Biochemical and immunohistochemical examination of the effects of ephedrine in rat ovary tissue

Purpose: It was aimed to investigate the biochemical and immunohistochemical effects of ephedrine (EPH) in bilateral ovariectomized rats. Methods: Twenty-four Sprague Dawley female rats were divided into three groups: control group: The abdomen was opened and closed without any treatment; ischemia-reperfusion (IR) group: 2 h of ischemia followed by 2 h of reperfusion were allowed to cause IR injury; IR+EPH group: oral EPH solution (5 mg/kg) was administered for 28 days. Results: Biochemical parameters were statistically significant in group comparisons. Increased interleukin-6 (IL-6) expression, degenerative preantral and antral follicle cells and inflammatory cells around blood vessels were seen in IR group. Negative IL-6 expression was observed in seminal epithelial cells, preantral and antral follicle cells in IR+EPH group. While caspase-3 activity increased in granulosa cells and stromal cells in IR group, caspase-3 expression was negative in preantral and antral follicle cells in the germinal epithelium and cortex in IR+EPH group. Conclusion: The effect of apoptosis, which occurs with the signaling that starts in the cell nucleus, caused the cessation of the stimulating effect at the nuclear level after EPH administration, and a decrease in the antioxidative effect in IR damage and inflammation in the apoptotic process.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.(AU)

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].(AU)

Deeper exploration of inflammatory cell populations in milk to monitor udder health in dairy cows

The present study explored the predictive values of milk leukocyte differentials (MLD) as a basis for improving the diagnosis of intramammary infections (IMIs) and subclinical mastitis. Quarter milk samples were collected for bacteriological analysis, quarter somatic cell count (qSCC), and MLD. The MLD were assessed using the cytospin technique, direct microscopic smears, and flow cytometry. The predictive values of each single leukocyte population and useful potential indices that could better reflect immune complexity were also calculated. Changes in the percentage of any leukocyte alone failed to substantially improve the predictive value of qSCC in diagnosing IMIs. Although certain parameters increased the area under the receiver operating characteristic curve (ROC curve) as a result of increased specificity values, a slight reduction in sensitivity was observed. The so-called CD8 complex was a unique parameter which improved both the sensitivity (78.79 %) and the specificity (80.77 %) in IMI diagnosis, resulting in the highest area under the ROC curve (0.87). To diagnose subclinical mastitis, the percentage of macrophages and the sum of the percentage PMNLs and T CD8+ cells divided by the percentage of macrophages showed the highest predictive values (sensitivity = 79.63, specificity = 73.68, and area under the ROC curve = 0.83) in the differentiation of the inflammatory condition status of cows. In conclusion, this study provides further insights into using T CD8+ lymphocytes in diagnosing bovine IMIs, combined with PMNLs and macrophages. The antidromic trend of macrophages vs. PMNLs and T CD8+ lymphocytes due to the increasing qSCCs was crucial to differentiating quarters under both inflammatory and non-inflammatory conditions.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis ​​pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

In vitro culture of parasitic stages of Haemonchus contortus / Cultivo in vitro de estádios parasitários de Haemonchus contortus

Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco's modified Eagle's medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes' reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.(AU)

Haemonchus contortus provoca grandes prejuízos à ovinocultura. Visando reduzir a utilização de ovinos hospedeiros e produzir estágios parasitários em larga escala, um protocolo para o cultivo in vitro por 42 dias de larvas de terceiro estádio de H. contortus foi realizado em meio Eagle, modificado por Dulbecco (DMEM). No cultivo sem células, as larvas foram mantidas a 39,6°C e incubadas em DMEM ácido (pH 6,1) por 3 ou 6 dias com 4Δ-ácido dafacrônico seguido por DMEM pH 7,4 suplementado ou não com reagente de Fildes. Em DMEM pH 7,4 a 37°C, a suplementação com células Caco-2 foi comparada à suplementação com Fildes. No Dia 14, as taxas de desenvolvimento até o quarto estádio larvar (L4) foram similares em meio ácido sem células suplementado (86,8-88,4%) ou não (74,4-77,8%) com Fildes e em co-cultura com células Caco-2 (92,6%), e superiores ao desenvolvimento em DMEM pH 7,4 com Fildes (0,0%). No Dia 21, o co-cultivo com células Caco-2 resultou em maior diferenciação (25,0%) e menor degeneração (13,9%) das larvas em comparação ao meio ácido (1,5-8,1% e 48,6-69,9%, respectivamente). Este é o primeiro relato de cultivo in vitro prolongado de H. contortus em meio comercial em co-cultivo com células Caco-2. Apesar da ausência de progressão até o estágio adulto, o co-cultivo com células Caco-2 resultou em diferenciação de L4 e manutenção da viabilidade das larvas de H. contortus por até 28 dias in vitro.(AU)

Genetic relationships of the Yucatan black hairless pig with Iberian breeds using single nucleotide polymorfisms / Relações genéticas de porcos pretos calvos de Yucatán com raças ibéricas usando Polimorfismo de nucleotídeos isolados

To conduct ex-situ creole pig conservation programs, it is essential to determine which breeding animals will be used, preferentially those with a more significant Iberian genetic component to preserve their origin. This study used a Yucatan black hairless pigs (YBHP) subpopulation to estimate its genetic diversity and population structure. One hundred four adult pigs were selected for the absence of hair, black skin (without spots), black hoof, and straight snout. The porcine-GGP-50K chip was used for SNP genotyping in YBHP, and information on Iberian and Yucatán hairless pigs from the United States (USYU) was taken from databases. All analysis was performed using PLINK v1.9 and v2.1 software. Inbreeding and fixation index values were lower in YBHP, with high observed heterozygosity and allogamy index values, which agree with those obtained in the populations of Canarias and Chato Murciano. According to the clusters generated by the "Genome-Wide Identity by State" analysis, four groups were identified, one of which included pigs from Guadyerbas, USYU, and YBHP. Between populations, YBHP was closely related to the hairless pigs from Guadyerbas, USYU, and Canarias. Principal component analysis showed the same result. According to the results obtained from the runs of homozygosity investigation, aimed to get pools consensus of regions of overlapping, 119 SNPs associated with genes and biological processes were identified. The BMP7 and NSUN2 genes were associated with epithelial cell differentiation, morphogenesis, and epithelial development. For nutrient metabolism: energy, the HADHA, PPARA, ADD1/SREBF1, and FAT 1genes were identified.(AU)

Para realizar programas de conservação ex-situ de suínos crioulos, é importante determinar quais animais serão criados, preferencialmente aqueles com maior componente de genética ibérica, para preservar sua origem. Uma subpopulação de porco preto calvo de Yucatán (YBHP) foi usada para estimar sua diversidade genética e estrutura populacional. Um total de 104 suínos adultos foram selecionados levando-se em consideração características como ausência de pelos, pele preta (sem manchas), casco preto e focinho reto. O painel GGP-50K foi utilizado para a genotipagem dos SNPs em animais YBHP, e informações de porcos sem pelos ibéricos e de Yucatán dos Estados Unidos (USYU) foram retiradas de bancos de dados. Todas as análises foram realizadas com o software PLINK v1.9 e v2.1. Os valores dos índices de endogamia e fixação foram menores em YBHP, com altos valores de índice de heterozigosidade e alogamia observados, que concordam com os obtidos nas populações de Canárias e Chato Murciano. De acordo com os clusters gerados pela análise "Genoma-Wide Identity By State", quatro grupos foram identificados, um dos quais incluiu porcos de Guadyerbas, USYU e YBHP. Entre as populações, YBHP estava intimamente relacionado com os porcos sem pelo de Guadyerbas, USYU e Canárias. A análise de componentes principais mostrou o mesmo resultado. De acordo com os resultados obtidos nas corridas de investigação de homozigose, visando obter consenso de pools de regiões de sobreposição, foram identificados 119 SNPs associados a genes e processos biológicos. Os genes BMP7 e NSUN2 foram associados à diferenciação de células epiteliais, morfogênese e desenvolvimento epitelial. Para metabolismo de nutrientes: energia, os genes HADHA, PPARA, ADD1/SREBF1 e FAT1 foram identificados.(AU)

Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins

The present review addresses the oocyte and the preimplantation embryo, and is intended to highlight the underlying principle of the "nature versus/and nurture" question. Given the diversity in mammalian oocyte maturation, this review will not be comprehensive but instead will focus on the porcine oocyte. Historically, oogenesis was seen as the development of a passive cell nursed and determined by its somatic compartment. Currently, the advanced analysis of the cross-talk between the maternal environment and the oocyte shows a more balanced relationship: Granulosa cells nurse the oocyte, whereas the latter secretes diffusible factors that regulate proliferation and differentiation of the granulosa cells. Signal molecules of the granulosa cells either prevent the precocious initiation of meiotic maturation or enable oocyte maturation following hormonal stimulation. A similar question emerges in research on monozygotic twins or multiples: In Greek and medieval times, twins were not seen as the result of the common course of nature but were classified as faults. This seems still valid today for the rare and until now mainly unknown genesis of facultative monozygotic twins in mammals. Monozygotic twins are unique subjects for studies of the conceptus-maternal dialogue, the intra-pair similarity and dissimilarity, and the elucidation of the interplay between nature and nurture. In the course of in vivo collections of preimplantation sheep embryos and experiments on embryo splitting and other microsurgical interventions we recorded observations on double blastocysts within a single zona pellucida, double inner cell masses in zona-enclosed blastocysts and double germinal discs in elongating embryos. On the basis of these observations we add some pieces to the puzzle of the post-zygotic genesis of monozygotic twins and on maternal influences on the developing conceptus.(AU)

Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI)

RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies.(AU)

Effects of systemic inflammation due to hepatic ischemia-reperfusion injury upon lean or obese visceral adipose tissue

Purpose: To evaluate how the induction of liver damage by ischemia and reperfusion affects the adipose tissue of lean and obese mice. Methods: Lean and diet-induced obese mice were subjected to liver ischemia (30 min) followed by 6 h of reperfusion. The vascular stromal fraction of visceral adipose tissue was analyzed by cytometry, and gene expression was evaluated by an Array assay and by RT-qPCR. Intestinal permeability was assessed by oral administration of fluorescein isothiocyanate (FITC)-dextran and endotoxemia by serum endotoxin measurements using a limulus amebocyte lysate assay. Results: It was found that, after liver ischemia and reperfusion, there is an infiltration of neutrophils, monocytes, and lymphocytes, as well as an increase in the gene expression that encode cytokines, chemokines and their receptors in the visceral adipose tissue of lean mice. This inflammatory response was associated with the presence of endotoxemia in lean mice. However, these changes were not observed in the visceral adipose tissue of obese mice. Conclusions: Liver ischemia and reperfusion induce an acute inflammatory response in adipose tissue of lean mice characterized by an intense chemokine induction and leukocyte infiltration; however, inflammatory alterations are already present at baseline in the obese adipose tissue and liver ischemia and reperfusion do not injure further.

Mfsd2a Promotes the Proliferation, Migration, Differentiation and Adipogenesis of Chicken Intramuscular Preadipocytes

Intramuscular fat (IMF) content is a crucial parameter for estimating meat quality. Growing evidence indicates that gene regulation plays an important role in IMF deposition. This study aimed to determine the function of Mfsd2a in chicken intramuscular preadipocytes. In the present study, high Mfsd2a mRNA levels were observed in the liver and adipose tissues of broilers. Subsequently, we synthesized small interfering RNAs to silence the expression of Mfsd2a in chicken intramuscular preadipocytes. The following results suggested that CDK2, PCNA, CCND1, CCND2 and MKI67 were inhibited, with CCK-8 and EdU assays revealing that cell proliferation was inhibited. Scratch test showed that cell migration ratios were declined. We also found that Mfsd2a silencing decreased the mRNA levels of PPARγ, RXRG and their target genes. The similar results were found in some key genes that contribute to lipid synthesis, including C/EBPα, C/EBPß, FABP4, FASN, ACACA and ACSL1. Finally, Oil red O staining showed that IMF accumulation was blocked after Mfsd2a silencing. In conclusion, our results implied that Mfsd2a promotes the proliferation and migration of chicken intramuscular preadipocytes, as well as the differentiation and adipogenesis through PPARγ signaling pathway, which may provide a potential target to improve chicken meat quality.(AU)

Triiodothyronine does not influence in vitro chondrogenic differentiation of adipose tissue-derived stem cells from young female rat / Triiodotironina não influencia na diferenciação condrogênica de células tronco do tecido adipose de ratas jovens

The objective of this study was to investigate the in vitro action of triiodothyronine (T3) on the chondrogenic differentiation of adipose tissue-derived stem cells (ASCs) of female rats, with different time periods and doses. ASCs were extracted from female Wistar rats and were cultured in chondrogenic medium with and without the presence of T3. Five groups were established: 1) ASCs without T3; and 2,3,4,5) ASCs with 0.01, 1, 100 and 1,000 nM T3, respectively). After 7, 14 and 21 days, cell morphology, chondrogenic matrix formation, and expression of Sox9, aggrecan, collagen II, and collagen X were evaluated. The Student-Newman-Keuls test was used. ASCs showed CD54, CD73, and CD90 before chondrogenic differentiation. The hormone treatment did not alter chondrogenic matrix formation, Sox9 expression at 14 or 21 days, or expression of collagen II or collagen X at any time. However, the 0.01, 1, and 1000 nM T3 doses decreased Sox9 expression at 7 days. In conclusion, chondrogenic differentiation of ASCs of female rats is not influenced by T3.

O objetivo do presente trabalho foi verificar o efeito in vitro da triiodotironina (T3) na diferenciação condrogênica de células tronco mesenquimais do tecido adiposo (CTM-TA) de ratas, durante vários períodos e em várias doses. CTM-TA foram coletadas de ratas Wistar e cultivadas em meio condrogênico com ou sem a presença de T3. Constitui-se cinco grupos: 1) CTM-TA sem T3; e 2,3,4,5) CTM-TA com T3 (0,01; 1; 100 e 1000 nM, respectivamente). Após sete, 14 e 21 dias, foram avaliados morfologia celular, formação de matriz condrogênica e expressão de Sox9, agrecano, colágeno II e colágeno X. Para as análises foi utilizado o teste de Student Newman Keuls. CTM-TA expressaram CD54, CD73 e CD90 antes da diferenciação condrogênica. O tratamento hormonal não alterou a formação de matriz condrogênica e a expressão de Sox9 aos 14 e 21 dias e expressão dos colágenos II e X em nenhum dos períodos avaliados. No entanto, as doses de 0,01; 1 e 1000 nM T3 diminuíram a expressão de Sox9 aos 7 dias. Conclui-se que a diferenciação condrogênica de CTM-TA de ratas não é influenciada pela T3.

Detection of respiratory viruses and expression of inflammatory cytokines in patients with acute exacerbation chronic obstructive pulmonary disease in Mongolia China / Detecção de vírus respiratórios e expressão de citocinas inflamatórias em pacientes com doença pulmonar obstrutiva crônica de exacerbação aguda na Mongólia, China

Chronic obstructive pulmonary disease (COPD) was estimated to be the third cause of global mortality by 2020. Acute exacerbation COPD (AECOPD) is a sudden worsening of COPD symptoms and could be due to virus/bacterial infections and air pollution. Increased expression of inflammatory markers in patients with AECOPD is associated with viral infection. This study aimed to detect different viruses and analyze the expression of various inflammatory markers associated with AECOPD patients. Three hundred and forty-seven patients diagnosed with COPD according to GOLD criteria were included in this study. Swab samples and blood were collected for the detection of viruses by RT-PCR and expression of inflammatory markers, respectively. Of the swab samples, 113 (32.6%) of samples were positive for virus detection. Of these, HRV (39.8%) was the predominant virus detected followed by FluB (27.4%) and FluA (22.1%). The presence of HRV was significantly higher (p=0.044) among the other detected viruses. When compared to healthy controls the expression levels of TNF-α, IL-6 and IL-8 were significantly higher (p<0.05) in virus-positive patients. The IL-6 and IL-8 were the next predominantly expressed in markers among the samples. The higher expression rate of IL-8 was significantly (p<0.05) associated with patients having COPD GOLD III severity level and smoking history. Although HRV was the predominant virus detected the combined prevalence of Influenza A and B surpassing the rate of HRV. The high-level expression of well known inflammatory markers of AECOPD, TNF-α, IL-6 and IL-8 indicates a chronic severe illness. These markers play an important role and could be used as a marker for determining the severity of AECOPD.(AU)

Estima-se que a doença pulmonar obstrutiva crônica (DPOC) seja a terceira causa de mortalidade global em 2020. A exacerbação aguda DPOC (AECOPD) é um agravamento súbito dos sintomas da DPOC e pode ser devido a infecções por vírus/bactérias e poluição do ar. O aumento da expressão de marcadores inflamatórios em pacientes com AECOPD está associado à infecção viral. Este estudo teve como objetivo detectar diferentes vírus e analisar a expressão de vários marcadores inflamatórios associados a pacientes com AECOPD. Trezentos e quarenta e sete pacientes com diagnóstico de DPOC de acordo com os critérios GOLD foram incluídos neste estudo. Amostras de swab e sangue foram coletadas para detecção de vírus por RT-PCR e expressão de marcadores inflamatórios, respectivamente. Das amostras de esfregaço, 113 (32,6%) amostras foram positivas para detecção de vírus. Nestas, o HRV (39,8%) foi o vírus predominante detectado, seguido do FluB (27,4%) e do FluA (22,1%). A presença de VFC foi significativamente maior (p = 0,044) entre os demais vírus detectados. Quando comparados a controles saudáveis, os níveis de expressão de TNF-α, IL-6 e IL-8 foram significativamente maiores (p <0,05) em pacientes com vírus positivo. A IL-6 e a IL-8 foram as próximas predominantemente expressas em marcadores entre as amostras. A maior taxa de expressão de IL-8 foi significativamente (p <0,05) associada a pacientes com grau de gravidade GOLD III da DPOC e história de tabagismo. Embora o HRV tenha sido o vírus predominante, a prevalência combinada de Influenza A e B ultrapassou a taxa de HRV. O alto nível de expressão de marcadores inflamatórios bem conhecidos de AECOPD, TNF-α, IL-6 e IL-8 indica uma doença crônica grave. Esses marcadores desempenham um papel importante e podem ser usados como um marcador para determinar a gravidade da AECOPD.(AU)

Uso de células-tronco no tratamento de sequelas neurológicas causadas pela cinomose / Use of stem cells in the treatment of sequelates neurological caused by cinomosis

A cinomose canina é uma doença infectocontagiosa causada pelo vírus Paramyxoviridae, que acomete cães de todas as idades, entretanto principalmente cães filhotes não vacinados. Uma das sequelas da doença é neurológica que, por muitas vezes, é de difícil tratamento. A terapia com células-tronco é uma alternativa para atenuar os sinais e sequelas neurológicas, propiciando melhor qualidade de vida para o animal. As células-tronco são células com alta capacidade de auto renovação e diferenciação, e, portanto, uma alternativa de tratamento para as sequelas neurológicas derivada pela cinomose.

Canine distemper is an infectocontagious disease caused by Paramyxoviridae virus, which affects dogs of all ages, however mainly unvaccinated puppies. Neurological manifestation are common in canine distemper which is often difficult to treat. Stem cell therapy is an alternative to attenuate neurological signs and sequel, providing better quality of life for the animal. Stem cells have high capacity for self-renewal and differentiation, and an alternative treatment for neurological sequel derived by distemper.

Knockdown of CPT1A Induce Chicken Adipocyte Differentiation to Form Lipid Droplets

Lipid metabolism dysfunction is closely related to obesity, inflammation, diabetes, lipodystrophy, cardiovascular disease. Along with having a positive effect on energy homeostasis during fasting or prolonged exercise through mitochondrial fatty acid oxidation (FAO), more than two dozen enzymes and transport proteins are involved in the activation and transport of fatty acids into the mitochondrial, providing insights into their critical roles in metabolism. CPT1A has been reported to be expressed ubiquitously in the body and associated with dire consequences affecting fat deposition as the key rate-limiting enzyme of FAO. However, there is a dearth of data on the specific role of CPT1A on adipogenic differentiation and adipocyte lipolysis on chicken. This study assessed CPT1A's function in adipocyte differentiation andadipocyte lipolysis, and the mechanisms were investigated. We found that CPT1A knockdown (KD) promotes the differentiation of chicken preadipocytes into mature adipocytes. CPT1A KD increased PPARγ protein expression level. Expression levels of lipid synthesis-related genes were increased, and lipolysis genes were reduced. Also, CPT1A KD can encourage the formation of lipid droplets. So our results confirmed that knockdown of CPT1A induced the lipid differentiation and inhibited the ß-oxidation process to promote the formation of lipid droplets. These findings may deepen our understanding on CPT1A function, especially its regulatory role in adipocyte biology.(AU)

Tanshinone IIA ameliorates myocardial ischemia/reperfusion injury in rats by regulation of NLRP3 inflammasome activation and Th17 cells differentiation

Purpose: Tanshinone IIA is a well-known lipophilic active constituent refined from traditional Chinese medicines, danshen. It has been previously demonstrated to possess various biological properties, including anti-inflammatory, antioxidant, promoting angiogenesis effect and so on. However, the mechanism of tanshinone IIA on myocardial ischemia-reperfusion injury (MI/RI) remains unclear. In this study, we investigated the effect of tanshinone IIA on MI/RI. Methods: MI/RI rat models were set up. Echocardiographic evaluation and hematoxylin and eosin staining were performed to analyze the cardiac function and morphology of MI/RI. Western blot was conducted for the detection of protein expression of pyrin domain containing 3 (NLRP3) and caspase-1 in heart tissues. Flow cytometry and real-time polymerase chain reaction were used for the detection of proinflammatory cytokines and Th17 cells differentiation. Results: We found that tanshinone IIA alleviated the myocardial damage of MI/RI, ameliorated the overall and local inflammatory reaction, and produced a cardioprotective effect by inhibiting of NLRP3 inflammasome activation and Th17/Treg cells differentiation. Conclusions: Our results highlighted the cardio-protective effect of tanshinone IIA on MI/RI and uncovered its underlying mechanism related to the NLRP3 inflammasome inhibition and the modulation of Th17/Treg cells differentiation.

Morphology and immunolocalization of intertubular steroidogenic cell in mesonephros of Podocnemis expansa during gonadal differentiation

Sex steroid hormones are critical in gonadal differentiation in turtles. The gonads are not the only organs responsible for producing these hormones during this phase. Mesonephros play an important role in steroidogenesis. The present study aimed to investigate the presence of steroidogenic cells in mesonephros of Podocnemis expansa during gonadal differentiation and to evaluate their morphology and ultrastructure. Ten embryos of P. expansa were collected from 5 nests on day 36 of incubation, during spawning period on an artificial beach. Embryos were extracted from eggs by slicing the shell and euthanized. They were dissected under a stereoscopic microscope to collect the gonad-mesonephro complex, in which were fixed and subsequently processed for light microscopy, immunohistochemistry and transmission electron microscopy analysis. During histological analysis was observed mesonephros has typical morphological structure. Immunohistochemistry showed immunoreaction to aromatase in cells of intertubular space. Confirming these findings, it was possible to observe a type of intertubular cell in several regions of mesonephro, being more predominant in region close to blood vessels, distal and proximal tubules. In ultrastructural analysis these cells were characterized by having a clear, large, and rounded nucleus with evident nucleolus and cytoplasm rich in electron-dense droplets. This study demonstrated for the first time the presence of cells with morphological, immunohistochemical and ultrastructural characteristics similar to steroid-producing cells in P. expansa mesonephrons, suggesting that this organ may contribute to gonadal differentiation in this species.(AU)

Oral adenocarcinoma observed in Boa constrictor maintained in captivity / Adenocarcinoma papilífero oral em jiboia (Boa constritor) mantida em cativeiro

Information on neoplasms in animals has increased over time, and these studies have helped in the management of reptiles that present tumors. There are similar incidences of neoplasms between homeothermic and ectothermic animals. Furthermore, there are usually more than one type of tumor present. The treatment of wild animals afflicted with cancer usually happens late, contributing to their low life expectancy. Thus, the present work aimed to describe an infrequent case of oral tumor in Boa constrictor. The tumor was observed in an adult female animal, raised in an exhibition area of the Zoo and Botanical Park of the Emílio Goeldi Museum, located in Belém, State of Pará, Brazil. Macroscopically, the mass presented morphologically with an irregular, multilobulated surface, color that varied from white to grey, hemorrhagic areas and its extension was 3.9 x 2.3 cm. The neoplasm was surgically removed, and the histopathological evaluation revealed an adenocarcinoma, with a papillary-like development pattern and a moderate degree of differentiation. The animal died three months after diagnosis due to starvation. The necropsy showed that there was tumor recrudescence and no metastases. Given the impossibility of surgical removal with a greater margin of safety, and adjuvant therapies, this condition favoured the resurgence of the neoplasm. This compromised the animal's ability to feed and consequently lead to death. Malignant neoplasms in reptiles may have an unfavourable clinical evolution for the maintenance of life, requiring specific therapeutic care such as chemotherapy. Scientific contributions on tumors in these animals are essential for the medical treatment of wild animals, and the conservation of wild species.(AU)

Estudos de neoplasias malignas e benignas em animais tem crescido ao longo do tempo, e os seus registros tem auxiliado no manejo de répteis que apresentam tumores. Descreve-se na literatura que há incidências semelhantes de neoplasmas entre animais homeotérmicos e ectotérmicos, e que estes geralmente apresentam mais de um tipo de tumoração. O tratamento de pacientes silvestres oncológicos geralmente acontece de forma tardia contribuindo para sua baixa sobrevida. Deste modo o presente trabalho objetivou descrever um caso infrequente de neoplasia oral em Boa constrictor. A casuística ocorreu num animal adulto, fêmea, criada em recinto de exposição do Parque Zoobotânico do Museu Emílio Goeldi, localizado em Belém, Estado do Pará, Brasil. Macroscopicamente a massa apresentou morfologicamente com superfície irregular, multilobulada, coloração que variou de branca a cinza, áreas hemorrágicas e sua extensão de 3,9 x 2,3cm. A neoplasia foi removida cirurgicamente e a avaliação histopatológica revelou um adenocarcinoma, com padrão de desenvolvimento do tipo papiliforme e grau moderado de diferenciação. O animal foi a óbito três meses pós-diagnóstico por inanição. A necropsia demonstrou que havia recrudescência do neoplasma e ausência de metástases. Diante da impossibilidade da remoção cirúrgica com maior margem de segurança, e terapias adjuvantes, tal condição favoreceu o ressurgimento da neoplasia, comprometendo a alimentação e consequentemente levando ao óbito. Conclui-se que neoplasias malignas em répteis podem ter uma evolução clínica desfavorável a manutenção da sua vida necessitando de cuidados terapêuticos específicos como quimioterapia. As contribuições cientificas sobre processos neoplásicos nesses animais são fundamentais para a clínica médica de animais silvestres além de contribuir para a conservação de espécies silvestres.(AU)