normal conditions in hepatocytes, was upregulated in YF patient samples. In vitroexperiments using the hepatic cell line HepG2 showed that YF infection is cytotoxic to this cells, wherein the cell death and cytotoxicity were more pronounced after RB infection compared to 17DD strain (61.0 and 31.4% respectively, p 2+ signaling was increased in the cells infected with YF compared to control. The amplitude and duration of mitochondrial calcium signaling were significantly increased in RB infected cells compared to control after ATP and TNF-a stimuli (2.2x and 1.7x respectively, p Conclusion: Based on these findings, we conclude that the disturb in the intracellular Ca 2+ signaling machinery and the activation of ER stress cascade play an important role in the genesis of hepatic lesions in severe yellow fever, by inducing a massive hepatocyte apoptosis.

Introduction:
The regenerative process of skeletal muscle is a process highly ordered. Several studies have suggested that proteolytic pathways such as ubiquitin-proteasome and autophagic are necessary on both phases, inflammatory (1-3 days post injury) and regenerative (3-21 days post injury). One critical step on differentiation of myogenic precursor cells, during the regeneration, is a metabolic shift from glycolytic to oxidative. At this point, we suggest that Parkin E3 ligase, required for mitochondrial turnover, may influence the process of muscle regeneration.
Objective: The aim of this study was to investigate whether Parkin is important for mitochondrial turnover of myogenic precursor cells, influencing the differentiation process during skeletal muscle regeneration.
Methodology: 10 wild-type animals (WT) and 10 knockout animals for Parkin (Parkin-/-) were used for the experiments. The injury was induced by cardiotoxin (CTX1 10μM) inoculated in the tibialis anterior muscle and the sacrifice of the groups was performed 3 days after injury (CTX3D) and 10 days after injury (CTX10D). The contralateral paw was used as control group (CTRL). All procedures were approved by CEUA (46871/2017). Data were compared between groups using One-Way ANOVA and Post-hoc Tukey test (p<0.05) and for two groups the T-test was used (p<0.05).
Results: Parkin-/-animals had a mean area 24.5% lower than CSA compared to WT animals at 10 days after injury (WT 1180μm 2 ± 695 vs Parkin-/-897 ± 641μm 2 n=4). We found a large increase of Parkin protein levels in the CTX3D group compared to the CTRL, which remained elevated until 10 days after the injury (n=3). In cell location, we detected Parkin colocalized with VDAC2 on 94.5% (±2.5) of myogenic precursor cells in the CTX3D group and 71.5% (±9.5 n=2) in the CTX10D group. We observed an important drop on protein content of VDAC2 in the WT and Parkin-/-animals, 3 days after injury compared to its related CTRL (n=3). At 10 days after the injury the levels of VDAC2 increased near the basal level in WT animals, but not in Parkin-/-animals (n=3). In the WT animals a large increase in LC3-I occurs 3 days after lesion and remained elevated until 10 days post injury (n=3); we also observed a gradual increase in LC3-II on both, 3 and 10 days after injury (n=3). In Parkin-/-animals, LC3-I and LC3-II only increased 10 days after injury (n=3). Using immunofluorescence, Parkin-/animals showed a higher number of LC3/VDAC2 positive cells at 10 days post-injury compared to WT animals (WT 7.6%± 0.9 vs Parkin-/-24.5% ±1.3, n=2). We also analyzed the p62 protein content and that protein was downregulated 3 days post injury and remained below baseline until 10 days after injury on both, WT mice as Parkin-/-animals (n=3).
Conclusion: At this point, our results point out that Parkin presence is important to mitochondrial turnover during the myogenic differentiation, and a deficiency in Parkin expression can lead to a weakened autophagic flow and a consequent loss of regenerative capacity of skeletal muscle.

Resumo
Introduction: The prostatic smooth muscle cells (SMCs) are surrounded by an external (basal) membrane and stromal type I collagen. The prostate is highly dependent on androgens. After the first hours of castration the endothelial cells suffer apoptosis and the resulting vascular permeability leads to the formation of a fibrin network. This also results in exposure of SMCs to serum-derived cytokines, such as TGFβ3 and TGFα, which are migratory stimuli for some cell types after tissue injury. The external stimuli produced by extracellular matrix (ECM) are translated through transmembrane receptors, the integrins, which bind specific ECM proteins. The integrin cytoplasmic domain interacts with several proteins in a complex called focal adhesion. One of these proteins is focal adhesion kinase (FAK), which is a 125-KDa protein-tyrosine kinase and acts as an integrator of the ECM and the cytoskeleton.
Objective: To identify how different (ECM) substrates and cytokines (TGFβ3 and TGFα) affect prostatic SMC phenotype and FAK content/distribution. Methodology: SMCs were obtained by culturing ventral prostate explants from male Wistar rats (Protocol: CEUA 4132-1), in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 10 nM dihydrotestosterone, and 5 µg/mL insulin. Subsequently, SMCs in the fourth-to-seventh passages were seeded on type I collagen, fibrin and laminin-rich (Matrigel) extracellular matrix 2D-substrates. After 48h in culture, cells were examined by immunofluorescence and Western blotting, using antibodies against α-SMA and total FAK, and by transmission electron microscopy. A wounding healing assay was used to study the effect of TGFβ3 and TGFα in the SMC migration.
Results: The morphology of the cells after 48h of culture on the substrates and FAK distribution was remarkably different. Western blotting and fluorescence techniques did not show changes in α-SMA or FAK levels. SMCs adhered very quickly to type I collagen. Their cytoskeleton showed several actin bundles and focal adhesions. When seeded on a fibrin matrix, the cells were very active, showing numerous processes, and promoted degradation of the fibrin matrix. It was observed that the cells on laminin-rich substrate had several processes, and formed cell clusters in the first hours of culture. Actin filaments in fibrin and laminin-rich substrate did not organize in thick bundles. The scrapping/wounding showed that TGFα, but not TGFβ3, stimulated cell migration.
Conclusion: These observations suggest that ECM components and cytokines regulate behavior, morphology and cytoskeleton, confirming the remarkable phenotypical plasticity of differentiated SMCs.