Effects of 25-hydroxycholecalciferol on performance, gut health, and bone quality of broilers fed with reduced calcium and phosphorus diet during Eimeria challenge.

This study evaluated the effects of 25-hydroxycholecalciferol (25-OHD) on performance, gut health, and bone quality of broilers fed with reduced calcium (Ca) and phosphorus (P) diet during Eimeria spp. challenge. A total of 576 fourteen-day-old Cobb 500 male chicks were randomly distributed in a 2 × 2 × 2 factorial arrangement, with 6 replicates of 12 birds each. The main factors were 25-OHD level (0 or 3,000 IU/kg of feed), mineral level (0.84% of Ca/0.42% of P, the levels recommended for the grower phase (NOR) or 0.64% of Ca/0.22% of P (RED), and mid-high mixed Eimeria challenge or nonchallenge. 25-OHD improved phosphorus retention (P = 0.019), bone ash weight (P = 0.04), cortical bone trabecular connectivity (P = 0.043) during coccidiosis. For birds fed with reduced mineral levels, 25-OHD supplementation increased bone ash weight (P = 0.04). However, 25-OHD did not improve bone ash weight when birds were challenged and fed with reduced mineral levels. The dietary 3,000 IU of 25-OHD supplementation did not improve performance or gut morphology but support bone health during coccidiosis. Future investigations are needed for better understand 25-OHD role on bone microarchitecture and oxidative metabolism during coccidiosis.

Oocytes of The Endangered Gray Catfish (Surubim-Do-Paraiba) Submitted to Different Conservation Protocols.

BACKGROUND: The gray catfish known as Surubim-do-Paraíba (Steindachneridion parahybae), which is endemic to the Paraíba do Sul river basin, is on the red list of Brazilian fauna threatened with extinction and the cryopreservation of germ cells of this fish is needed in support of conservation. OBJECTIVE: We aimed to assess the effect of storage temperature on S. parahybae mature and immature oocytes. METHODS: Two trials were carried out. Trial I (TI.1-3) used 30 mature oocytes (diameter >1.8 mm) placed in cryoprotectant solutions and submitted to three different techniques. Trial II (TII.1-3) used 30 immature oocytes (diameter <1.6 mm) placed in cryoprotectant solutions and submitted to three storage temperatures (i.e., TII.1 at room temperature for 120 min; TII.2 in the freezer for 120 min; TII.3 in liquid nitrogen for 24 hours. RESULT: The mature oocytes were sensitive to every protocol used, including at room temperature. In contrast, the immature oocytes had increased sensitivity according to the temperature reduction to which they were submitted, with the treatment in liquid nitrogen causing greater damage. CONCLUSION: The immature stages exhibit more promising results, encouraging further studies using the combination of different CPSs, mainly penetrating ones, in oocyte cryopreservation protocols.

Misreport of energy intake assessed with food records and 24-h recalls compared with total energy expenditure estimated with DLW.

BACKGROUND/OBJECTIVES: The accuracy of dietary assessment methods has rarely been validated using precise techniques. The objective of this work was to evaluate the validity of energy intake (EI) estimated with food records (FRs) and 24-h recalls (24hRs) against total energy expenditure (EE) estimated by the doubly labeled water (DLW) method. In addition, the magnitude of EI under-reporting was assessed along with its associated characteristics. SUBJECTS/METHODS: The studied group included 83 adults between 20 and 60 years of age who were recruited from a population-based sample. Within-person variation-adjusted means of EI estimated from two FRs and three 24hRs were compared with EE estimated using the DLW method multiple-point protocol. The Wilcoxon signed-rank test was used to assess the differences between EI and EE, whereas Bland-Altman and survival-agreement plots assessed the agreement between the estimates. RESULTS: The mean EE (2540 kcal) was greater than the mean reported EI for both dietary assessment methods (FR: 1774 kcal; 24hR: 1658 kcal, P<0.01). The frequency of under-reporting was lower (20%) for EI estimated with the 24hR than that estimated with the FR (32%). Men presented lower magnitude of under-reported EI than women did. For women, differences between EI and EE were lower with FR than with 24hR. Overall, FR and 24hR showed similar performance. The mean under-reported EI was ~30% for both methods. CONCLUSIONS: Irregular meal habits, smoking and low education were associated with the under-report of EI. Both FR and 24hR are subjected to bias suggesting the need of refining the procedures applied in dietary assessment methods.

Transcranial direct current stimulation and exercises for treatment of chronic temporomandibular disorders: a blind randomised-controlled trial.

To evaluate the effect of adding transcranial direct current stimulation (tDCS) to exercises for chronic pain, dysfunction and quality of life in subjects with temporomandibular disorders (TMD). Participants were selected based on the RDC/TMD criteria and assessed for pain intensity, pressure pain threshold over temporomandibular joint and cervical muscles and quality of life. After initial assessment, all individuals underwent a 4-week protocol of exercises and manual therapy, together with active or sham primary motor cortex tDCS. Stimulation was delivered through sponge electrodes, with 2 mA amplitude, for 20 min daily, over the first 5 days of the trial. A total of 32 subjects (mean age 24.7 ± 6.8 years) participated in the evaluations and treatment protocol. Mean pain intensity pre-treatment was 5.5 ± 1.4 for active tDCS group, and 6.3 ± 1.2 for sham tDCS. Both groups showed a decrease in pain intensity scores during the trial period (time factor--F(4.5,137.5) = 28.7, P < 0.001; group factor--F(1.0,30.0 = 7.7), P < 0.05). However, there were no differences between the groups regarding change in pain intensity (time*group interaction--F(4.5,137.5) = 1.5, P = 0.137). This result remained the same after 5 months (t-test t = 0.29, P > 0.05). Pressure pain thresholds decrease and improvement in quality of life were also noticeable in both groups, but again without significant differences between them. Absolute benefit increase was 37.5% (CI 95%: -15.9% to 90.9%), and number needed to treat was 2.66. This study suggests that there is no additional benefit in adding tDCS to exercises for the treatment of chronic TMD in young adults.

Monitoramento da variabilidade genética de pacu, Piaractus mesopotamicus, do programa de aumento de estoque do rio Paranapanema / Monitoring of the genetic variability of pacu, Piaractus mesopotamicus, of the stock enhancement program of the Paranapanema River

Avaliou-se a variabilidade genética dos estoques de reprodutores e dos peixes jovens de Piaractus mesopotamicus de três pisciculturas do estado do Paraná, utilizadas no programa de aumento de estoque de peixes no rio Paranapanema. Foi utilizado o marcador RAPD para avaliar as amostras do estoque de reprodutores e dos peixes jovens das pisciculturas de Palotina, Cambará e Andirá. A porcentagem de fragmentos polimórficos e o índice de diversidade genética de Shannon dos estoques de reprodutores variaram de 75,0 por cento a 71,4 por cento e de 0,434 a 0,376, respectivamente. Os peixes jovens das pisciculturas apresentaram valores mais elevados para ambos os parâmetros, com exceção da piscicultura de Palotina, na qual o índice de diversidade genética de Shannon foi semelhante. Os estoques de reprodutores apresentaram alta variabilidade genética, e esta foi mantida nos peixes jovens.

The genetic variability of broodstocks and juveniles of Piaractus mesopotamicus raised in three hatchery stations in Parana state, that were used in the fish stock enhancement program of the Paranapanema River, was estimated. The RAPD marker was used to evaluate samples taken from broodstocks and juveniles in the hatchery stations of Palotina, Cambará, and Andirá cities. The percentage of polymorphic fragments and the Shannon genetic diversity index of broodstocks ranged from 75.0 percent to 71.4 percent and from 0.434 to 0.376, respectively. The juveniles of the hatchery stations presented higher values for both parameters, except in the hatchery station of Palotina in which the Shannon genetic diversity index was similar. The broodstocks presented high genetic variability, and this was maintained in the juveniles.

Diversidade genética de estoques de reprodutores de Colossoma macropomum / Genetic diversity of Colossoma macropomum broodstocks

Analisou-se a diversidade genética de estoques de reprodutores de Tambaqui (Colossoma macropomum), mediante o uso de marcador RAPD, utilizando-se 10 primers para analisar 30 amostras do estoques de reprodutores das pisciculturas de Boa Esperança e Vale Verde, localizadas no Estado de Rondônia. A porcentagem de fragmentos polimórficos e o índice de diversidade genética de Shannon foram altos nos dois estoques de reprodutores. O estoque de reprodutores de Boa Esperança apresentou um fragmento exclusivo. A diferenciação genética foi baixa e o número de migrantes por geração foi alto entre os estoques de reprodutores. O dendrograma não separou os indivíduos dos estoques de reprodutores em grupos distintos. Há alta variabilidade genética nos estoques de reprodutores, um pouco inferior no estoque de Vale Verde, e há grande proximidade genética entre os indivíduos dos estoques de reprodutores.

The genetic diversity of tambaqui (Colossoma macropomum) broodstocks from two hatchery station in Rondônia State was studied by the RAPD marker. Ten primers were used to analyze 30 broodstocks samples from the hatchery stations of Boa Esperança and Vale Verde. The polymorphic fragments percentage and Shannon genetic diversity index were high in the two broodstocks. The Boa Esperança broodstock presented an exclusive fragment. The genetic differentiation was low and the number of migrants per generation was high among the broodstocks. The dendrogram did not separate the broodstocks individuals in different groups. The results indicate a high genetic variability in the broodstocks, being a little bit lower in the Vale Verde broodstock. Besides, there is a genetic proximity among the broodstocks.

Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae.

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with high mitotic stability. The pMIRY integrative vector system, based upon targeted integration into the yeast rDNA locus, developed in our laboratory satisfies these criteria. However, insertion of a (foreign) gene drastically reduced its mitotic stability of the resulting vector in comparison to its parent. In this paper we have investigated a number of possible reasons for this reduction in stability. The results demonstrate that plasmid size is an important, but not the only, determinant of mitotic stability. Stable maintenance is only observed when the complete plasmid has a size no larger than that of the rDNA unit (9.1 kb). In addition stability depends upon the nature of the rDNA fragment present in the plasmid, required for targeting its integration. On the other hand, it turned out to be irrelevant for mitotic stability whether the heterologous gene was expressed or not. These findings will be important in the design of a pMIRY vector suitable for industrial production of heterologous proteins.

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae.

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression.

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.

[Prevalence of caries in first permanent molars in students living in regions with and without fluoridated drinking water (comparative study based on clinical and radiographic examinations)]. / Prevalência de lesão cariosa em primeiros molares permanentes de escolares residentes em regiões com flúor e sem flúor na água de abastecimento. (Estudo comparativo baseado em exames clínico e radiográfico).

To analyze the prevalence of dental caries by means of the DMF-S index and verify their distribution on the occlusal, buccal, lingual, mesial and distal surfaces using the intrabuccal interproximal technique, this comparative study included a sample of 360 seven to twelve year old boys and girls, without distinction of race, from the low social-economic class who, since birth, have lived in the cities of Teresina and Barras located in the state of Piauí in Brazil. The average level of fluor in first; city's public water supply is 0.68 ppm, while the second city lacks this halogen. A statistical analysis of the results verified that children in Teresina presented a DMF-S index 26.10% less than the children in Barras. The "T" Test of Student was significant for the seven, eight, nine, 10, and 12 years olds and for the sample as a whole in Teresina as well as for the types of surfaces regardless of age and sex. Children in the same community showed no significant difference when analyzed according to sex. In the two communities, the annual increase of the DMF-S index for all ages shows the cumulative and progressive aspect of dental caries; however, the increase was less in the city with fluoridated water.