Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

Effect of dietary phytase suplementation on the performance, bone densitometry, and phosphorus and nitrogen excretion of broilers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reforma a vapor catalítica do metano: Otimização da produção e seletividade em hidrogênio por absorção in situ do CO2 produzido

Topics of research related to energy and environment have significantly grown in recent years, with the need of its own energy as hydrogen. More particularly, numerous researches have been focused on hydrogen as energy vector. The main portion of hydrogen is presently obtained by reforming of methane or light hydrocarbons (steam, oxy, dry or auto reforming). During the methane steam reforming process the formation of CO2 undesirable (the main contributor to the greenhouse effect) is observed. Thus, an oxide material (sorbent) can be used to capture the CO2 generated during the process and simultaneously shifting the equilibrium of water gas shift towards thermodynamically more favorable production of pure hydrogen. The aim of this study is to develop a material with dual function (catalyst/sorbent) in the reaction of steam reforming of methane. CaO is well known as CO2 sorbent due to its high efficiency in reactions of carbonation and easy regeneration through calcination. However the kinetic of carbonation decreases quickly with time and carbonation/calcination cycles. A calcium aluminate (Ca12Al14O33) should be used to avoid sintering and increase the stability of CaO sorbents for several cycles. Nickel, the industrial catalyst choice for steam reforming has been added to the support from different manners. These bi-functional materials (sorbent/catalyst) in different molar ratios CaO.Ca12Al14O33 (48:52, 65:35, 75:25, 90:10) were prepared by different synthesis methodologies, among them, especially the method of microwave assisted self-combustion. Synthesis, structure and catalytic performances of Ni- CaO.Ca12Al14O33 synthesized by the novel method (microwave assisted selfcombustion) proposed in this work has not being reported yet in literature. The results indicate that CO2 capture time depends both on the CaO excess and on operating conditions (eg., temperature and H2O/CH4 ratio). To be efficient for CO2 sorption, temperature of steam reforming needs to be lower than 700 °C. An optimized percentage corresponding to 75% of CaO and a ratio H2O/CH4 = 1 provides the most promising results since a smaller amount of water avoids competition between water and CO2 to form carbonate and hydroxide. If this competition is most effective (H2O/CH4 = 3) and would have a smaller amount of CaO available for absorption possibly due to the formation of Ca(OH)2. Therefore, the capture time was higher (16h) for the ratio H2O/CH4 = 1 than H2O/CH4 = 3 (7h) using as catalyst one prepared by impregnating the support obtained by microwave assisted self-combustion. Therefore, it was demonstrated that, with these catalysts, the CO2 sorption on CaO modifies the balance of the water gas-shift reaction. Consequently, steam reforming of CH4 is optimized, producing pure H2, complete conversion of methane and negligible concentration of CO2 and CO during the time of capture even at low temperature (650 °C). This validates the concept of the sorption of CO2 together with methane steam reforming

Involvement of L-Type Calcium Channel and SERCA2a in Myocardial Dysfunction Induced by Obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca2+) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca2+ channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca2+ channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca2+ was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca2+ channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca2+ channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca2+ protein levels. J. Cell. Physiol. 226: 2934-2942, 2011. (C) 2011 Wiley-Liss, Inc.

Valores séricos de cálcio, fósforo, sódio, potássio e proteínas totais em caprinos fêmeas da raça Parda Alpina

Por ser de grande importância o conhecimento de valores séricos referenciais do mineralograma e do proteinograma, a sua utilização na medicina veterinária, e a escassez de trabalhos brasileiros relacionados à espécie caprina, este estudo objetivou determinar e comparar os níveis séricos de cálcio, fósforo, sódio, potássio e proteínas (proteína total, albumina, globulina e a relação albumina/globulina) em caprinos fêmeas da raça Parda Alpina, em três idades diferentes. Foram utilizadas 30 fêmeas caprinas da raça Parda Alpina criadas em regime intensivo, sendo separadas em três grupos: Grupo 1 - 10 fêmeas jovens com idade de quatro meses; Grupo 2 - 10 fêmeas adultas jovens com idade de 18 meses; Grupo 3 - 10 fêmeas adultas lactantes com idade de 24 a 60 meses. Os animais foram avaliados por meio de exames clínico e coproparasitológico. Foram encontrados os seguintes valores médios de cálcio, fósforo, sódio e potássio no grupo G1: 10,10± 1,70, 10,13± 2,04, 151± 3,30 e 5,16± 0,41; no grupo G2: 10,36± 0,94, 7,49± 1,34, 145,9± 3,35 e 4,56± 0,48; e para o grupo G3: 9,47± 1,45, 5,90± 1,21, 153,3± 5,17 e 4,20± 0,55. Para as variáveis proteína total sérica, albumina, globulina e relação albumina/globulina foram encontrados os seguintes valores médios, para o grupo G1: 6,29± 0,94, 3,5± 10,41, 2,78± 0,95 e 1,40± 0,48; para o grupo G2: 7,27± 0,73, 4,92± 1,06, 3,06± 0,56, 1,43± 0,44; e para o grupo G3: 7,14± 0,84, 3,70± 0,20, 3,44± 0,79 e 1,14± 0,30. A análise estatística dos dados demonstra que existe influência da faixa etária nas variáveis fósforo, sódio, potássio, proteína total e albumina.

Effect of the calcium level and limestone particle size on the performance of semi-heavy layers in the second cycle of egg production

An experiment was carried out at the Research and Development Unit of Brotas aiming at evaluating dietary calcium level and limestone particle size on the production performance of commercial (Hy-Line Brown) layers in the second lay cycle. Experiment duration was 112 days. A total number of 288 hens, with 83 weeks of age in the beginning of the experiment, were used in a completely randomized experimental design in a factorial arrangement of 2x3, with two calcium levels (3.5 and 4.0%) and three limestone particle size compositions: 100% fine limestone (FL), 30% coarse limestone (CL) + 70% fine limestone (FL), and 50% (CL) + 50% (FL), with six replicates of eight birds each. Egg weight (g), egg production (%), egg mass (%), feed intake (g), feed conversion ratio (kg/dz and kg/kg), mortality (%), and egg loss (%) were evaluated. The analysis of variance did not detect significant differences (p>0.05) among treatments on any of the evaluated performance parameters. It was concluded that the tested calcium levels and limestone particle composition did not influence the performance of semi-heavy layers in second production cycle.

Desempenho ponderal de bovinos Nelore suplementados com fontes alternativas de fósforo

O desempenho produtivo e a possível interferência do flúor sobre a saúde dos animais foram investigados em bovinos Nelore suplementados, por 866 dias, com distintas fontes alternativas de fósforo com diferentes relações fósforo:fluor (P:F). Os tratamentos experimentais foram: Controle negativo (CONTNEG, sem qualquer suplementação com P), fosfato bicálcico (FB 120:1, FB 30:1 e FB 10:1), fosfato monobicálcico (FMBC 60:1), superfosfato triplo (SFT 30:1) e fosfato de rocha de Cajati (FR 10:1). Foram utilizados 49 novilhos, desmamados aos oito meses de idade, castrados e com 230 kg de peso médio, distribuídos em sete piquetes com água e mistura mineral formulada sem P. A dieta padrão foi feita com bagaço de cana (0,03% de P) como volumoso e um concentrado contendo 0,239 % de P oferecido na base de 1% do peso dos animais para permitir um ganho de peso aproximado de 0,50 kg/dia. Até o dia 134, não houve diferença estatística entre os diversos lotes, inclusive para o tratamento CONTNEG, que não recebeu fósforo suplementar na dieta e ganhou 71,6 kg de peso ou 0,633 kg/dia. Após 866 dias de confinamento (2,37 anos), os animais suplementados com o fosfato bicálcico padrão (120:1) ganharam menos peso que os suplementados com as fontes FMCB 60:1, FB 30:1 e SFT 30:1. Até um ano de suplementação fosfórica com fosfato bicálcico padrão (120:1) artificialmente fluoretado com NaF ou com o fosfato de rocha não se detectou danos à saúde ou ao ganho de peso dos animais. As análises de fósforo nos ossos mostraram diferença estatística apenas entre o tratamento CONTNEG e os que tinham fosfato bicálcico. As concentrações de flúor nos ossos se mostraram intimamente associadas à quantidade de flúor disponível nas fontes utilizadas. Conforme a proporção P:F na dieta foi diminuindo, características relacionadas à fluorose dentária ficaram mais evidentes, sendo que os animais que receberam fontes com relação 10:1, apresentaram, ao final do experimento, dentes incisivos permanentes mal formados, quebradiços e com manchas esbranquiçadas.

Osteoconductive Properties of beta-Tricalcium Phosphate Matrix, Polylactic and Polyglycolic Acid Gel, and Calcium Phosphate Cement in Bone Defects

Extensive bone defects in maxillofacial region can be corrected with autogenous grafts; otherwise, the disadvantages of the therapeutics modality take the research for new bone substitutes. The aim of the study was to evaluate and compare the osteoconductive properties of 3 commercial available biomaterials. A total of 30 calvarial defects (5-mm diameter) were randomly divided into 5 treatment groups, with a total of 6 defects per treatment group (n = 6). The treatment groups were as follows: 500 to 1000 Km beta-tricalcium phosphate (beta-TCP), polylactic and polyglycolic acid (PL/PG) gel, calcium phosphate cement, untreated control, and autograft control. The evaluations were based on histomorphometric analysis at 60 postoperative days. The results have shown that beta-TCP and autograft control supported bone formation at 60 postoperative days. beta-Tricalcium phosphate showed the highest amount of mineralized area per total area and statistically significant compared with PL/PG, calcium phosphate cement, and untreated control groups. The PL/PG gel does not have osteoconductive properties and performed similar to empty control. Calcium phosphate cement showed higher number of multinucleated giant cells around the sites of the biomaterial and showed newly formed bone only at the edges of the biomaterial, without bone formation within the biomaterial. The findings presented herein indicate that bone formation reached a maximum level when rat calvarial defects were filled with beta-TCP at 60 postoperative days. Further studies should be conducted with beta-TCP to understand the potential of this biomaterial in bone regeneration.

Phosphorus magnetic resonance spectroscopy in malformations of cortical development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desempenho de bezerros da raça Nelore e cruzados desmamados recebendo concentrado em pastagem adubada de Cynodon dactylon cv. Coastcross

Em três anos de pesquisa, avaliou-se o desempenho de bezerros desmamados de quatro grupos genéticos (Nelore; Canchim × Nelore; Angus × Nelore; e Simental × Nelore) em pastagem de Cynodon dactylon, cv. Coastcross, adubada, sob manejo rotacionado, recebendo mistura mineral ou concentrado. O concentrado foi fornecido na quantidade de 3 kg/animal/dia e continha 18,8% de proteína bruta e 81,5% de nutrientes digestíveis totais na matéria seca. A quantidade de forragem disponível diferiu com a utilização de mistura mineral e concentrado (2.961 kg e 3.383 kg de matéria seca (MS) por hectare, respectivamente). A oferta de MS/animal/dia foi 9,9 kg/dia (3,9% PV) para mistura mineral e 9,0 kg/dia (3,3% PV) para concentrado. A forragem disponível possuía, na matéria seca, 13,6% de proteína bruta, 79,8% de fibra em detergente neutro; 62,3% de digestibilidade in vitro da matéria seca, 3,9 g/kg de cálcio, 2,0 g/kg magnésio, 2,5 g/kg de fósforo, 22,7 g/kg de potássio, 2,8 g/kg de enxofre, 9,9 mg/kg de cobre, 22,5 mg/kg de zinco, 98 mg/kg de manganês e 188 mg/kg de ferro. Foram observadas interações grupo genético × suplementação e ano × suplementação para ganho diário de peso e taxa de lotação das pastagens e ciclo de pastejo × suplementação para a taxa de lotação. O ganho diário de peso nos animais Nelore, Canchim × Nelore, Angus × Nelore e Simental × Nelore criados com suplementação do pasto com mistura mineral foi de 0,48; 0,63; 0,68 e 0,50 kg, enquanto naqueles que receberam concentrado foi de 0,87; 0,95; 0,99 e 0,95 kg, respectivamente. As médias estimadas das taxas de lotação das pastagens foram 6,1 para todos os grupos genéticos que receberam mistura mineral e 7,5; 7,6; 8,8 e 9,0 unidades animais/ha para os grupos genéticos Nelore, Canchim × Nelore, Angus × Nelore e Simental × Nelore que receberam concentrado, respectivamente. O grupo genético influencia o desempenho de bovinos em pastagem de capim-coastcross e interage com a suplementação com concentrado.

CONCERTED ACTION OF THE HIGH-AFFINITY CALCIUM-BINDING SITES IN SKELETAL-MUSCLE TROPONIN-C

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC complex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

Establishing Standards for the Integrated Recommendation and Diagnosis System (DRIS) for Irrigated Bean Crops

This article addresses the establishment of integrated diagnostics and recommendation system (DRIS) standards for irrigated bean crops (Phaseolus vulgaris) and compares leaf concentrations and productivity in low- and high-productivity populations. The work was carried out in Santa Fe de Goias, Goias State, Brazil, in the agricultural years 1999/2000 and 2000/2001. For the nutritional diagnosis, leaf samples were collected, and leaf concentrations of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) were established in 100 commercial bean crops. A database was set up listing the leaf nutrient content and the respective productivities, subdivided into two subpopulations, high and low productivity, using a bean yield value of 3000 kg ha-1 to separate these subpopulations. Sufficiency values found in the high-productivity population matched only for the micronutrients B and Zn. The nutritional balance among the populations studied was coherent and was lower in the high-productivity population. The DRIS standards proposed for irrigated bean farming were efficient in evaluating the nutritional status of the crop areas studied. Calcium, Cu, and S were found to be the least available nutrients, indicating high response potential for the fertilizing using these nutrients.

Application of impedance spectroscopy to evaluate the effect of different setting accelerators on the developed microstructures of calcium phosphate cements

The main goal of the present study was to evaluate the effect of different setting accelerator agents on the developed microstructures of calcium phosphate cements (CPCs) by employing the impedance spectroscopy (IS) technique. Six compositions of CPCs were prepared from mixtures of commercial dicalcium phosphate anhydrous (DCPA) and synthesized tetracalcium phosphate (TTCP) as the solid phases. Two TTCP/DCPA molar ratios (1/1 and 1/2) and three liquid phases (aqueous solutions of Na(2)HPO(4), tartaric acid (TA) and oxalic acid (OA), 5% volume fraction) were employed. Initial (I) and final (F) setting times of the cement pastes were determined with Gillmore needles (ASTM standard C266-99). The hardened samples were characterized by X-ray powder diffraction (XRD), Fourier transformed infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and apparent density measurements. The IS technique was employed as a non-destructive tool to obtain information related to porosity, tortuosity and homogeneity of the cement microstructures. The formulation prepared from a TTCP/DCPA equimolar mixture and OA as the liquid phase presented the shortest I and F (12 and 20 min, respectively) in comparison to the other studied systems. XRD analyses revealed the formation of low-crystallinity hydroxyapatite (HA) (as the main phase) as well as the presence of little amounts of unreacted DCPA and TTCP after 24 h hardening in 100% relative humidity. This was related to the proposed mechanisms of dissolution of the reactants. The bands observed by FTIR allowed identifying the presence of calcium tartrate and calcium oxalate in the samples prepared from TA and OA, in addition to the characteristic bands of HA. High degree of entanglement of the formed crystals was observed by SEM in samples containing OA. SEM images were also correlated to the apparent densities of the hardened cements. Changes in porosity, tortuosity and microstructural homogeneity were determined in all samples, from IS results, when the TTCP/DCPA ratio was changed from 1/1 to 1/2. The cement formulated from an equimolar mixture of TTCP/DCPA and OA as the liquid phase presented setting times, degree of conversion to low-crystallinity HA and microstructural features suitable to be used as potential bone cement in clinical applications. The IS technique was shown to be a very sensitive and non-destructive tool to relate the paste composition to the developed microstructures. This approach could be very useful to develop calcium phosphate bone cements for specific clinical demands.

Determination of calcium by atomic absorption spectrophotometry applied to the preparation of alginate:chitosan complexes

Polysaccharicles, as alginate and chitosan, have been used to obtain modified release dosage forms. Alginate, due to its property of building gels during the complex formation with calcium ions, allows the building of capsules containing a core constituted by calcium alginate. This work had for objective to determine the appropriate calcium concentration for the preparation of alginate-chitosan capsules, by means of calcium quantification using atomic absorption spectrophotometry. The methodology of calcium quantification was validated through analysis of the limit of detection, precision, accuracy and recovery of the method. The capsules, containing or not the drug, were prepared by the complex coacervation/ionotropic gelification method. Calcium was quantified after samples mineralization and dilution in lantanium solution. The results showed that the amount of calcium incorporated into the capsules depends on the amount of calcium added to the medium, and this ratio increases until the concentration of 1.5% of initial calcium chloride and above this concentration there is a decrease in the proportion of calcium bonded. It was observed that the proportion of calcium that links to the polymer is inversely proportional to the amount of calcium added. The calcium amount incorporated depends on the concentration of the polymeric dispersions used as well as on the ratio between the two polymers.

Sealing ability of retrograde obturation materials containing calcium hydroxide or MTA

Newly available materials for retrograde obturation should have their sealing properties evaluated. The goal of this study was to evaluate the sealing ability of Endo CPM sealer, an MTA-based endodontic cement. Single-rooted extracted human teeth were endodontically treated. After apical sectioning, retrograde cavities were prepared. Teeth were divided into five experimental groups (n=12), in which the following materials were used: Sealer 26 (S26), white Mineral Trioxide Aggregate (MTA), Endo CPM Sealer (CPM1), Endo CPM Sealer in thicker consistency (CPM 2), and zinc oxide and eugenol cement (ZOE), and two control groups (n=3). After retrograde obturation, the teeth were immersed in 0.2% rhodamine B dye for 48 hours in a vacuum chamber Marginal leakage data were subjected to ANOVA and Tukey tests at 5% significance level. S26 presented greater sealing ability (p<0.05) than ZOE, MTA, CPM1, and CPM2, all of which had similar results (p>0.05). We concluded that Sealer 26 has the greatest sealing ability. Endo CPM Sealer, with sealing ability similar to MTA, could be used as a retrograde obturation material.