In vitro effects of lapachol and ß-lapachone against Leishmania amazonensis.

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and ß-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 µg/mL and for ß-lapachone was 0.65, 1.24, and 0.71 µg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for ß-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

In vitro effects of lapachol and β-lapachone against Leishmania amazonensis

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for β-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

Larval trematodes hosted by Biomphalaria straminea in the Brazilian semiarid region: implications for schistosomiasis control.

The present study aimed to identify larval trematodes shed by snails found in water bodies used by urban communities in a former schistosomiasis endemic area in the state of Piauí, in the Brazilian semiarid region. A malacological survey was performed followed by analysis of the cercariae shed by the snails after light exposure. Biomphalaria straminea specimens (n=1,224) were obtained from all seven collection sites. Cercariae shed by snails were i) single tailed, in which one type of cercariae was identified ( Echinostoma cercariae), and ii) with bifurcated tail (brevifurcate apharyngeate distome, brevifurcate pharyngeate distome, and longifurcate pharyngeate distome [strigeocercaria]). Brevifurcate apharyngeate distome were further examined and the presence of spikes in swimming membranes enabled the identification of Spirorchiidae cercariae in all individuals, demonstrating the absence of cercariae compatible with Schistosoma mansoni . Nevertheless, the accurate diagnosis of S. mansoni circulation in former endemic areas is still necessary.

Larval trematodes hosted by Biomphalaria straminea in the Brazilian semiarid region: implications for schistosomiasis control

@#The present study aimed to identify larval trematodes shed by snails found in water bodies used by urban communities in a former schistosomiasis endemic area in the state of Piauí, in the Brazilian semiarid region. A malacological survey was performed followed by analysis of the cercariae shed by the snails after light exposure. Biomphalaria straminea specimens (n=1,224) were obtained from all seven collection sites. Cercariae shed by snails were i) single tailed, in which one type of cercariae was identified (Echinostoma cercariae), and ii) with bifurcated tail (brevifurcate apharyngeate distome, brevifurcate pharyngeate distome, and longifurcate pharyngeate distome [strigeocercaria]). Brevifurcate apharyngeate distome were further examined and the presence of spikes in swimming membranes enabled the identification of Spirorchiidae cercariae in all individuals, demonstrating the absence of cercariae compatible with Schistosoma mansoni. Nevertheless, the accurate diagnosis of S. mansoni circulation in former endemic areas is still necessary.

Combined effects of caloric restriction and fish oil attenuated anti-depressant and anxiolytic-like effects of fish oil: association with hippocampal BDNF concentrations.

Omega-3-enriched fish oil (FO) and caloric restriction (CR) are nutritional therapeutic approaches that exert an important impact on brain function, behavior, memory, and neuroprotection. Here, we investigate the synergic effects of both therapeutic approaches combined (CR + FO) on behavior (memory, anxiety-like behavior, antidepressant-like behavior), as well as its association with hippocampal brain-derived neurotrophic factor (BDNF) concentrations. Adult male Wistar rats were divided into four dietary groups: Control group (C) - chow ad libitum; CR group - 30 % CR, considering C group food intake; FO group - FO-enriched chow ad libitum; and CR + FO group - FO-enriched 30 % CR chow. After 12 weeks of dietary treatment, behavioural analysis set was conducted, and hippocampal BDNF concentrations were measured. FO group presented anxiolytic-like and antidepressant-like behaviors as well as improved memory in the Morris' water maze. These effects were attenuated by the combined CR + FO treatment. FO group also presented higher BDNF concentrations. There was a positive association between the number of entries in the platform quadrant in the MWM and hippocampal BDNF concentrations (ß = 0.39; R² = 0.15; p = 0.042) and an inverse association between forced swim immobility time and BDNF concentrations (ß = -0.39; R² = 0.15; p = 0.041). Taken together, our data showed that the 12-week FO dietary treatment promoted anxiolytic-like and antidepressant-like behaviors as well as memory improvement, and these effects were associated with BDNF concentrations. Synergic effects of interventions attenuated FO-related behavioral responses and BDNF concentrations and probably reduced hippocampal neuroplasticity.

The comparative advantages of ethanol and sucrose as co-substrates in the degradation of an anionic surfactant: microbial community selection.

The efficiency of linear alkylbenzene sulfonate (LAS) removal from laundry wastewater and the related microbial community was investigated in an anaerobic fluidized bed reactor (AFBR). The AFBR was operated in three stages, in addition to the biomass adaptation stage without LAS (stage I). The stages were differentiated by their supplementary co-substrates: stage II had sucrose plus ethanol, stage III had only ethanol, and stage IV had no co-substrate. The replacement of sucrose plus ethanol with ethanol only for the substrate composition favored the efficiency of LAS removal, which remained high after the co-substrate was removed (stage II: 52 %; stage III: 73 %; stage IV: 77 %). A transition in the microbial community from Comamonadaceae to Rhodocyclaceae in conjunction with the co-substrate variation was observed using ion sequencing analysis. The microbial community that developed in response to an ethanol-only co-substrate improved LAS degradation more than the community that developed in response to a mixture of sucrose and ethanol, suggesting that ethanol is a better option for enriching an LAS-degrading microbial community.

Levels of mannose-binding lectin in individuals with visceral leishmaniasis in the northeast region of Brazil.

Visceral leishmaniasis (VL) is one of the seven priority endemic diseases in the world. The clinical outcome of many infections is not only dependent on the pathogenic organism, but also on the genetic variability of the host susceptibility to infection. Mannose-binding lectin (MBL) is a protein that plays an important role in the innate immune system. The aim of this study was to compare the serum levels of MBL between healthy controls and carriers of VL. The VL cases were recruited randomly from the main hospitals and referral outpatient clinics for VL in São Luís, and from home visits. Determination of MBL protein levels was performed by enzyme-linked immunosorbent assay. Of the 161 patients with VL and the 161 healthy controls, 60.9 and 67.1% had high levels of MBL, respectively. There was no significant difference in MBL levels between cases and controls. Low socioeconomic status and living conditions are conducive to the occurrence of VL. Owing to the small number of existing studies, it is extremely important to conduct further studies on MBL levels and susceptibility to VL, especially in regions where the disease is endemic, such as Maranhão, Brazil.

Novel electrochemical method of fast and reproducible fabrication of metallic nanoelectrodes.

A novel electrochemical wire etching method of fabrication of ultrasharp nanoelectrodes is reported. Tungsten wires can be sharpened to less than 10 nm tip radius in a reproducible manner in less than 1 min by using controllable hydrodynamic electrolyte flow combined with optimized electrochemical etching parameters. The method relies on the variations of the electric field at the surface of a metal wire, while the electrolyte solution is in motion, rather than on the ionic gradient generated in a static solution.

Avaliação histomorfométrica do efeito do extrato aquoso de urucum (norbixina) no processo de cicatrização de feridas cutâneas em ratos / Histomorphometric evaluation of the effects of the aqueous extract of annatto (norbixin) on wound skin healing in rats

A norbixina é um carotenóide dicarboxílico hidrofílico presente no pericarpo das sementes do urucuzeiro (Bixa orellana L.). O urucum é comumente utilizado na indústria alimentícia e cosmética como corante natural. O objetivo deste trabalho foi avaliar o processo de reparo cicatricial de feridas cutâneas abertas de ratos tratadas com extrato aquoso de urucum contendo 2,5% de norbixina através de análise histomorfométrica. Para tanto, realizou-se feridas cutâneas no dorso de 32 ratos Wistar machos. Estas foram tratadas com extrato de urucum (grupo experimental, n = 16) e solução salina 0,9% (grupo controle, n = 16). Aos 3, 7, 14 e 21 dias após realização do procedimento cirúrgico, os animais foram sacrificados. Os preparados histológicos obtidos foram submetidos à técnica de coloração pela Hematoxilina-Eosina (H.E.) para contagem de células inflamatórias e de fibroblastos; corados pelo azul de toluidina 1% para contagem dos mastócitos e com picrossirius-red para avaliação das fibras colágenas totais. As imagens histológicas destas lâminas foram capturadas por câmera digital acoplada ao microscópio óptico, sob foco fixo e clareza de campo, obtendo-se 10 campos por lâmina com aumento final de 400X. As fotomicrografias foram avaliadas através do software ImageJ. Os resultados obtidos foram submetidos ao teste t de student sendo o valor de p considerado significativo para p< 0,05. O tratamento tópico com extrato de urucum utilizado aumentou o infiltrado inflamatório durante o 3º, 14º e 21º dia pós-operatório. O grupo tratado apresentou maior densidade vascular quando comparado ao controle a partir do 7º e menor quantidade de fibroblastos até o 14º dia pós-cirúrgico. Conclui-se que o extrato de urucum contendo 2,5% de norbixina não é inócuo aos tecidos cutâneos e possui efeitos pró-inflamatórios e pró-angiogênicos durante o processo de reparo tecidual cutâneo em ratos, interferindo no processo fisiológico de cicatrização.

Norbixin is a dicarboxylic water-soluble carotenoid present in the pericarp of the achiote (Bixa orellana L.) seed. Annatto is commonly used by the food and cosmetic industries as a natural pigment. The aim of this study was to evaluate the healing process of open dermal wounds of rats treated with an aqueous solution of annatto containing 2.5% of norbixin carried out by histomorphometric analysis. For this end, cutaneous wounds were made on the back of 32 male Wistar rats. The wounds were treated with annatto solution (experimental group, n = 16) and saline solution 0.9% (control group, n = 16). At 3, 7, 14 and 21 days after the surgical procedure, the animals were sacrificed. After histological preparation, the histological material was submitted to the staining technique with hematoxylin-eosin for the counting of inflammatory cells and fibroblasts, with toluidine blue 1% for mast cell counting and with picrosirius red for the total collagen fibers. The images of those histological slides were captured by a digital camera connected to an optical microscope, with fixed focal length and clear field; 10 fields were captured from each slide with final zoom of 400X. The photomicrographs were analyzed by the ImageJ software. The results were submitted to the Student's t-test and the value of p < 0.05 was considered as relevant. The topical treatment with the annatto solution increased the inflammatory infiltrate during the 3rd, 14th and 21st days after surgery. The experimental group presented increased vascular density compared to the control group after the 7th day and a smaller amount of fibroblasts up to the 14th day after surgery. We concluded that the annatto solution containing 2.5% of norbixin is not innocuous to skin tissues and has proinflammatory and proangiogenic effects during the process of skin wound healing in rats, interfering in the physiological healing process.

Characterization and genome organization of a repetitive element associated with the nucleolus organizer region in Leporinus elongatus (Anostomidae: Characiformes).

Chromosome mapping and studies of the genomic organization of repetitive DNA sequences provide valuable insights that enhance our evolutionary and structural understanding of these sequences, as well as identifying chromosomal rearrangements and sex determination. This study investigated the occurrence and organization of repetitive DNA sequences in Leporinus elongatus using restriction enzyme digestion and the mapping of sequences by chromosomal fluorescence in situ hybridization (FISH). A 378-bp fragment with a 54.2% GC content was isolated after digestion with the SmaI restriction enzyme. BLASTN search found no similarity with previously described sequences, so this repetitive sequence was named LeSmaI. FISH experiments were conducted using L. elongatus and other Anostomidae species, i.e. L. macrocephalus, L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii, S. isognathus, and Abramites hypselonotus which detected signals that were unique to male and female L. elongatus individuals. Double-FISH using LeSmaI and 18S rDNA showed that LeSmaI was located in a nucleolus organizer region (NOR) in the male and female metaphases of L. elongatus. This report also discusses the role of repetitive DNA associated with NORs in the diversification of Anostomidae species karyotypes.

Influence of the traditional Brazilian drink Ilex paraguariensis tea on glucose homeostasis.

In this study we examined the acute in vivo effect and short- and long-term in vitro effects of samples from native and commercial Ilex paraguariensis on glucose homeostasis. Also, the potential effect of I. paraguariensis on serum insulin secretion was investigated. The chemical identification and quantification of methyl xanthines and polyphenols in CH2Cl2, EtOAc and n-BuOH fractions of native I. paraguariensis as well as infusions of green and roasted I. paraguariensis from a commercial source was verified by high-performance liquid chromatography. The results for the serum glucose-lowering indicated that both fractions and both infusions were able to improve significantly the oral glucose tolerance curve. Additionally, both the EtOAc and n-BuOH fractions induced-insulin secretion, but EtOAc induced an early (at 15 min) and late (at 60 min) biphasic peak of insulin secretion similar to glipizide stimulatory effect. Both fractions increased liver glycogen content compared with fasted normal rats. Also, EtOAc and n-BuOH fractions inhibited in vitro disaccharidases activities after an acute treatment. The maximum inhibitory effect of the EtOAc and n-BuOH fractions on maltase activity (at 5 min) was around 35%. The evident reduction of protein glycation by glucose or fructose with EtOAc and n-BuOH fractions increased from 7 to 28 days of in vitro incubation. Inhibition of bovine serum albumin glycation by glucose and fructose, by around 50% and 90%, respectively, was observed. Additionally, the green and roasted mate infusions reduced the formation of AGEs in a characteristic long-term effect. In conclusion, this study shows that I. paraguariensis has an anti-hyperglycemic potential role able to improve the diabetic status and is probably a source of multiple hypoglycemic compounds.

Factors associated with oxidative stress in women with breast cancer.

OBJECTIVE: To assess the association between physiological, physical, lifestyle and nutritional variables and oxidative stress biomarkers in women with breast cancer. METHODS: This cross-sectional study was conducted on 55 women newly diagnosed with breast cancer. The extent of oxidative stress was analyzed by the measurement of plasma lipid hydroperoxides (LH), thiobarbituric acid reactive substances (TBARS), protein carbonyl, whole blood reduced glutathione (GSH) and serum antioxidant capacity (AC). Diet data were obtained from food frequency questionnaire. Linear regression was used to determine the association between the variables studied and oxidative stress biomarkers. The protein carbonyl data was not included in the linear regression analyses since the data did not show a normal distribution, even after logarithmic and other transformations. RESULTS: After adjusting for energy intake, the intake of chicken and high-fat dairy products was associated with increased levels of LH, while vitamin E intake was associated with decreased LH levels (R² = 23.8%). Intake of oils was associated with increased levels of TBARS (R² = 6.82%). Positive axillary lymph node status was associated with decreased levels of GSH (R² = 9.31%). Increasing age was directly associated with levels of AC, while animal fat, dairy product, and sweet food intakes were associated with low levels of AC (R² = 41.42%). CONCLUSION: Intake of chicken, vitamin E, dairy products (particularly high-fat dairy products), oils, animal fat, and sweet foods, along with axillary lymph node status and age, may be important determinants of oxidative stress in women with breast cancer.

Electronic structure of Zn, Cu and Ni impurities in germanium.

We present a density functional modelling study of Zn, Cu and Ni impurities in hydrogen-terminated germanium clusters. Their electronic structure is investigated in detail, especially their Jahn-Teller instabilities and electrical levels. Interstitial and substitutional defects were considered and the latter were found to be the most stable defect form for nearly all Fermi level positions. Relative formation energies are estimated semi-empirically with the help of the measured formation energy of the single Ge vacancy. We find that while Zn is a double shallow acceptor, Cu and Ni are deep acceptors with levels close to the available experimental data. Donor levels were only found for interstitial Cu and Zn.

Dietary intake and oxidative stress in breast cancer: before and after treatments.

OBJECTIVE: The aim of this study was to investigate changes in dietary intake, anthropometric parameters and markers of oxidative stress in 40 women who underwent surgery, chemotherapy or radiation therapy for breast cancer. METHODS: Pretreatment and post-treatment measurements included data collected through a food frequency questionnaire, weight and height to calculate the body mass index (BMI) and oxidative stress markers assessed from blood reduced glutathione (GSH), serum antioxidant capacity (AC), plasma thiobarbituric acid reactive substances (TBARS), serum lipid hydroperoxides (LH) and plasma carbonyls. Differences were compared using paired Student's t-test or paired Wilcoxon's test. RESULTS: A significant increase (P < 0.05) in the intake of the food groups: meat and eggs, dairy products, beans, oils and fats, as well as food from the subgroups: red meat, milk and other dairy products rich in fat, fruit rich in vitamin C and vegetable fats was found after treatments. There was a significant increase in body weight (P < 0.05), BMI (P < 0.05), levels of TBARS (P < 0.0001), LH (P < 0.005) and carbonyls (P < 0.0001) and a significant decrease of levels of AC (P < 0.005) and GSH (P < 0.0001). CONCLUSION: Breast cancer diagnosis and treatments were associated with dietary intake changes and increased body weight, BMI and oxidative stress. These potential changes have important implications for preventive nutrition counseling.

Aetiological treatment with itraconazole or ketoconazole in individuals with Trypanosoma cruzi/HIV co-infection.

Two Brazilian cases of Trypanosoma cruzi/HIV co-infection have recently been treated with azole derivatives. Benznidazole, the drug generally used for the treatment of Chagas disease, was initially used in one case but discontinued because of an adverse effect (retrobulbar neuritis) and replaced by itraconazole. The other case had oesophageal candidiasis, which was treated with ketoconazole, a drug that had already been shown to be effective in the treatment of Chagas disease. Since the medications were effective in reducing the T. cruzi parasitaemia in both patients, they probably helped prevent the severe morbidity sometimes associated with Chagas disease, although the HIV infections still proved fatal in both cases.

Anxiolytic-like effect of chronic treatment with Erythrina velutina extract in the elevated plus-maze test.

INTRODUCTION: In Brazil, Erythrina velutina (Fabaceae) is widely used as a tranquilizer and/or sedative, and its extract exerts an anxiolytic-like effect profile in animal models, although these results may be caused by its sedative or amnesic effects. AIMS, MATERIALS AND METHODS: Thus, this study evaluated the effect of acute and chronic (23-26 days) administrations of the hydroalcoholic extract of the stem bark of Erythrina velutina (orally) in mice submitted to the following tests: elevated plus-maze, forced swim, spontaneous locomotor activity, and habituation to active chamber. Chlordiazepoxide and imipramine were used as standard drugs. RESULTS: In the elevated plus-maze test, chronic, but not acute, Erythrina velutina (100mg/kg) administration increased the percentage of open arm entries, an effect also seen in both acute and chronic treatments with chlordiazepoxide (7.5mg/kg). In the forced swim test, only imipramine (25mg/kg) decreased immobility time. Impairment of habituation was seen only with acute imipramine administration and with the lowest doses of Erythrina velutina extract tested in acute (10mg/kg) and chronic (50mg/kg) administrations. CONCLUSIONS: These results suggest that chronic administration of the hydroalcoholic extract of the stem bark of Erythrina velutina exerts an anxiolytic-like effect on mice, and it could serve as a new approach for the treatment anxiety, although it may have an amnesic effect at low doses.

Magnetic carriers: a promising device for targeting drugs into the human body.

Suboptimal disposition behavior of drugs requires innovative delivery approaches. Magnetic drug targeting seems to be a promising one. Magnetic particles develop magnetic polarization and magnetophoretic mobility, and because of such unique properties, these carriers may be eligible candidates for delivering drugs to specific locations within the body. Their special properties also allow other uses, such as those in magnetic separation, hyperthermia, and magnetic resonance imaging. This review focuses on a brief discussion of magnetic drug targeting, the properties and fate of magnetic carriers, the methods used to produce and characterize them, and their other uses in biotechnology.

Antiinflammatory and chronic toxicity study of the leaves of Ageratum conyzoides L. in rats.

The hydroalcoholic extract (HAE) of Ageratum conyzoides leaves was studied for its antiinflammatory effect on subacute (cotton pellet-induced granuloma) and chronic (formaldehyde-induced arthritis) models of inflammation in rats. The absence or presence of toxicity by prolonged use of HAE was also evaluated through biochemical and hematological analysis of rats blood samples using daily oral doses of 250 or 500 mg/kg body wt., during 90 days. The results showed that the group of rats treated with HAE (250 mg/kg body wt.; p.o.) had a 38.7% (p < 0.05) reduction in cotton-pellet granuloma. The development of chronically induced paw edema was also reduced significantly (p < 0.05) by the plant extract. The toxicity study did not show any treatment-related abnormalities in biochemical and hematological parameters. The biochemical analysis from blood samples drawn from group of rats treated orally with 500 mg/kg body wt. did, however, present 30.2% (p < 0.05) reduction of SGPT activity as compared to the corresponding control group. These results confirm the antiinflammatory properties of A. conyzoides, with no apparent hepatotoxicity.

Chagas' disease susceptibility/resistance: linkage disequilibrium analysis suggests epistasis between major histocompatibility complex and interleukin-10.

Association between the major histocompatibility complex (MHC) and the susceptibility/resistance to acquire Chagas' disease has been largely demonstrated. To study the role of candidate genes in this susceptibility/resistance to Chagas, we designed a population-genetic-based case-control approach (chagasic n = 104 and controls n = 60) and tested the presence of genotype and linkage disequilibrium on microsatellite loci establishing specific landmarks for the MHC, interleukin (IL)-2, IL-2Rbeta chain, IL-4, IL-10, and natural resistance-associated mactophage protein 1 (NRAMP1). After demonstrating no genetic stratification among cases and controls (F(st) were not different from 0), we found significant allelic differences among chagasic patients and controls at microsatellite locus D6S291 (MHC) and at the microsatellite pointing out the IL-10. At the MHC, we found significant differences between patients and controls in Hardy-Weinberg equilibrium-expected genotype proportions. Additionally, MHC II-locus-inferred haplotypes in chagasic patients exhibited strong significant departures from the expected proportions predicted by the second Mendelian law. The linkage disequilibrium pattern at MHC involves a region of approximately 10 cM. These results replicate previous analyses and suggest that presence of epistasis between MHC with humoral systems, such as IL-10, could be underlying the susceptibility/resistance to Chagas' disease.

Otimização da técnica para coloração de ferro (PERLS)

A coloração de Perls é de grande utilidade para identificar ferro em forma de grânulos no interior dos EritrobLastos (denominados sideroblastos), nos histiócitos e macrófagos, além do ferro intersticial. É muito útil no diagnóstico diferencial de várias patologias hematológicas. Assim, propusemos-nos a modificar algumas fases desta técnica a fim de otimizar a coloração do ferro nas células e interstício. Na primeira fase, o esfregaço sanguíneo que era fixado em vapor de formol por 30 minutos e lavado várias vezes em água, passou a ser fixado por 40 minutos sem lavagem subsequente em água, diminuindo o risco de danos (lise) ao esfregaço. A fase de coloração com ferrocianeto ácido contracoloração com Fast Red Nuclear, aumentamos a concentração...