Belo Horizonte, Brazil
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de Arruda M.M.,University of Brasilia | Figueiredo F.B.,Institute Pesquisa Clinica Evandro Chagas | Cardoso F.A.,Fundacao Ezequiel Dias FUNED | Hiamamoto R.M.,University of Sao Paulo | And 5 more authors.
PLoS ONE | Year: 2013

Background: American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. Methods: A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. Results: The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. Interpretation: ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil. © 2013 de Arruda et al.


Salceda E.,Autonomous University of Puebla | Zaharenko A.J.,Butantan Institute | Peigneur S.,Catholic University of Leuven | Lopez O.,Autonomous University of Puebla | And 4 more authors.
Peptides | Year: 2014

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50 ∼ 100 nM), and also voltage-gated K+ currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na+ voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected. © 2013 Elsevier Inc.


PubMed | Fundacao Ezequiel Dias FUNED, Butantan Institute, Autonomous University of Puebla, Catholic University of Leuven and University of Ulm
Type: | Journal: Peptides | Year: 2014

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.


Gadelha I.C.N.,Rural University | Fonseca N.B.S.,Rural University | Oloris S.C.S.,Fundacao Ezequiel Dias FUNED | Melo M.M.,Federal University of Minas Gerais | Soto-Blanco B.,Federal University of Minas Gerais
Scientific World Journal | Year: 2014

Gossypol is a phenolic compound produced by pigment glands in cotton stems, leaves, seeds, and flower buds (Gossypium spp.). Cottonseed meal is a by-product of cotton that is used for animal feeding because it is rich in oil and proteins. However, gossypol toxicity limits cottonseed use in animal feed. High concentrations of free gossypol may be responsible for acute clinical signs of gossypol poisoning which include respiratory distress, impaired body weight gain, anorexia, weakness, apathy, and death after several days. However, the most common toxic effects is the impairment of male and female reproduction. Another important toxic effect of gossypol is its interference with immune function, reducing an animal's resistance to infections and impairing the efficiency of vaccines. Preventive procedures to limit gossypol toxicity involve treatment of the cottonseed product to reduce the concentration of free gossypol with the most common treatment being exposure to heat. However, free gossypol can be released from the bound form during digestion. Agronomic selection has produced cotton varieties devoid of glands producing gossypol, but these varieties are not normally grown because they are less productive and are more vulnerable to attacks by insects. © 2014 Ivana Cristina N. Gadelha et al.


PubMed | Lutheran University of Brazil, Fundacao Ezequiel Dias FUNED, Federal University of Santa Maria, Federal University of Minas Gerais and Institute Ensino e Pesquisa da Santa Casa
Type: | Journal: Pharmacology, biochemistry, and behavior | Year: 2014

The effects of intrathecal administration of the toxins Ph1 and -conotoxin MVIIA were investigated in visceral nociception induced by an intraperitoneal injection of acetic acid and an intracolonic application of capsaicin. The pretreatments for 2h with the toxins reduced the number of writhes or nociceptive behaviors compared with the control mice. Ph1 administration resulted in an Imax of 846 and an ID50 of 12 (5-27), and -conotoxin MVIIA resulted in an Imax of 829 and an ID50 of 11 (4-35) in the contortions induced by the intraperitoneal injection of acetic acid. The administration of Ph1 resulted in an Imax of 644 and an ID50 of 18 (9-38), and -conotoxin MVIIA resulted in an Imax of 719 and an ID50 of 9 (1-83) in the contortions induced by intracolonic capsaicin administration. Ph1 (100/site) or -conotoxin MVIIA (30pmol/site) pretreatments caused a reduction in CSF glutamate release in mice intraperitoneally injected with acetic acid or treated with intracolonic capsaicin. The toxin pretreatments reduced the ROS levels induced by intraperitoneal acetic acid injection. Ph1, but not -conotoxin MVIIA, reduced significantly the ROS levels induced by intracolonic capsaicin administration.Ph1 is a -toxin with high therapeutic index and a broader action on calcium channels. It shows analgesic effect in several rodents models of pain, including visceral pain, suggesting that this toxin has the potential to be used in clinical setting as a drug in the control of persistent pathological pain.


Pereira L.S.A.,Federal University of Minas Gerais | Carneiro M.F.,Fundacao Ezequiel Dias FUNED | Botelho B.G.,Federal University of Minas Gerais | Sena M.M.,Federal University of Minas Gerais | Sena M.M.,Instituto Nacional Of Ciencia E Tecnologia Em Bioanalitica
Talanta | Year: 2016

Calibration transfer is commonly used for spectra obtained in different spectrometers or other conditions. This paper proposed the use of calibration transfer between spectra recorded for the same samples in different physical forms. A new method was developed for the direct determination of nevirapine in solid pharmaceutical formulations based on diffuse reflectance near infrared spectroscopy (NIRS) and partial least squares (PLS). This method was developed with 50 powder mixtures and then, successfully extended to the quantification in intact tablets by using calibration transfer with double window piecewise direct standardization (DWPDS). This chemometric strategy provided good results with a small number of tablet transfer samples, only seven, prepared out of the narrow range of active principle ingredients (API) content around the nominal value of the formulation (100%). The method was fully validated in the working range of 83.0-113.9% of nevirapine and the use of DWPDS allowed to significantly decreasing the root mean square error of prediction (RMSEP) from 4.8% (tablets predicted by a model built with only powder samples) to 2.6%. The range of relative errors decreased from -5.1/8.7% to -4.6/3.3%. Considering that the amount of raw materials demanded for preparing tablets is up to ten times higher than for powder mixtures, this type of application is of particular interest in pharmaceutical analysis. In the context of process analytical technology (PAT), the use of the same multivariate model in different steps of the production is very advantageous, saving time and labor. © 2015 Elsevier B.V. All rights reserved.


Bastos E.M.A.F.,Fundacao Ezequiel Dias FUNED | Santana R.A.,Fundacao Ezequiel Dias FUNED | Calaca-Costa A.G.F.,Federal University of Minas Gerais | Thiago P.S.,Fundacao Ezequiel Dias FUNED
Brazilian Journal of Biology | Year: 2011

In Minas Gerais, green propolis is produced from the collection of resinous substance found in shoot apices of Baccharis dracunculifolia. The aim of this study was to evaluate the biological parameters associated with the interaction Apis mellifera x Baccharis dracunculifolia, to elucidate the supply of resin for green propolis production in Minas Gerais. We selected male and female individuals of two populations of Baccharis dracunculifolia located on São Judas Tadeu Farm - FSJT, in the municipality of Betim, MG and the Experimental Garden of the Ezequiel Dias Foundation - HORTO, located in an urban area in Belo Horizonte, MG. We made weekly observations, from June 2007 to June 2008, and evaluated in both populations: richness and abundance of insect visitors; resin collecting visits of Apis mellifera; presence of Baccharopelma dracunculifoliae galls; growth of individuals and phenological phases. Statistical analyses were made using R software. The rainy season showed the highest number of visitors. A. mellifera collected resin in shoot apices of Baccharis dracunculifolia from August to April, only in the FSJT population, where galls of B. dracunculifoliae were also present. Ovoposition of gall inductor on host plants occurs during the rainy season, when there is a peak of visitants and resin collecting visits of honeybees. This fact stimulates plant defense strategies against parasitoids and predators, which includes the production of several secondary metabolites, and ultimately reduces competition for food by inhibiting the attack of other phytophagous insects, not adapted to the chemical environment of plant tissues. Green propolis production in Minas Gerais is related to the abundant supply of resin by Baccharis dracunculifolia, when they are parasitised by B. dracunculifoliae galls. They induce plant production of defense exudates, which attract Apis mellifera bees to collect resin and consequently favour the production of green propolis.


PubMed | Fundacao Ezequiel Dias FUNED, São Paulo State University and Federal University of Minas Gerais
Type: | Journal: Toxicon : official journal of the International Society on Toxinology | Year: 2016

Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named , and . In the present study, we identified, characterized and performed structural and evolutionary analyses of sbPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources.


PubMed | Fundacao Ezequiel Dias FUNED and Federal University of Minas Gerais
Type: | Journal: Talanta | Year: 2015

Calibration transfer is commonly used for spectra obtained in different spectrometers or other conditions. This paper proposed the use of calibration transfer between spectra recorded for the same samples in different physical forms. A new method was developed for the direct determination of nevirapine in solid pharmaceutical formulations based on diffuse reflectance near infrared spectroscopy (NIRS) and partial least squares (PLS). This method was developed with 50 powder mixtures and then, successfully extended to the quantification in intact tablets by using calibration transfer with double window piecewise direct standardization (DWPDS). This chemometric strategy provided good results with a small number of tablet transfer samples, only seven, prepared out of the narrow range of active principle ingredients (API) content around the nominal value of the formulation (100%). The method was fully validated in the working range of 83.0-113.9% of nevirapine and the use of DWPDS allowed to significantly decreasing the root mean square error of prediction (RMSEP) from 4.8% (tablets predicted by a model built with only powder samples) to 2.6%. The range of relative errors decreased from -5.1/8.7% to -4.6/3.3%. Considering that the amount of raw materials demanded for preparing tablets is up to ten times higher than for powder mixtures, this type of application is of particular interest in pharmaceutical analysis. In the context of process analytical technology (PAT), the use of the same multivariate model in different steps of the production is very advantageous, saving time and labor.


Rabelo L.S.,Federal University of Uberlandia | Vilhena A.M.G.F.,Federal University of Uberlandia | Bastos E.M.A.F.,Fundacao Ezequiel Dias FUNED | Augusto S.C.,Federal University of Uberlandia
Journal of Natural History | Year: 2012

Pollen analysis of larval food allows identification of plants used as nest provision sources. Considering the importance of Centris analis in pollination, we aimed to identify its plant sources, food niche width in Cerrado, and analyze the use of sources between cool/dry and warm/wet seasons. We sampled 22 nests collected in two areas from February 2008 to October 2009. Heteropterys spp. (Malpighiaceae) were the main pollen sources, but Byrsomia spp. (Malpighiaceae), Banisteriopsis malifolia (Malpighiaceae), Solanum lycocarpum (Solanaceae) and Machaerium spp. (Leguminosae) were also important food sources. There was no difference in the use of the sources between the seasons (F 1,100 = 0.001; p = 0.979). Despite the high number of pollen types (n = 14) the food niche widths were low, H = 0.777 and H = 0.949, which can be explained by the dominance of some food items. Hence, C. analis use, profusely, a few plants as food sources that could be considered conservation targets. © 2012 Taylor and Francis Group, LLC.

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