Malnutrition and nutritional deficiencies in children with cerebral palsy: a systematic review and meta-analysis.

OBJECTIVES: This systematic review study and meta-analysis sought to estimate the prevalence of malnutrition and nutritional deficiencies in children with cerebral palsy (CP). STUDY DESIGN: This is a systematic review and meta-analysis. METHODS: The systematic review was conducted according to the PRISMA guidelines. The articles were chosen using the PubMed, Embase, Scopus, Web of Science, Cochrane Library, SciELO, and Lilacs databases and the bibliographical reference lists of the articles. No limitations were placed on the time of publication, but the articles had to include children from 0 to 18 years old with CP who presented the prevalence of malnutrition and nutritional deficiencies. The methodological quality of the articles was assessed using the verification list for analytical cross-sectional studies, the Newcastle-Ottawa scale, and the Cochrane Collaboration tool for randomized studies. The meta-analysis of proportions was conducted based on the prevalence data for malnutrition or nutritional deficiencies. The study is registered in PROSPERO under CRD number 42020175068. RESULTS: Sixty-seven articles (N = 453,804) published between 1986 and 2019 were included. Most of the articles presented a low risk of bias and no publication was excluded for quality reasons. The most widely used anthropometric index for diagnosing nutritional status was weight-to-age and the estimated prevalence of malnutrition was 40% (95% CI = 28.0-53.0). Nutrient deficiency was investigated by nine publications, with hypocalcemia and reduced serum concentrations of zinc, copper, and vitamin D being reported the most. CONCLUSIONS: We found a high rate of malnutrition in the population in this review, moreover, we suggest that some nutritional deficiencies are associated with food deficit and that the socio-economic and age factors of these children may relate with the poor nutritional outcome. This makes monitoring and personalized nutritional management necessary, in accordance with the characteristics and particularities of children with CP.

Anisakidae Skrjabin & Karokhin, 1945 and Raphidascarididae Hartwich, 1954 nematodes in lutjanidae (pisces: perciformes) from the Brazilian Northeast Coast / Nematodas Anisakidae Skrjabin & Karokhin, 1945 e Raphidascarididae Hartwich, 1954 em Lutjanidae (Pisces: Perciformes) do litoral nordeste brasileiro

Abstract The present study aimed at describing and evaluating the fauna of Anisakidae and Raphidacarididae nematodes of lutjanid fish (snappers) from the Brazilian northeastern coast unloaded at the city of Aracaju, capital of the State of Sergipe. A total of 186 lutjanids of 5 different species were analyzed including 69 Lutjanus analis, 51 L. vivanus , 29 Ocyurus chrysurus, 23 L. synagris, and 14 L. jocu. Nematode specimens found in the viscera of these fish were clarified with lactophenol. Illustrations of each of these specimens were prepared to help identify these samples and compose their taxonomic description and classification. At necropsy, 3,183 nematodes belonging to two families were collected from fish viscera which included the following: Anisakidae: Anisakis, Terranova , Contracaecum, and Goezia; Raphidascarididae: Raphidascaris (Ichthyascaris) and Hysterothylacium . A total of six genera of nematodes were found at postmortem examination of which adult worms of only three genera (Goezia, Raphidascaris and Hysterothylacium) were detected in the fish examined. Larvae of Terranova sp. were found only in the musculature of O. chrysurus . Most of the nematode larvae were present in the mesentery and organs of the fish necropsied. Therefore, the zoonotic potential of these nematodes cannot be ruled out. There was a significant negative correlation between the intensity of parasitism by anisakids and the total body length of O. chrysurus; the intensity of parasitism was greater in smaller and shortef fish. There was a significant positive correlation between the abundance of these parasites and the length of L. synagris; the presence of these parasites increased according to the fish's body length. To the authors' knowledge, these are the first records of Raphidascaris (Ichthyascaris ) sp. and Hysterothylacium sp. in L. vivanus , of Terranova sp. in O. chrysurus and L. jocu, and of Goezia sp. in L. analis. These novel findings add the Brazilian northeast coast and the State of Sergipe in the geographic distribution of these parasites in the country.

Resumo O objetivo deste trabalho é descrever e avaliar a fauna de Anisakidae e Raphidacarididae de peixes lutjanídeos do litoral nordeste do Brasil desembarcados na cidade de Aracaju, capital do estado de Sergipe. Foram analisados 186 lutjanídeos das espécies Lutjanus analis 69, L. vivanus 51, Ocyurus chrysurus 29, L. synagris 23 e L. jocu 14. Exemplares dos nematodas encontrados nas vísceras foram clarificados em lactofenol e ilustrados para auxiliar na identificação e compor a descrição taxonõmica. Foram coletados 3.183 nematodas das vísceras dos peixes, pertencentes a duas famílias: Anisakidae: Anisakis, Terranova, Contracaecum e Goezia; e Raphidascarididae: Raphidascaris (Ichthyascaris) e Hysterothylacium. Totalizando seis gêneros encontrados, apenas nos três últimos gêneros foram encontrados vermes em estágio adulto. Apenas em O. chrysurus foram encontradas larvas de Terranova sp. na musculatura. A maioria das larvas estava restrita ao mesentério e órgãos dos peixes, e em grande intensidade não podendo se descartar o potencial zoonótico. Observou-se uma correlação significativa negativa entre a intensidade anisakídeos e o comprimento total de O. chrysurus, onde a intensidade dos parasitas é maior em peixes de menor comprimento e uma correlação significativa positiva entre a abundância desses mesmos parasitos com o comprimento de L. synagris, onde a presença dos parasitas aumenta conforme o comprimento do peixe. Esse é o primeiro registro de Raphidascaris ( Ichthyascaris) sp. e Hysterothylacium sp. em L. vivanus ; de Terranova sp. em O. chrysurus e L. jocu e Goezia sp. em L. analis, adicionando o litoral nordeste do Brasil e o estado de Sergipe na distribuição geográfica desses parasitos.

Anisakidae Skrjabin & Karokhin, 1945 and Raphidascarididae Hartwich, 1954 nematodes in lutjanidae (pisces: perciformes) from the Brazilian Northeast Coast.

The present study aimed at describing and evaluating the fauna of Anisakidae and Raphidacarididae nematodes of lutjanid fish (snappers) from the Brazilian northeastern coast unloaded at the city of Aracaju, capital of the State of Sergipe. A total of 186 lutjanids of 5 different species were analyzed including 69 Lutjanus analis, 51 L. vivanus , 29 Ocyurus chrysurus, 23 L. synagris, and 14 L. jocu. Nematode specimens found in the viscera of these fish were clarified with lactophenol. Illustrations of each of these specimens were prepared to help identify these samples and compose their taxonomic description and classification. At necropsy, 3,183 nematodes belonging to two families were collected from fish viscera which included the following: Anisakidae: Anisakis, Terranova , Contracaecum, and Goezia; Raphidascarididae: Raphidascaris (Ichthyascaris) and Hysterothylacium . A total of six genera of nematodes were found at postmortem examination of which adult worms of only three genera (Goezia, Raphidascaris and Hysterothylacium) were detected in the fish examined. Larvae of Terranova sp. were found only in the musculature of O. chrysurus . Most of the nematode larvae were present in the mesentery and organs of the fish necropsied. Therefore, the zoonotic potential of these nematodes cannot be ruled out. There was a significant negative correlation between the intensity of parasitism by anisakids and the total body length of O. chrysurus; the intensity of parasitism was greater in smaller and shortef fish. There was a significant positive correlation between the abundance of these parasites and the length of L. synagris; the presence of these parasites increased according to the fish's body length. To the authors' knowledge, these are the first records of Raphidascaris (Ichthyascaris ) sp. and Hysterothylacium sp. in L. vivanus , of Terranova sp. in O. chrysurus and L. jocu, and of Goezia sp. in L. analis. These novel findings add the Brazilian northeast coast and the State of Sergipe in the geographic distribution of these parasites in the country.

Dengue infection in mice inoculated by the intracerebral route: neuropathological effects and identification of target cells for virus replication.

Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7th day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.

Estudo comparativo entre métodos manual e digital no cálculo do ângulo do platô tibial em cães / Comparative study of manual and digital method in the calculation of the tibial plateau angle in dogs

São poucos os dados publicados sobre a aplicabilidade ou a confiabilidade dos métodos digitais para mensuração do ângulo do platô tibial (APT) em comparação à mensuração manual. O objetivo deste estudo foi comparar o APT obtido pelo cálculo manual padrão, em filmes radiográficos, com os valores obtidos por cálculos em imagens digitais, por meio do software do computador. Foram analisadas radiografias digitais do membro pélvico, direito e esquerdo, em perfil verdadeiro, de 20 cães saudáveis, com idade superior a um ano. Três observadores com graus de experiência semelhantes em cálculo do APT realizaram as mensurações em cada radiografia. Primeiramente, foram calculados os ângulos dos platôs tibiais manualmente nas películas radiográficas e, posteriormente, os cálculos foram realizados na imagem digital, por meio do software para medicina veterinária do sistema de raios X digital. Os resultados do presente estudo demonstraram diferenças significativas entre os métodos, com valor das médias do cálculo digital 20,48±3.71 e do cálculo manual 23.90±4.02. Valor de P foi < 0,01 na comparação entre médias dos dois grupos. Na avaliação interobservadores, houve diferença significativa no cálculo manual no perfil direito, o que sugere maior variação no cálculo manual quando comparado ao digital. O cálculo realizado pelo software apresentou menor variação nos valores interobservadores. Acredita-se que a menor variação obtida nos resultados aqui apresentados se dê em razão das facilidades proporcionadas pelo software. Conclui-se que houve diferença entre os valores obtidos por meio dos métodos convencional e digital no cálculo do ângulo do platô tibial, sendo os valores digitais menores entre os observadores, e que o método digital no cálculo do ângulo do platô tibial levou à menor variação quando comparado ao método manual.(AU)

There are few published data on the applicability and reliability of digital methods for measuring the TPA compared with manual measurement. The objective of this study was to compare the TPA obtained by calculating standard manual on conventional radiographs, with the values obtained by calculations in digital images through a computer program. Bilateral digital radiographs of hind limb were analyzed in true profile for 20 healthy dogs older than two years. Three observers with similar levels of experience in calculating the TPA conducted measurements on each radiograph. First we calculated the angle of the tibial plateaus manually in x-ray film and then the calculations were made through the program offered by the computer program in the digital image. The results of this study showed significant differences between the methods, with average value of 20.48±3.71 digital calculating average and 23.90±2.4 in the manual calculation. P value was < 0.01 when comparing averages. No significant difference in manual calculation in right profile were found between interobserver assessments, suggesting greater variation in manual calculation compared to digital. The calculation performed by the software showed less variation in values. The slight variation obtained in our results is believed to be due to the facilities provided by the software. There was a difference between the values obtained by means of conventional and digital methods for calculating the angle of the tibial plateau, and the smaller digital values between the observers and the digital method in the calculation of the tibial plateau angle led to less variation when compared to the manual method.(AU)

Anisakidae Skrjabin & Karokhin, 1945 and Raphidascarididae Hartwich, 1954 nematodes in lutjanidae (pisces: perciformes) from the Brazilian Northeast Coast

Abstract The present study aimed at describing and evaluating the fauna of Anisakidae and Raphidacarididae nematodes of lutjanid fish (snappers) from the Brazilian northeastern coast unloaded at the city of Aracaju, capital of the State of Sergipe. A total of 186 lutjanids of 5 different species were analyzed including 69 Lutjanus analis, 51 L. vivanus , 29 Ocyurus chrysurus, 23 L. synagris, and 14 L. jocu. Nematode specimens found in the viscera of these fish were clarified with lactophenol. Illustrations of each of these specimens were prepared to help identify these samples and compose their taxonomic description and classification. At necropsy, 3,183 nematodes belonging to two families were collected from fish viscera which included the following: Anisakidae: Anisakis, Terranova , Contracaecum, and Goezia; Raphidascarididae: Raphidascaris (Ichthyascaris) and Hysterothylacium . A total of six genera of nematodes were found at postmortem examination of which adult worms of only three genera (Goezia, Raphidascaris and Hysterothylacium) were detected in the fish examined. Larvae of Terranova sp. were found only in the musculature of O. chrysurus . Most of the nematode larvae were present in the mesentery and organs of the fish necropsied. Therefore, the zoonotic potential of these nematodes cannot be ruled out. There was a significant negative correlation between the intensity of parasitism by anisakids and the total body length of O. chrysurus; the intensity of parasitism was greater in smaller and shortef fish. There was a significant positive correlation between the abundance of these parasites and the length of L. synagris; the presence of these parasites increased according to the fishs body length. To the authors knowledge, these are the first records of Raphidascaris (Ichthyascaris ) sp. and Hysterothylacium sp. in L. vivanus , of Terranova sp. in O. chrysurus and L. jocu, and of Goezia sp. in L. analis. These novel findings add the Brazilian northeast coast and the State of Sergipe in the geographic distribution of these parasites in the country.

Resumo O objetivo deste trabalho é descrever e avaliar a fauna de Anisakidae e Raphidacarididae de peixes lutjanídeos do litoral nordeste do Brasil desembarcados na cidade de Aracaju, capital do estado de Sergipe. Foram analisados 186 lutjanídeos das espécies Lutjanus analis 69, L. vivanus 51, Ocyurus chrysurus 29, L. synagris 23 e L. jocu 14. Exemplares dos nematodas encontrados nas vísceras foram clarificados em lactofenol e ilustrados para auxiliar na identificação e compor a descrição taxonõmica. Foram coletados 3.183 nematodas das vísceras dos peixes, pertencentes a duas famílias: Anisakidae: Anisakis, Terranova, Contracaecum e Goezia; e Raphidascarididae: Raphidascaris (Ichthyascaris) e Hysterothylacium. Totalizando seis gêneros encontrados, apenas nos três últimos gêneros foram encontrados vermes em estágio adulto. Apenas em O. chrysurus foram encontradas larvas de Terranova sp. na musculatura. A maioria das larvas estava restrita ao mesentério e órgãos dos peixes, e em grande intensidade não podendo se descartar o potencial zoonótico. Observou-se uma correlação significativa negativa entre a intensidade anisakídeos e o comprimento total de O. chrysurus, onde a intensidade dos parasitas é maior em peixes de menor comprimento e uma correlação significativa positiva entre a abundância desses mesmos parasitos com o comprimento de L. synagris, onde a presença dos parasitas aumenta conforme o comprimento do peixe. Esse é o primeiro registro de Raphidascaris ( Ichthyascaris) sp. e Hysterothylacium sp. em L. vivanus ; de Terranova sp. em O. chrysurus e L. jocu e Goezia sp. em L. analis, adicionando o litoral nordeste do Brasil e o estado de Sergipe na distribuição geográfica desses parasitos.

Impact of insulin concentration and mode of FSH addition on the in vitro survival and development of isolated bovine preantral follicles.

UNLABELLED: The insulin and FSH are two important substances in the folliculogenesis process. Thus, the hypothesis of this experiment is that insulin concentration and the form of FSH addition affect the in vitro survival, growth, and estradiol production after culture of isolated bovine preantral follicles. The effects of insulin concentration (experiment 1) and the influence of both fixed and sequential concentrations of FSH (experiment 2) on the in vitro survival and development of bovine preantral follicles were investigated in this study by IVC for 18 days. In experiment 1, on Day 18 of culture, the addition of insulin at all concentrations promoted follicular survival rates significantly higher than that of the control, with the 10-ng/mL insulin treatment showing values significantly higher than the other treatments. The addition of 5- and 10-ng/mL insulin promoted higher follicular growth than the control and other treatments. In experiment 2, FSH 100 had a higher percentage of follicular viability compared with the control. FSH 100 produced follicle diameters significantly higher than those of the control and FSH seq. TREATMENT: Estradiol levels in the presence of FSH (fixed concentration) were significantly higher than the other treatments. In conclusion, the association of insulin (10 ng/mL) and fixed concentration FSH (100 ng/mL) provides high rates of survival, growth, and estradiol production in bovine preantral follicles.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue.

OBJECTIVE: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.

Peripheral effects induced in BALB/c mice infected with DENV by the intracerebral route.

The lack of an immunocompetent animal model for dengue mimicking the disease in humans is a limitation for advances in this field. Inoculation by intracerebral route of neuroadapted dengue strains in mice is normally lethal and provides a straightforward readout parameter for vaccine testing. However, systemic effects of infection and the immune response elicited in this model remain poorly described. In the present work, BALB/c mice infected by the intracerebral route with neuroadapted DENV2 exhibited several evidences of systemic involvement. DENV-inoculated mice presented virus infective particles in the brain followed by viremia, especially in late stages of infection. Infection induced cellular and humoral responses, with presence of activated T cells in spleen and blood, lymphocyte infiltration and tissue damages in brain and liver, and an increase in serum levels of some pro-inflammatory cytokines. Data highlighted an interplay between the central nervous system commitment and peripheral effects under this experimental condition.

Is the mouse follicle culture a good model for the goat with respect to the development of preantral follicles in vitro?

The present study evaluated the efficiency of using 2 culture media developed for mice and for goats in the in vitro preantral follicle culture of each species. Murine and caprine secondary follicles were cultured in vitro with human recombinant follicle-stimulating hormone (murine medium) or with bovine recombinant follicle-stimulating hormone in association with growth hormone (caprine medium). The results showed that murine follicles cultured in caprine medium had lower (P < 0.05) rates of follicular survival and growth, whereas for caprine follicles, these variables were not affected by the type of medium used (P > 0.05). After in vitro maturation, a higher (P < 0.05) number of oocytes that resumed meiosis were observed in the murine medium for both species. In contrast, only in the caprine species estradiol production was significantly superior when the caprine medium was used. Higher progesterone production was observed in the presence of the murine medium only for murine follicles (P < 0.05). In conclusion, murine and caprine preantral follicles cultured under the same in vitro culture medium conditions respond differently; caprine oocytes grown in vitro in the presence of the murine medium show the greatest developmental competence among the tested combinations. Therefore, under the present experimental conditions, the mouse follicle culture has proved be a good model for the development of new culture media for caprine preantral follicles.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

Role of nerve growth factor (NGF) and its receptors in folliculogenesis.

Summary Nerve growth factor (NGF) is a prototype member of the neurotrophins family and has important functions in the maintenance of viability and proliferation of neuronal and non-neuronal cells, such as certain ovarian cells. The present review highlights the role of NGF and its receptors on ovarian follicle development. NGF initiates its multiple actions through binding to two classes of receptors: the high affinity receptor tyrosine kinase A (TrkA) and the low-affinity receptor p75. Different intracytoplasmic signalling pathways may be activated through binding to NGF due to variation in the receptors. The TrkA receptor activates predominantly phosphatidylinositol-3-kinase (PI3K) and mitogenic activated protein kinase (MAPK) to promote cell survival and proliferation. The activation of the phospholipase type Cγ (PLCγ) pathway, which results in the production of diacylglycerol (DAG) and inositol triphosphate (IP3), culminates in the release of calcium from the intracytoplasmic cellular stocks. However, the details of activation through p75 receptor are less well known. Expression of NGF and its receptors is localized in ovarian cells (oocyte, granulosa, theca and interstitial cells) from several species, which suggests that NGF and its receptors may regulate some ovarian functions such as follicular survival or development. Thus, the use of NGF in culture medium for ovarian follicles may be of critical importance for researchers who want to promote follicular development in vitro in the future.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture.

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

How the concentration of insulin affects the development of preantral follicles in goats.

This study investigated the effect of adding different insulin concentrations to the culture medium for goat preantral follicle development in vitro. The ovarian fragments were immediately fixed or cultured for 7 days in MEM with insulin (0, 5, 10 ng/ml and 5 or 10 µg/ml). The results showed that, after 7 days of culture, insulin at 10 ng/ml was the best concentration to preserve follicular viability and ultrastructure, resulting in the highest rates of normal follicles. After 7 days, only treatments with 10 ng/ml and 5 µg/ml of insulin increased follicular activation when compared to other concentrations. Regarding follicular and oocyte growth, the presence of 10 ng/ml of insulin promoted a larger diameter than other treatments. In conclusion, this study shows that addition of 10 ng/ml of insulin to the culture medium improved the survival and stimulated growth of goat preantral follicles.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles.

The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.

Nerve growth factor promotes the survival of goat preantral follicles cultured in vitro.

The aim of this study was to investigate the effects of nerve growth factor (NGF) on the in vitro culture of goat preantral follicles. Ovarian cortex fragments were cultured in α-MEM+ supplemented with 0, 1, 10, 50, 100 or 200 ng/ml NGF for 1 or 7 days. Small fragments of noncultured ovarian tissue as well as those cultured for 1 or 7 days were processed for histology and transmission electron microscopy. The results showed that after 1 or 7 days of culture at all concentrations of NGF, except at 1 ng/ml after 1 day of culture, there was a significant reduction in the percentage of normal follicles compared to noncultured tissues. At higher NGF concentrations (100 and 200 ng/ml) after 7 days of culture, there was a significant reduction in the percentage of normal follicles compared to tissues cultured in α-MEM+ alone or at the other concentrations of NGF. It is important to note that ultrastructural and fluorescent analyses confirmed only the integrity of follicles cultured with 1 ng/ml of NGF after 7 days. In contrast to noncultured control tissues, the percentage of developing follicles was significantly increased at all concentrations of NGF after 1 or 7 days of culture. We observed that follicular diameter was greater at 1 and 10 ng/ml NGF after culture for 7 days than at the other concentrations but was similar to follicles cultured in α-MEM+ alone. In conclusion, NGF improved the survival of goat preantral follicles cultured in vitro in a dose-dependent manner.

DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice.

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.

Morphoquantitative aspects of NADH-diaphorase myenteric neurons in the ileum of diabetic rats treated with acetyl-L-carnitine.

In this work, we investigated the effect of the acetyl-L-carnitine (ALC) supplementation (200 mg/kg/day) on the myenteric neurons of the ileum of rats made diabetic by streptozotocin (35 mg/kg, i.v.). Four groups were used: diabetic (D), diabetic supplemented with ALC (DC), control (C) and control supplemented with ALC (CC). After 15 weeks of diabetes induction the animals were killed and the ileum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The density of neurons seen in 12.72 mm2 of ileum showed no difference among the groups, although in group D it was 22% smaller than in group C, while group DC was 9% smaller to group CC. The profiles of the cell bodies (PC) of 1000 neurons per group were analysed. The neurons PC in group D decreased (P < 0.0001) when compared with other groups and increased (P < 0.0001) when compared with group DC. The incidence of neurons with a PC inferior to 200 microm2 was larger in group D. The frequency of neurons with a PC higher than 200 microm2 in group DC was close to those seen in groups C and CC. We concluded that ALC eases the loss of neurons and makes the incidence of myenteric neurons with a PC higher than 200 microm2 similar to the control rats.

DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus.

Dengue is one of the most important mosquito-borne viral disease causing dengue fever and/or dengue shock syndrome/haemorrhagic fever. In some reports, the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. In order to investigate the potential of a DNA vaccine based on the NS1 protein against DENV, we used the plasmid pcTPANS1, which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. All Balb/c mice intramuscularly inoculated with the pcTPANS1 presented high levels of NS1-specifc antibodies. Vaccinated animals were challenged with intracerebral DENV-2 virus inoculations and a 100% survival was observed. In general, results demonstrate that the pcTPANS1 plasmid is able to induce protection in mice, and then may be used as a vaccination approach against DENV in further assays.

Ex vivo evaluation of the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment.

AIM: To evaluate ex vivo the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment. METHODOLOGY: The root canals of 62 maxillary and mandibular canines were prepared to a length 1 mm short of the apical foramen, to an apical size 35 using 1% sodium hypochlorite as an irrigant. Once prepared, the length of each tooth was measured directly using a size 15 K-Flexofile introduced in the canal until the tip was visible at the apical foramen. After the file was removed, its length was recorded to a precision of 0.01 mm using a calliper. These direct lengths (DL) became the 'gold standard' for comparison with the electronic lengths (EL) derived from the Tri Auto ZX. After direct measurement, the tooth was measured electronically (EL1) and the canals were filled using lateral condensation of gutta-percha cones and sealer. Seven days later the root filling was removed using solvent, Gates-Glidden burs, and K-files, and new electronic lengths determined (EL2). The electronic lengths (EL1 and EL2) were compared with the DL, and the differences were analysed statistically using the proportions test and Student's t-test. RESULTS: At a tolerance limit of +/-0.5 mm, EL1 coincided with the DL in 76% of cases. Lengths obtained in the presence of remnant of filling material (EL2) coincided in 81% of cases. The proportions test used to compare these percentages showed no statistically significant difference between EL1 and EL2 (P > 0.05). The Student's t-test revealed a statistically significant difference (P < 0.05) between the means of the differences between DL/EL1 (-0.36 mm) and DL/EL2 (-0.04 mm). CONCLUSION: The Tri Auto ZX was accurate to +/-0.5 mm in more than 80% of teeth following removal of root fillings.