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Chagas disease (CD) is a neglected disease, prevalent and endemic in Latin America, but also present in Europe and North America. The main treatment used for this disease is benznidazole, but its efficacy is variable in the chronic phase and presents high toxicity. So, there is a need for the development of new therapeutic agents. The five-membered heterocyclic 1,2,4-oxadiazole ring has received attention for its unique properties and a broad spectrum of biological activities and is therefore a potential candidate for the development of new drugs. Thus, the aim of this study was to evaluate the activity of the N-cyclohexyl-3-(3-methylphenyl)-1,2,4-oxadiazol-5-amine (2) on the evolutionary forms of Trypanosoma cruzi strain Y, as well as its mechanisms of action and in silico theoretical approach. The results by computational method showed an interaction of the 1,2,4-oxadiazole (2) with TcGAPDH, cruzain, and trypanothione reductase, showing good charge distribution and affinity in those three targets. Furthermore, cytotoxicity in LLC-MK

Empresa júnior (EJ) é uma associação civil, sem fins lucrativos, inserida no ambiente universitário, constituída e gerida por estudantes da graduação; atuam em atividades de consultoria, que se caracterizam como uma oportunidade de desenvolver uma visão global do funcionamento empresarial, fomentando o empreendedorismo, permitindo o aprendizado dentro de cenários práticos. É fundamental que esse desenvolvimento ocorra no cerne das universidades, conferindo um teor científico e culminando na criação de parques tecnológicos e incubadoras de empresas. A temática do empreendedorismo torna relevante a implantação de EJ em cursos da saúde, destacando o Curso de Farmácia, uma vez que o profissional farmacêutico atua na gestão em seu campo profissional, traçando planos estratégicos, frente às infinitas necessidades e aos recursos limitados. O Curso de Farmácia da UFC dispõe de uma EJ, a IPHARMA JR – Consultoria Farmacêutica, a qual tem como missão ser o impacto no mercado farmacêutico e na vida das pessoas, formando profissionais diferenciados por meio da vivência empresarial. No funcionamento da EJ, prioriza-se a estruturação de processos em detrimento do qualitativo de contratos ou faturamento, o que permite um amadurecimento pessoal e coletivo, deixando claro que o planejamento estratégico faz parte do processo de crescimento. Tudo isso propicia um posicionamento privilegiado no mercado, conferindo uma melhor condição de empregabilidade. Na IPHARMA, o planejamento estratégico como ferramenta de educação para o empreendedorismo tem posicionado muito bem os egressos do projeto, visto que os profissionais egressos da EJ se colocaram rapidamente no mercado de trabalho e muitos abriram suas próprias empresas.

Chagas disease is a leading public health problem.

Acute Kidney Injury (AKI) can be defined as a spectrum of diseases associated with a sudden onset of a renal failure status, the feline patient has azotemia, the disorder in fractional electrolyte excretion (FE), and shedding of epithelial cells from renal tubular segments observed in the urinary sediment. Thus, the objective of the present study was to evaluate the FE, plasma and urinary osmolality, and urinary specific gravity (USG) in cats that spontaneously developed AKI due to urethral obstruction (UO) and healthy cats. Blood and urine samples were collected from a group of 20 cats diagnosed with AKI secondary to urethral obstruction (GAKI; n=20) and clinically healthy cats (GC; n=15). The serum creatinine (sCre) and urinary creatinine (uCre), were measured by spectrophotometry, serum and urinary analyzes of sodium, potassium and chloride by ion selective electrode device and serum and urinary osmolarity by osmometer. The GAKI results were statistically compared with those of the CG using Student's t tests to assess normal data, while the Maan-Whitney test was used for non-normal data. A significant increase in the sCr, sK, FENa, FECl and RFI parameters of the GAKI cats when compared to the GC (p < 0.05). The sCl, USG, uCr, uK and uOSM parameters decreased significantly when compared between the two groups. Thus, given the established methodology and the results found, it is possible to infer that an increase in EFNa, EFCl in addition to the RFI and a decrease in USG and uOSM were associated with cats with AKI and can serve as markers of kidney damage, as well as monitoring the prognosis.

Chagas disease infects approximately seven million people worldwide. Benznidazole is effective only in the acute phase of the disease, with an average cure rate of 80% between acute and recent cases. Therefore, there is an urgent need to find new bioactive substances that can be effective against parasites without causing so many complications to the host. In this study, the triterpene 3β-6β-16β-trihydroxilup-20 (29)-ene (CLF-1) was isolated from Combretum leprosum, and its molecular structure was determined by NMR and infrared spectroscopy. The CLF-1 was also evaluated in vitro and in silico as potential trypanocidal agent against epimastigote and trypomastigote forms of Trypanosoma cruzi (Y strain). The CLF-1 demonstrated good results highlighted by lower IC50 (76.0 ± 8.72 µM, 75.1 ± 11.0 µM, and 70.3 ± 45.4 µM) for epimastigotes at 24, 48 and 72 h, and LC50 (71.6 ± 11.6 µM) for trypomastigotes forms. The molecular docking study shows that the CLF-1 was able to interact with important TcGAPDH residues, suggesting that this natural compound may preferentially exert its effect by compromising the glycolytic pathway in T. cruzi. The ADMET study together with the MTT results indicated that the CLF-1 is well-absorbed in the intestine and has low toxicity. Thus, this work adds new evidence that CLF-1 can potentially be used as a candidate for the development of new options for the treatment of Chagas disease. Communicated by Ramaswamy H. Sarma

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic cells in the substantia nigra pars compacta. PD patients' brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The present study aims to evaluate the neuroprotective activity of VD3 on astrocytes after their exposure to rotenone (ROT) a natural pesticide known to exhibit neurotoxic potential via the inhibition of mitochondrial complex I. Cell viability parameters were evaluated by the MTT test and staining with 7-AAD in cultures of astrocytes treated and untreated with VD3 (0.1, 0.5, and 1.0 ng/mL) and/or ROT (10 µg/mL or 5 µg/mL), and the cytoplasmic production of ROS and the cell death profile were measured by flow cytometry. Glutathione accumulation and ultrastructural changes were evaluated and immunocytochemistry assays for NF-kB and Nrf2 were also carried out. The results showed that VD3 improved the viability of cells previously treated with VD3 and then exposed to ROT, reducing the occurrence of necrotic and apoptotic events. Furthermore, cells exposed to ROT showed increased production of ROS, which decreased significantly with previous treatment with VD3. Importantly, the decrease by ROT in the mitochondrial transmembrane potential was significantly prevented after treating cells with VD3, especially at a concentration of 1 ng/mL. Therefore, treatment with VD3 protected astrocytes from damage caused by ROT, decreasing oxidative stress, decreasing NF-kB and Nrf2 expressions, and improving mitochondrial function. However, further investigation is needed regarding the participation and mechanism of action of VD3 in this cellular model of PD focusing on the crosstalk between Nrf2 and NF-kB.

Resveratrol (RSV), a phytoalexin from grapes and peanuts, has been reported to exhibit antiproliferative effects on various cancer cell lines. In breast cancer, RSV has been demonstrated to exert an antiproliferative effect on both hormone-dependent and hormone-independent breast cancer cell lines. However, RSV is a lipophilic drug, and its therapeutic effect could be improved through nanoencapsulation. Functionalizing polymeric nanoparticles based on polycaprolactone (PCL) with polyethylene glycol 1000 tocopheryl succinate (TPGS) has been reported to prolong drug circulation and reduce drug resistance. However, the effect of TPGS on the physicochemical properties and biological effects of breast cancer cells remains unclear. Therefore, this study aimed to develop RSV-loaded PCL nanoparticles using nanoprecipitation and investigate the effect of TPGS on the nanoparticles’ physicochemical characteristics (particle size, zeta potential, encapsulation efficiency, morphology, and release rate) and biological effects on the 4T1 breast cancer cell line (cytotoxicity and cell uptake), in vitro and in vivo. The optimized nanoparticles without TPGS had a size of 138.1 ± 1.8 nm, a polydispersity index (PDI) of 0.182 ± 0.01, a zeta potential of −2.42 ± 0.56 mV, and an encapsulation efficiency of 98.2 ± 0.87%, while nanoparticles with TPGS had a size of 127.5 ± 3.11 nm, PDI of 0.186 ± 0.01, zeta potential of −2.91 ± 0.90 mV, and an encapsulation efficiency of 98.40 ± 0.004%. Scanning electron microscopy revealed spherical nanoparticles with low aggregation tendency. Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR) identified the constituents of the nanoparticles and the presence of drug encapsulation in an amorphous state. In vitro release studies showed that both formulations followed the same dissolution profiles, with no statistical differences. In cytotoxicity tests, IC50 values of 0.12 µM, 0.73 µM, and 4.06 µM were found for the formulation without TPGS, with TPGS, and pure drug, respectively, indicating the potentiation of the cytotoxic effect of resveratrol when encapsulated. Flow cytometry and confocal microscopy tests indicated excellent cellular uptake dependent on the concentration of nanoparticles, with a significant difference between the two formulations, suggesting that TPGS may pose a problem in the endocytosis of nanoparticles. The in vivo study evaluating the antitumor activity of the nanoparticles confirmed the data obtained in the in vitro tests, demonstrating that the nanoparticle without TPGS significantly reduced tumor volume, tumor mass, maintained body weight, and improved survival in mice. Moreover, the biochemical evaluation evidenced possible hepatotoxicity for formulation with TPGS.

The sanitary emergency generated by the pandemic COVID-19, instigates the search for scientific strategies to mitigate the damage caused by the disease to different sectors of society. The disease caused by the coronavirus, SARS-CoV-2, reached 216 countries/territories, where about 20 million people were reported with the infection. Of these, more than 740,000 died. In view of the situation, strategies involving the development of new antiviral molecules are extremely important. The present work evaluated, through molecular docking assays, the interactions of 4′-acetamidechalcones with enzymatic and structural targets of SARS-CoV-2 and with the host’s ACE2, which is recognized by the virus, facilitating its entry into cells. Therefore, it was observed that, regarding the interactions of chalcones with Main protease (Mpro), the chalcone N-(4′[(2E)-3-(4-flurophenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPF) has the potential for coupling in the same region as the natural inhibitor FJC through strong hydrogen bonding. The formation of two strong hydrogen bonds between N-(4[(2E)-3-(phenyl)-1-(phenyl)-prop-2-en-1-one]) acetamide (PAAB) and the NSP16-NSP10 heterodimer methyltransferase was also noted. N-(4[(2E)-3-(4-methoxyphenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPM) and N-(4-[(2E)-3-(4-ethoxyphenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPE) chalcones showed at least one strong intensity interaction of the SPIKE protein. N-(4[(2E)-3-(4-dimetilaminophenyl)-1-(phenyl)-prop-2-en-1-one]) acetamide (PAAPA) chalcone had a better affinity with ACE2, with strong hydrogen interactions. Together, our results suggest that 4′-acetamidechalcones inhibit the interaction of the virus with host cells through binding to ACE2 or SPIKE protein, probably generating a steric impediment. In addition, chalcones have an affinity for important enzymes in post-translational processes, interfering with viral replication., Graphical abstract Image 1, Highlights • Chalcones derivatives interacting with SARS-CoV-2 SPIKE protein. • Strong affinity with the ACE2 human host cells. • The 4′-acetamidechalcones can interfering with the virus replication.

Staphylococcus aureus is a highly virulent pathogen, capable of biofilm formation and responsible for thousands of deaths each year. The prevalence of Methicillin-Resistant S. aureus (MRSA) strains has increased in recent years and thus, the development of new antibiotics has become necessary. Antimicrobial Peptides (AMPs) are effective against a variety of multidrug-resistant bacteria and low levels of resistance have been reported regarding these molecules. Dinoponera quadriceps ant venom (DqV) has been described regarding its effect against S. aureus. In this study, we have evaluated the antibacterial effect of DqV-AMPs, the dinoponeratoxins (DNTxs), against Methicillin-Sensitive and a Methicillin-Resistant S. aureus strains. Our results show DNTx M-PONTX-Dq3a as a potent inhibitor of both strains, being able to prevent biofilm formation at low micromolar range (0.78–3.12 μM). It also showed a short-time effect through membrane disruption. M-PONTX-Dq3a opens up new perspectives for the prevention of biofilm formation through the development of anti-adhesive surface coatings on medical devices, as well as the treatment of resistant strains in skin or soft tissue infections.

Acute kidney injury (AKI) is a complex condition which has an intricate pathology mostly involving hemodynamic, inflammatory, and direct toxic effects at the cellular level with high morbidity and mortality ratios. Renal ischemic reperfusion injury (RIRI) is the main factor responsible for AKI, most often observed in different types of shock, kidney transplantation, sepsis, and postoperative procedures. The RIRI-induced AKI is accompanied by increased reactive oxygen species generation together with the activation of various inflammatory pathways. In this context, plant-derived medicines have shown encouraging nephroprotective properties. Evidence provided in this systemic review leads to the conclusion that plant-derived extracts and compounds exhibit nephroprotective action against renal ischemic reperfusion induced-AKI by increasing endogenous antioxidants and decreasing anti-inflammatory cytokines. However, there is no defined biomarker or target which can be used for treating AKI completely. These plant-derived extracts and compounds are only tested in selected transgenic animal models. To develop the results obtained into a therapeutic entity, one should apply them in proper vertebrate multitransgenic animal models prior to further validation in humans.

BACKGROUND AND AIMS The aim of the study was to evaluate the early association of urinary cystatin-C (uCysC) (proximal tubular injury biomarkers) with sepsis and longer hospital stay in premature newborns (NBs) with neonatal infection. METHOD This is a prospective study with NBs from Neonatal Intensive Care Unit (nUTI), between August 2019 and September 2020. Three groups were constructed: healthy NBs, NBs with neonatal infection without sepsis and NBs with sepsis. Premature newborns with kidney disease, renal malformation, insufficient data in the medical record, or samples with scarce volume were excluded. Urine samples were collected for biomarkers measurements. Urinary cystatin-C was quantified in the urine and expressed with or without ratio by urinary creatinine to investigate the urinary concentration bias. RESULTS In total, 62 NBs were included: 27 healthy, 24 with neonatal infection without sepsis and 11 with sepsis. No acute kidney injury (AKI) was found in NBs and 22 (63%) had longer hospital stay (˃ 30 days). Higher levels of urinary cystatin-C, both adjusted for urinary creatinine, were elevated in NBs's sepsis group. Moreover, in NBs with a longer hospital stay, there was an elevated level of urinary cystatin-C, which was capable to predict the need for ˃ 30 days of hospital stay (AUC-ROC = 0.783, P = .01). CONCLUSION Our study demonstrated that kidney tubular damage biomarker, such as uCysC, was associated with early sepsis in premature NBs and was useful to prognosis evaluation.

The extract from leaves of Genipa americana L., Rubiaceae, has antichagasic effect. The major components of this extract, arabinogalactan-glycoconjugate fractions (PFI and PFII), have shown similar biological effects of the crude extract, becoming interesting to evaluate the antichagasic effect of PFI and PFII. This work aimed to access the trypanocidal effect of PFI and PFII against the benznidazole-resistant Trypanosoma cruzi Y strain and to investigate the mechanism of action. PFI and PFII were evaluated on epimastigote (24, 48, 72 h), trypomastigote (24 h), and amastigote (24 and 48 h) forms. The cytotoxicity on LLC-MK2 cells was made by MTT assay. Flow cytometry analysis with PE-annexin V (Ax), 7-AAD, and DCFH-DA staining and scanning electron microscopy (SEM) were performed in epimastigote forms for evaluation of the cell death pathway. PFI showed effect against epimastigote forms (EC50/24 h = 580 ± 0.17 μg/ml; EC50/48 h = 530 ± 0.13 μg/ml; EC50/72 h = 500 ± 0.14 μg/ml), while PFII did not show effect on any tested concentrations. In trypomastigotes, the PFI and PFII showed effect with EC50 values of 100 ± 0.09 and 23 ± 0.06 μg/ml respectively. PFI and PFII were also able to decrease amastigotes/100 cells parameter. PFI and PFII were not cytotoxic in LLC-MK2 cells at the highest tested concentration, resulting in selectivity index (SI) higher than 15 for PFI and higher than 65 for PFII. The increase in labeling of 7-AAD and DCFH-DA fluorescence on cytometry flow assays suggested necrosis as the cell death pathway of PFI in epimastigotes of T. cruzi, being confirmed by pores in the membrane observed on SEM. PFI and PFII are partially responsible by the trypanocidal effect of the crude extract of G. americana, with PFI showing the same action mechanism of crude extract and PFII being the most selective and potent fraction, presenting the biotechnological potential for antichagasic natural products.

Gentamicin is an aminoglycoside antibiotic used in the treatment of gram-negative bacterial infections. However, its prolonged use is limited due to a high risk of nephrotoxicity. This study aimed to investigate the protective effects of a lipid transfer protein isolated from Morinda citrifolia L., Rubiaceae, seeds against gentamicin-induced nephrotoxicity in rats, and in a proximal tubular epithelial cell line from Rhesus monkey (LLC-MK2). Treatment with 6.25–25 μg/ml of the protein protected cells against cytotoxicity induced by 2 mM gentamicin. In the in vivo study, protein treatment (8 mg/kg, orally administered daily for 7 days 2 h after intraperitoneal injection of 100 mg/kg gentamicin) improved the levels of creatinine, urea, and uric acid. Additionally, it alleviated oxidative stress and inflammation induced by gentamicin, including a reduction in myeloperoxidase activity and cytokine modulation. Data were corroborated with histological analysis showing attenuated alterations by protein treatment. These findings suggest that this protein protects against gentamicin-induced nephrotoxicity. In addition, this natural product may be as an adjunctive tool to reduce kidney toxicity and improve the clinical usage profile of gentamicin.

Interleukin-18 (IL-18), a proinflammatory and proatherogenic cytokine, has been associated with type 2 diabetes, metabolic syndrome, stroke and coronary artery disease. Some studies have indicated that the IL-18 promoter -137 G/C polymorphism seems to be associated with changes in the IL-18 expression and may contribute to the development of cardiovascular disease (CVD). The aim of this study was to evaluate the association between -137 G/C polymorphism and the levels of IL-18, biochemical markers for cardiovascular disorders, anthropometric profile and cardiovascular disease in Brazilian patients with type 2 diabetes (T2DM).Study subjects comprised 125 T2DM patients undergoing follow-up at a reference endocrinology service in northeastern Brazil. The -137G/C polymorphism in the IL-18 gene and serum IL-18 levels were determined by using allele-specific polymerase chain reaction (PCR) and enzyme-linked immune assay (ELISA), respectively. The anthropometric parameters were assessed using a Body Composition Monitor with Scale, and the laboratory data were measured using an automatic analyzer as well as spectrophotometric analysis.The genotype distribution of IL-18 -137 G/C genetic polymorphism was significantly different among T2DM patients with and without CVD. The results show an association between the CC genotype of -137G/C polymorphism and CVD in T2DM patients (p 0.001). Serum levels of IL-18 were significantly higher in CC carriers (843.1 pg/mL) compared with GG or GC carriers (303.6 pg/mL and 292.0 pg/mL, respectively). In addition, the present study showed that carriers of the CC genotype also had significantly higher concentrations of creatinine and albuminuria than carriers of the GG or GC genotypes (p 0.05 in both).These results suggest that Brazilian T2DM patients with the CC genotype seem to show a predisposition to CVD, as well as an elevation in markers of renal function.

Parkinson's disease (PD) is characterized by dopaminergic cell loss in the substantia nigra, and PD brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The study evaluated the neuroprotective activity of 1α,25-dihydroxy vitamin D3 (VD3), on the rotenone (ROT)-induced cytotoxicity in PC12 cells. The viability parameters were assessed by the MTT and flow cytometry, on cells treated or not with VD3 and/or ROT. Besides, ROS production, cell death, mitochondrial transmembrane potential, reduced GSH, superoxide accumulation, molecular docking (TH and Keap1-Nrf2), and TH, Nrf2, NF-kB, and VD3 receptor protein contents by western blot were evaluated. VD3 was shown to improve the viability of ROT-exposed cells. Cells exposed to ROT showed increased production of ROS and superoxide, which decreased after VD3. ROT decrease in the mitochondrial transmembrane potential was prevented, after VD3 treatment and, VD3 was shown to interact with tyrosine hydroxylase (TH) and Nrf2. While ROT decreased TH, Nrf2, and NF-kB expressions, these effects were reversed by VD3. In addition, VD3 also increased VD3 receptor protein contents and values went back to those of controls after ROT exposure. VD3 protects PC12 cells against ROT damage, by decreasing oxidative stress and improving mitochondrial function. One target seems to be the TH molecule and possibly an indirect Nrf2 activation could also justify its neuroprotective actions on this PC12 cell model of PD.

Amphotericin B (AmB), used for systemic fungal infections, has a limited clinical application because of its high nephrotoxicity. Natural antioxidant and anti-inflammatory substances have been widely studied for protection against drug-induced nephrotoxicity. α-Bisabolol (BIS) has demonstrated a nephroprotective effect on both in vitro and in vivo models.The aim of this work was to evaluate the effect of BIS against AmB-induced nephrotoxicity in vitro.LLC-MK2 cells were pre- and post-treated with non-toxic BIS concentrations and/or AmB IC50 (13.97 μM). Cell viability was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. Flow cytometry analyses were used to assess cell death mechanism, production of reactive oxidative stress (ROS) and mitochondrial transmembrane potential. Kidney Injury Molecule-1 (KIM-1) levels were measured via ELISA.The present work showed that BIS pretreatment (125; 62.5 and 31.25 μM) increased cell viability when compared to the group treated only with AmB IC50. AmB treatment induced both necrosis (7-AAD-labeled cells) and late apoptosis (AnxV-labeled). BIS was able to prevent the occurrence of these events. These effects were associated with a decrease of ROS accumulation, improving transmembrane mitochondrial potential and protecting against tubular cell damage, highlighted by the inhibition of KIM-1 release after BIS treatment.BIS presented a potential effect on model of renal cytotoxicity induced by AmB, bringing perspectives for the research of new nephroprotective agents.

Background:Scorpion venom causes renal injury and affects vascular ion-channels function. Centruroides margaritatus scorpion is found in Colombia and is frequently the cause of envenomation accidents; however, its renal impact has never been investigated.Objective:To evaluate the effects of C. margaritatus venom (CmV) on renal parameters using isolated rat kidney and renal cell culture models.Methods:Wistar rats (n = 5, weighing 240-300 g) were first perfused with Krebs-Henseleit solution containing 6 g 100 mL-1 bovine serum albumin. After 30 minutes, the kidneys were perfused with CmV to a final concentration of 10 μgmL-1; evaluation was performed by measuring Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Moreover, kidney histological analyses and cell cytotoxicity in renal tubule epithelial cells (MDCK) and proximal tubular cells (LLC-MK2) were assessed.Results:CmV increased PP and RVR 60 min after perfusion. On the other hand, UF, GFR, and the percentages of sodium, potassium and chloride tubular transport decreased after experimental envenomation. UF dropped after 120 min, while GFR and percentage of electrolyte tubular transport diminished after 60, 90 and 120 min. CmV was not toxic to MDCK cell line but reduced the viability of LLC-MK2 cells at concentrations ranging from 6.25 to 200 μgmL-1. Histological analyses disclosed hydropic degeneration, edema, and protein deposits. Flow cytometry disclosed that cell death occurred predominantly by necrosis.Conclusion:Our results suggest that C. margaritatus venom can trigger renal impairment, mainly in the proximal kidney tubule.

Acute Kidney Injury (AKI) is associated with high morbidity and mortality. Ischemia and reperfusion (I/R) are events that lead to AKI through hypoxia, reactive oxygen species (ROS) production, oxidative stress and apoptosis. We aimed to evaluate the mechanism of nephroprotection mediated by Bisabolol in human tubular kidney cells after injury by I/R in vitro. HK2 cells were exposed to I/R and treated with Bisabolol. Cell viability was accessed by MTT assay. Cells were submitted to flow cytometry to evaluate necrotic/apoptotic cells, reactive oxygen species production and mitochondrial transmembrane depolarization. TBARS and GSH were used as parameters of redox balance. Also, KIM-1 supernatant levels were measured. In order to identify an interaction between bisabolol and NOX4, molecular docking and enzymatic assays were performed. Expression of isoform NOX4 on treated cells was examined by western-blot. Finally, cells were visualized by scanning electron microscopy. Bisabolol improved cell viability and prevented cell death by apoptosis, indicated also by the decreased levels of KIM-1. It was observed a decrease on reactive oxygen species production and mitochondrial depolarization, with antioxidant regulation by increased GSH and decreased lipid peroxidation. It was also demonstrated that bisabolol treatment can inhibit NOX4. Finally, SEM images showed that bisabolol reduced I/R-induced cell damage. Bisabolol treatment protects HK2 cells against oxidative damage occasioned by I/R. This effect is related to inhibition of apoptosis, decrease on KIM-1 release, reactive oxygen species accumulation and mitochondrial dysfunction. Bisabolol inhibited NOX4 activity in the tubular cells, impairing reactive oxygen species synthesis.

The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwestern India and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a growing body of evidence indicating that these envenomings may be associated with systemic alterations, including acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these experimental models, in agreement with clinical observations.

Chagas disease is a disease that is emerging in North America and Europe countries. Benznidazole is the main drug available, but it has high toxicity and low efficacy in the chronic phase. In this way, researching new antichagasic agents is necessary. Thus, the aim of this study is to evaluate the effect of novel chalcones and the influence of chlorine substitutions on Trypanosoma cruzi and host cells. Unsubstituted (1), 4-chlorine substituted (2) and 2,4-chlorine substituted (3) chalcones were synthesized by Claisen-Schmidt condensation, characterized, and electrical distribution was assessed by Density Fuctional Theory (DFT). The host cells toxicity (LLC-MK2) was performed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) reduction assay. The effect on epimastigote (24, 48 and 72h), trypomastigote (24h) and amastigotes (24 h) was evaluated. Flow cytometry assays were performed with 7-Aminoactinomycin D (7-AAD) and Annexin-PE, Dichlorofluorescein diaceteate (DCFH-DA) and Rhodamine123 (Rho123). Finally, molecular docking predicted interactions between chalcones and cruzain (TcCr) and trypanothione reductase (TcTR). The toxicity on host cells was reduced almost twenty times on chlorine substituted molecules. On epimastigote and trypomastigote forms, all substances presented similar effects. After treatment with molecule 3, it was observed a decrease in infected cells and intracellular amastigotes. Their effect is related to necrotic events, increase of cytoplasmic Reactive Oxygen Species (ROS) and mitochondrial dysfunction. Also, this effect might be associated with involvement of TcCr and TcTR enzymes. Therefore, the results showed that chlorine substitution on chalcones reduces the host cell's toxicity without compromising the effect on Trypanosoma cruzi Y strain forms, and it occurs over membrane damage, oxidative stress and possible interactions with TcCr and TcTR.

Background The sanitary emergency installed in the world, generated by the pandemic of COVID-19, instigates the search for scientific strategies to mitigate the damage caused by the disease to different sectors of society. The disease caused by the coronavirus, SARS-CoV-2, reached 216 countries/territories, where about 199 million people were reported with the infection. Of these, more than 4 million died. In this sense, strategies involving the development of new antiviral molecules are extremely important. The main protease (Mpro) from SARS-CoV-2 is an important target, which has been widely studied for antiviral treatment. This work aims to perform a screening of pharmacodynamics and pharmacokinetics of synthetic hybrids from thymoquinone and artemisin (THY-ART) against COVID-19. Results Molecular docking studies indicated that hybrids of artemisinin and thymoquinone showed a relevant interaction with the active fraction of the enzyme Mpro, when compared to the reference drugs. Furthermore, hybrids show an improvement in the interaction of substances with the enzyme, mainly due to the higher frequency of interactions with the Thr199 residue. ADMET studies indicated that hybrids tend to permeate biological membranes, allowing good human intestinal absorption, with low partition to the central nervous system, potentiation for CYP-450 enzyme inhibitors, low risk of toxicity compared to commercially available drugs, considering mainly mutagenicity and cardiotoxicity, low capacity of hybrids to permeate the blood–brain barrier, high absorption and moderate permeability in Caco-2 cells. In addition, T1–T7 tend to have a better distribution of their available fractions to carry out diffusion and transport across cell membranes, as well as increase the energy of interaction with the SARS-CoV-2 target. Conclusions Hybrid products of artemisinin and thymoquinone have the potential to inhibit Mpro, with desirable pharmacokinetic and toxicity characteristics compared to commercially available drugs, being indicated for preclinical and subsequent clinical studies against SARS-CoV-2. Emphasizing the possibility of synergistic use with currently used drugs in order to increase half-life and generate a possible synergistic effect. This work represents an important step for the development of specific drugs against COVID-19.

Background and objectives: Single Nucleotide Polymorphisms (SNP) are promising atherosclerosis indicators. The APOAI gene polymorphism variant rs670 shows that G (Guanine) allele is the most frequent and the A (Adenine) allele is the rarest, with both being associated with HDL-c variations in different ethnicities. In the APOB variant rs693, the C (Cytosine) allele is the most frequent, while the T (Timine) allele is the rarest and associated with dyslipidemia and cardiovascular risk. This study aims to evaluate biochemical and genetic risk factors for CVD development in young Brazilians and Africans.Methods: Anthropometric parameters were used for measurement. Blood Samples were collected to evaluate Total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, Apolipoproteins A-I and B, ApoB/ApoA-I ratio, and high-sensitive C-reactive protein (hs-CRP) quantification. The antioxidant capacity of HDL was evaluated by the LagTime assay and the serum enzyme Paraoxonase 1 (PON1) activity. Genetic Analysis was made through DNA extraction from whole blood leukocytes and genotyping of the rs670 and rs693 polymorphisms using the real-time PCR (q-PCR) technique. Results: G allele and homozygous GG genotype of rs670 were the most frequent in both ethnicities. Among Brazilians, the A allele and GA genotype were associated with higher cardiovascular risk. Regarding the rs693 polymorphism, the C allele was the most frequent in both ethnicities, and the T allele was more frequent between Africans. Also, the CC genotype was the most frequent among Brazilians, and the CT genotype the most frequent among Africans, which presented higher TT frequency. T allele and TT genotype were associated with higher cardiovascular risk factors in both populations. Conclusion: Brazilians are doubly affected by these variables, while Africans are more susceptible to risks due to changes in the rs693 polymorphism.

Ischemia-reperfusion (I/R) plays an important role in the process of acute kidney injury (AKI) due to the generation of reactive oxygen species (ROS). Substances of natural origin have been studied in the prevention of oxidative damage related to I/R. Quercetin is a flavonoid with antioxidant potential and modulate enzymes, such the inhibition of the Rennin-Angiotensin System (RAS). The aim of this study is to evaluate the nephroprotective effect of quercetin against the I/R and analyze the inhibition of RAS. Rhesus monkey Kidney Epithelial Cells (LLC-MK2 line) were submitted to an in vitro ischemia/reperfusion model. After the reperfusion cells were treated with quercetin, the cell viability was accessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Tubular cell damage was assessed by the Kidney Injury Molecule-1 (KIM-1) measurement. Oxidative stress was evaluated through Thiobarbituric Acid Reactive Substances (TBARS) and reduced glutathione (GSH). The evaluation of cell death and the mitochondrial depolarization were analyzed by flow cytometry. Quercetin prevents cell death reducing oxidative stress and preventing mitochondrial membrane depolarization. Molecular docking showed that quercetin prevents cell damage better than losartan and lisinopril, inhibitors of RAS. Quercetin has a potential to interact with type 1 angiotensin II receptor (AT1) with greater affinity through the formation of five hydrogen bonds of strong intensity.

Clinical Background: Mental disorders, especially depression, are associated with several comorbidities in the kidneys. Depression is the psychiatric disorder that mostly affects individuals with chronic kidney disease (CKD) and end-stage kidney disease. Epidemiology: The mainly prescribed drugs involved in overdose cases are opioids, benzodiazepines, and antidepressants. Antidepressants are the main psychiatric drugs that lead to kidney injury, mainly the second-generation ones. However, the prevalence of depression in dialysis patients varies from 22.8 to 39.3%. Therefore, psychiatric patients have 1.5-3 times more hospitalization compared to patients having only CKD. Challenges: Randomized clinical studies should be encouraged. Studies have shown an association between depression and progression of kidney disease. The mechanisms are not completely clear, but changes on neurotransmitter release and endocrine functions appear to be related to it. Additionally, the use of antidepressant and other psychoactive drugs can induce kidney injury. Hyponatremia induced by second-generation antidepressant drugs is an important feature and can be a risk factor for elderly or patients with comorbidities such as cerebral edema, brain damage or coma. Besides this class, drugs used for anxiety and bipolar disorders or sympathomimetic drugs of abuse can trigger acute kidney injury, possibly due to endothelial dysfunction and thromboembolic and ischemic events. Prevention and Treatment: The early detection of renal impairment and the prescription of nephroprotective strategies has been a clinical challenge. Some studies aim to describe the biochemical mechanisms involved and develop clinical management strategies for these patients. This chapter brings attention to this topic, discussing the major mechanisms and clinical features of kidney injury associated with mental illness, and the most relevant clinical strategies.

Clinical Background: Mental disorders, especially depression, are associated with several comorbidities in the kidneys. Depression is the psychiatric disorder that mostly affects individuals with chronic kidney disease (CKD) and end-stage kidney disease. Epidemiology: The mainly prescribed drugs involved in overdose cases are opioids, benzodiazepines, and antidepressants. Antidepressants are the main psychiatric drugs that lead to kidney injury, mainly the second-generation ones. However, the prevalence of depression in dialysis patients varies from 22.8 to 39.3%. Therefore, psychiatric patients have 1.5-3 times more hospitalization compared to patients having only CKD. Challenges: Randomized clinical studies should be encouraged. Studies have shown an association between depression and progression of kidney disease. The mechanisms are not completely clear, but changes on neurotransmitter release and endocrine functions appear to be related to it. Additionally, the use of antidepressant and other psychoactive drugs can induce kidney injury. Hyponatremia induced by second-generation antidepressant drugs is an important feature and can be a risk factor for elderly or patients with comorbidities such as cerebral edema, brain damage or coma. Besides this class, drugs used for anxiety and bipolar disorders or sympathomimetic drugs of abuse can trigger acute kidney injury, possibly due to endothelial dysfunction and thromboembolic and ischemic events. Prevention and Treatment: The early detection of renal impairment and the prescription of nephroprotective strategies has been a clinical challenge. Some studies aim to describe the biochemical mechanisms involved and develop clinical management strategies for these patients. This chapter brings attention to this topic, discussing the major mechanisms and clinical features of kidney injury associated with mental illness, and the most relevant clinical strategies.

Background and Aims Drug-induced acute kidney injury (AKI) is a health problem that increases morbidity and mortality rates as well as costs to health systems. Amphotericin B (AnB) is a widely used antifungal, which its therapy is closely associated with nephrotoxicity. The mechanisms of nephrotoxicity are not yet fully understood and there is a need to search for substances that prevent and / or reverse the associated renal damage. (-) - α-Bisabolol (BIS) has been demonstrated nephroprotective effect due to its antioxidant action. Thus, the aim of this study was to evaluated the effect of BIS as a protective substance on renal cell injury induced by amphotericin B. Method LLC-MK2 tubular renal cells were treated with different concentrations of BIS and AnB and its viability was measured by MTT assay to define the working concentrations of BIS and AnB. Then, cells were pretreated or post treated with BIS (31.25 - 125 μM) and AnB (13.97 μM) for 24 hours to evaluate the protective effect of BIS. Control (CT) was treated with sterile PBS. The experimental groups were further submitted to flow cytometry assays for membrane integrity evaluation using 7-AAD and measurement of cytoplasmic ROS production using DCFH-DA. The experiments were performed in triplicate (n = 3), and the data analyzed by One-Way ANOVA with Bonferroni post-test. Results BIS concentrations from 31.25 to 125 μM were selected, as well as the IC50 for AnB (13.97 ± 1.45 μM) for the furthers assays. Pretreatment with BIS increased cell viability by about 10% (31.25 – 63.31 ± 2.04%; 62.5 – 64.75 ± 2.10%; 125 – 63.92 ± 2.72%) compared to the AnB-only group (49.79 ± 1.25%), which had its cell viability reduced by about 50% (CT – 100.70 ± 0.83%). BIS was also able to increase the whole cell population at concentrations of 62.5 (83.10 ± 1.10%) and 125 μM (84.67 ± 0.98%) when compared to the AnB group (76.54 ± 1.18%). In DCF labeling, it was observed that AnB increased the production of ROS in 20% (IFR = 1.21 ± 0.02) when compared to the control (IFR = 1.00 ± 0.01), and BIS (31.25 μM) was able to reduce in 41% (IFR = 0.79 ± 0.01) when compared to the AnB group. Conclusion The (-)-α-bisabolol was able to prevent the renal cytotoxicity induced by amphotericin.

Caffeic acid (CA) is a hydroxycinnamic acid, a group of natural organic phenolic compounds used in folk medicine and in foods for the prevention and treatment of obesity. CA is present in a variety of plants, as it is an intermediate in secondary metabolism. It is found in coffee, wine and olive oil. The aim of this work was to study the influence of caffeic acid (CA) on the reduction of intracellular lipid accumulation, intracellular reactive oxygen species (ROS) formation and in mitochondrial transmembrane potential alterations in differentiated 3T3-L1 cells. The pre-adipocyte cell line 3T3-L1 was differentiated using a differentiation cocktail. Cells were treated with CA, and the MTT assay was performed to assess the effect of CA on pre-adipocytes. The quantification of lipids accumulated within the mature cells was performed using Oil Red O dye. Flow cytometry was used to evaluate the production of ROS through 2',7'-dichlorofluorescein diacetate (DCFH-DA) oxidation and changes in mitochondrial transmembrane potential by Rhodamine 123 dye (Rho123). CA did not exhibit significant toxicity on the 3T3-L1 cell line at the studied concentrations. Caffeic acid causes significant reduction of lipid content in the cells submitted to the post- and co-treatment, being more effective in co-treatment. Caffeic acid results in an absolute decrease in the formation of intracytoplasmic reactive oxygen species. The treatment with caffeic acid protects against oxidative stress caused in the mitochondria by the adipocyte differentiation process. Thus, CA acts on adipogenesis, reducing intracellular lipid accumulation in the 3T3-L1 cells. It reduces intracellular ROS formation and mitochondrial transmembrane potential alterations in differentiated cells. Key words: Caffeic acid, adipocytes, reactive oxygen species, oxidative stress.

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.

Ethnopharmacological relevance The parts of the Genipa americana (Rubiaceae) tree, also known as “jenipapo” or “jenipapeiro”, has been used in traditional Medicine in parasitic and bacterial infections. Thus, the experimental evolution of the antiparasitic activity of polysaccharide extracts from Genipa americana leaves, and correlation with antiparasitic and popular use is important. Aim of the study To evaluate the effect of polysaccharide extract obtained from Genipa americana leaves on all Trypanosoma cruzi (Y strain: benznidazole-resistant) developmental forms, a protozoan that causes Chagas’ disease. Materials and methods An extract rich in polysaccharides was obtained from the leaves of Genipa americana (GaEPL) by associating depigmentation in methanol followed by extraction of polysaccharides in NaOH and precipitation with ethanol. Cytotoxicity to mammalian cells (LLC-MK2) was determined using an MTT assay. Antiparasitic activity was evaluated against epimastigote, trypomastigote and amastigote forms of T. cruzi. Cell-death mechanism was determined in epimastigote forms by flow cytometry analysis after FITC-annexin V (Ax), 7-AAD, and H2DCFDA staining. Striking morphological changes were observed by scanning electron microscope. Results GaEPL (6.5% yield; 54.6% total carbohydrate; 21.1% uronic acid and 12% protein), inhibited all T. cruzi developmental forms, epimastigotes after periods of 24 h (IC50 = 740 ± 0.075 µg/mL), 48 h (IC50 = 710 ± 0.053 µg/mL) and 72 h (IC50 = 870 ± 0.052 µg/mL) of incubation; trypomastigotes (IC50 = 470 ± 0.082 µg/mL) after periods of 24 h and intracellular amastigotes (IC50/2 = 235 or IC50 = 470 µg/mL) after periods of 24 and 48 h of incubation, with no toxicity on LLC-MK2 cells at the used concentrations. Analysis of the possible action mechanism in the parasites suggested cell death by necrosis with the involvement of reactive oxygen species (ROS). The scanning electron microscopy (SEM) confirmed T. cruzi death by necrosis. Conclusions GaEPL showed significant activity against the epimastigote, trypomastigote and amastigote forms of T. cruzi, strain Y, suggesting cell death by necrosis with involvement of reactive oxygen species.

COVID-19 pandemic is the biggest public health problem of the century so far.The main protease (Mpro) is one of the main enzymes studied as a pharmacological target. In this context, the present work aimed to perform a virtual screening of possible inhibitors against the enzyme Mpro, having limonoids as the main object of research as supposed inhibitors. Molecular docking simulations indicated that limonoids have an affinity to complex with M-pro.However, Limonine and Nimoliciol showed nonspecific and low affinity interactions. In conclusion, Limonoids are substances of natural origin that can be used in the study of new pharmacological tools designed to combat and understand COVID-19., Graphical abstract Image 1

Cathelicidins are antimicrobial peptides produced by humans and animals in response to various pathogenic microbes. Crotalicidin (Ctn), a cathelicidin-related vipericidin from the South American Crotalus durissus terrificus rattlesnake's venom gland, and its fragments have demonstrated antimicrobial and antifungal activity, similarly to human cathelicidin LL-37. In order to provide templates for the development of modern trypanocidal agents, the present study evaluated the antichagasic effect of these four peptides (Ctn, Ctn[1-14], Ctn[15-34] and LL-37). Herein, Ctn and short derived peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Ctn inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease. Moreover, Ctn showed a high selective index against trypomastigote forms (>200). Ctn induced cell death in T. cruzi through necrosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as loss of membrane integrity and cell shrinkage, as observed through scanning electron microscopy. Overall, Ctn seems to be a promising template for the development of antichagasic agents.

Summary Cardiovascular diseases are among the main causes of mortality worldwide, and dyslipidemia is a principal factor risk. Hence the study of biochemical markers is necessary for early diagnosis. Objectives Evaluate biomarkers to diagnose the risks of cardiovascular diseases in healthy Brazilian and African young adults. Design & Methods Weight, height, waist circumference, percentage of body fat and systemic blood pressure were measured; and fasting blood samples were taken for biochemical analysis. Triglycerides, total cholesterol, HDL-c, and apolipoproteins A-I and B were measured on automated equipment using commercially available kits, in addition to the tests of antioxidant capacity of HDL and the enzymatic activity of Paraoxonase 1. Results After statistical analysis, it was found that BMI, WC, fat (%), triglycerides, ApoB/ApoA-I ratio and Vmax were higher in Brazilians, while HDL-c, ApoA-I, Lag Time, Vmax and PON1 activity were higher in Africans. In Brazilians, the ApoB/ApoA-I ratio was related to obesity factors and lipid profile, but in Africans it was related only to lipids. The antioxidant capacity of HDL and PON1 activity was better in Africans. Through independence testing, we observed an association with moderate risk of myocardial infarction with gender in Africans. In the binary logistic regression analysis, it was found that men in general – and particularly African men – have higher risk of myocardial infarction than women; Odds Ratio 2144 (CI95%: 1343–3424) and 2281 (CI95%: 1082–4811), respectively. Conclusions The anthropometric and biochemical parameters of Brazilians, especially men, predispose them to greater risks of cardiovascular diseases.

Made available in DSpace on 2020-12-10T19:40:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Research Support Foundation of Ceara State (FUNCAP/CE) Background: Envenomation caused by Bothrops alternatus is common in Southern Brazil. Acute Kidney Injury occurs after Bothrops snakebite and more information is necessaryrequired to understand its mechanism. Objective: The objective was to evaluate the effect of Bothrops alternatus venom (BaV) on renal cells and rat isolated kidney function. Methods: Wistar rats (n = 6, weighing 260-320 g) were perfused with a Krebs-Henseleit solution containing 6 g 100 mL(-1) of bovine serum albumin. After 30 minutes, the kidneys were perfused with BaV to a final concentration of 1 and 3 mu gmL(-1); and subsequently were evaluated for Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Renal histological analysis, cytokine release, oxidative stress and cytotoxicity in renal proximal tubular cells were assessed. Results: BaV reduced PP, RVR, GFR, UF, total and proximal sodium transport (% TNa+), and chloride (%TCl-) in the isolated kidney perfusion model. Histological analysis of perfused kidneys disclosed the presence of proteinaceous material in the glomeruli and renal tubules, vacuolar tubular epithelial cell degeneration, Bowman's capsule degeneration, swelling of glomerular epithelial cells, glomerular atrophy and degeneration, and the presence of intratubular protein. Cytokine release (TNF-alpha, IL-1 beta, IL-10) and oxidative stress were increased in the kidneys. The viability of LLC-MK2 cells (IC50: 221.3 mu g/mL) was decreased by BaV and necrosis was involved in cell death. Conclusion: These findings indicate that BaV modifies functional parameters in an isolated perfused kidney model and has cytotoxic effects on renal lineage cells. Univ Fed Ceara, Fac Med, Dept Physiol & Pharmacol, BR-60430270 Fortaleza, Ceara, Brazil Univ Fed Ceara, Dept Clin & Toxicol Anal, Fortaleza, Ceara, Brazil Univ Fed Ceara, Fac Med Vet, Fortaleza, Ceara, Brazil Univ Estadual Paulista, CEVAP, Botucatu, SP, Brazil Univ Estadual Paulista, CEVAP, Botucatu, SP, Brazil