Effect of duration of estradiol exposure on embryo survival and endometrial gene expression in anestrous embryo recipient mares.

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P = .09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P > .05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19, IGF1, FGF2 and PGR gene expression, more conducive to embryo survival and further development.

Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABAB receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5's interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER.

Energy and mineral metabolism of peripartum mares and foals of the Quarter Horse breed.

BACKGROUND: Several studies have demonstrated variations in peripartum blood biochemical analytes used to monitor the health status of mares and their foals of different breeds. OBJECTIVES: We aimed to characterize the physiologic changes of peripartum energy and mineral metabolism in healthy Quarter Horse mares and their neonatal foals. METHODS: Blood samples were initially collected from 17 mares on days 60 and 30 prepartum, and from mares and their foals on the day of parturition (after colostrum ingestion), and on days 15, 30, and 60 postpartum. The serum concentrations of calcium, phosphorus, magnesium, total cholesterol (T-Cho), triglyceride, beta-hydroxybutyrate (BHBA), and nonesterified fatty acids (NEFA) were measured using commercial kits. Statistical differences were analyzed using one-way ANOVA followed by Tukey's test (parametric variables) or the Kruskal-Wallis test followed by Dunn's multiple comparison test (nonparametric variables). RESULTS: Fourteen of 17 healthy Quarter Horse mares and their neonatal foals remained in the study until termination. Serum BHBA, T-Cho, and calcium concentrations in mares showed significant differences during the observation period. Serum BHBA concentrations increased from 60 days prepartum to 60 days postpartum. Postpartum serum T-Cho and calcium concentrations significantly decreased until day-30 postpartum. In the foals, NEFA, T-Cho, calcium, and magnesium concentrations significantly differed between sampling times. In the foals, serum NEFA concentrations decreased from parturition until day-60 postpartum, while serum T-Cho, calcium, and magnesium concentrations were lowest at day-30 postpartum. CONCLUSIONS: The temporal changes observed in the blood biochemical analytes of this study could be used to improve the clinical evaluation of periparturient mare and neonatal foal Quarter Horses.

Influence of Organic Matter in Sorption of the Saflufenacil in Ferralsols.

The saflufenacil herbicide has been applied in the agricultural areas as an efficient alternative for the control of resistant weeds to glyphosate. However, the environmental risks from the use of saflufenacil, especially in tropical soil, is not yet clearly known. We evaluated if the organic matter addition into the soil influences the sorption of saflufenacil in samples of a sandy and clayey texture soils. The sugarbeet was used as a species indicating the presence of saflufenacil in the solution of the substrates. We estimated the required dose of saflufenacil responsible for causing 50% of sugarbeet intoxication (C50) and the sorption ratio (SR) of this herbicide. The addition of organic matter increased the C50 and SR of saflufenacil in both soils. Here it is demonstrated that the soil organic matter content increases saflufenacil sorption in tropical soils and, consequently influences the dose of this herbicide to be applied in pre-emergence of weeds.

Therapeutic effect of uridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

Teratoma Associated With Testicular Tissue in a Female-Like Horse With 64,XY (SRY-Positive) Disorder of Sex Development.

A four-year-old female-like Quarter Horse was admitted for clinical evaluation because of masculinized-aggressive behavior and lack of estrous signs. On external inspection, a hypertrophied clitoris and prominent muscles were observed. On gynecological examination, apparently normal mammary glands, vulva, vagina, and cervix were noted. On the other hand, the uterus had no tone and was smaller than normal. The left gonad was very soft on palpation and the right gonad was mostly firm, irregular, and wider than the left gonad. On ultrasound examination, there were no signs of regular ovarian structure or follicular activity. Because of the different shapes and consistencies of the gonads and a suspicion of increased testosterone production, a bilateral gonadectomy was recommended. Blood was collected for testosterone levels quantification and for cytogenetic and molecular investigations. After removal, gonads were analyzed macroscopically and sections were sent for histopathological examination. A final diagnosis of benign adult teratoma associated with seminiferous tubules and Leydig cells was made. On cytogenetic and molecular analyses, a normal diploid number of 64 chromosomes and the presence of the XY sex chromosomes were seen in all cells, as well as the SRY gene. Testosterone levels were higher than normal before surgery and were reduced after gonads removal. In conclusion, the masculinized behavior was probably caused by increased testosterone levels produced by testicular tissue, in a female-like horse with 64,XY SRY-positive disorder of sex development, which was associated with a teratoma.

CPBMF65, a synthetic human uridine phosphorylase-1 inhibitor, reduces HepG2 cell proliferation through cell cycle arrest and senescence.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

Octyl gallate reduces ATP levels and Ki67 expression leading HepG2 cells to cell cycle arrest and mitochondria-mediated apoptosis.

Octyl gallate (OG) is an antioxidant that has shown anti-tumor, anti-diabetic and anti-amyloidogenic activities. Mitochondria play an important role in hepatocellular carcinoma, mainly by maintaining accelerated cellular proliferation through the production of ATP. Thus, the mitochondria may be a target for antitumor therapies. Here, we investigated the effects of OG in the hepatocarcinoma cell line (HepG2) and the mechanisms involved. We report, for the first time, that treatment with OG for 24h inhibited HepG2 cell growth by decreasing mitochondrial activity and mass, which led to the reduction of ATP levels. This reduction in the energy supply triggered a decrease in Ki67 protein expression, leading cells to cycle arrest. In addition, treatment with two doses of OG for 48h induced loss of mitochondrial functionality, mitochondrial swelling and apoptosis. Finally, we report that HepG2 cells had no resistance to treatment after multiple doses. Collectively, our findings indicate that metabolic dysregulation and Ki67 protein reduction are key events in the initial anti-proliferative action of OG, whereas mitochondrial swelling and apoptosis induction are involved in the action mechanism of OG after prolonged exposure. This suggests that OG targets mitochondria, thus representing a candidate for further research on therapies for hepatocarcinoma.

Liraglutide, a glucagon-like peptide-1 analog, induce autophagy and senescence in HepG2 cells.

It has been reported that glucagon-like peptide-1 (GLP-1) agents have been associated with both the increased risk of cancer and inhibition of tumor growth and metastases. The aim of this study is to evaluate the effect of liraglutide on hepatocellular carcinoma cells - HepG2. Cytometry was used to evaluate mechanism related to decreased cell proliferation. Nuclear staining and morphometric analysis were also used to verify the process that was taking place after treatment with liraglutide, and in order to better understand the mechanism, TGF-ß1 was performed. HepG2 cells decreased proliferation after liraglutide treatment without altering oxidative stress levels. Liraglutide was able to induce autophagy and senescence through the increase of TGF-ß1 which possibly explains the growth decrease. We have demonstrated that liraglutide has an antiproliferative effect in HepG2 cells inducing autophagy and senescence by the increase of TGF-ß1.

Transferência de embriões em éguas receptoras anovulatórias / Embryo transfer in anovulatory recipient mares / Transferencia de embriones en yeguas receptoras anovulatorias

Resumo: Este artigo discute os principais aspectos do uso de protocolos hormonais baseados na aplicação de estrógeno seguido de progestágenos em receptoras anovulatórias o acíclicas, seja em anestro ou transição nos protocolos de transferência de embriões. A maioria das éguas apresentam comportamento poliéstrico estacional, com o que manifestam estro e ciclos ovulatórios nos períodos de maior luminosidade diária. O ciclo reprodutivo anual das éguas é caracterizado pela presença de quatro fases definidas pela dinâmica folicular: de anestro, de transição de primavera, ovulatória e de transição de outono. Durante as fases de transição e anestro, a incidência das ovulações diminui ou é nula, ou que dificulta a sincronização das ovulações entre doadoras e receptoras durante a preparação para a transferência de embriões. Alguns estudos mostram que as receptoras anovulatórias/acíclicas mantidas com protocolos com base em esteroides apresentam mudanças uterinas similares às que acontecem nas éguas gestantes. Contudo, não há pesquisas suficientes que permitam esclarecer o melhor protocolo de esteroides, no que se refere à dose, tempos de tratamento e vias de administração.

Abstract: This article discusses the main aspects of using hormonal protocols based on the application of estrogen followed by progestogen in anovulatory or acyclic recipients, either in anestrous or transition in embryo transfer protocols. Most mares demonstrate seasonally polyestrous behavior, by which they manifest estrus and ovulatory cycles during periods of higher daily luminosity. The annual reproductive cycle of mares is characterized by the presence of four phases defined by follicular dynamics: anestrous, spring transition, ovulatory, and autumn transition. During the phases of transition and anestrous, the incidence of ovulations decreases or is zero, making it difficult to synchronize ovulations between donors and recipients in preparation for embryo transfer. Some studies have shown that anovulatory/acyclic recipient mares managed with steroid-based protocols have similar uterine changes to those observed in pregnant mares. However, there is no sufficient research to allow to clarify the best steroid protocol with respect to dose, treatment times, and routes of administration.

Resumen: Este artículo discute los principales aspectos del uso de protocolos hormonales basados en la aplicación de estrógeno seguido de progestágeno en receptoras anovulatorias o acíclicas, sea en anestro o transición en los protocolos de transferencia de embriones. La mayoría de las yeguas presentan comportamiento poliéstrico estacional, con lo que manifiestan estro y ciclos ovulatorios en los periodos de mayor luminosidad diaria. El ciclo reproductivo anual de las yeguas es caracterizado por la presencia de cuatro fases definidas por la dinámica folicular: de anestro, de transición de primavera, ovulatoria y de transición de otoño. Durante las fases de transición y anestro, la incidencia de las ovulaciones disminuye o es nula, lo que dificulta la sincronización de las ovulaciones entre donadoras y receptoras durante la preparación para la transferencia de embriones. Algunos estudios han mostrado que las receptoras anovulatorias/acíclicas mantenidas con protocolos con base en esteroides presentan cambios uterinos similares a los que pasan en las yeguas gestantes. Sin embargo, no hay investigaciones suficientes que permitan aclarar el mejor protocolo de esteroides, con respecto a dosis, tiempos de tratamiento y vías de administración.

Rapamycin and fructose-1,6-bisphosphate reduce the HEPG2 cell proliferation via increase of free radicals and apoptosis.

Hepatocellular carcinoma is the most prevalent type of tumor among primary tumors affecting the liver. Rapamycin is currently used as a basis for chemotherapy in the treatment of cancers, including the liver. Because it shows several adverse effects, minimizing these effects without compromising efficacy is important. In this sense other drugs may be used concomitantly. One of these drugs is fructose-1,6-bisphosphate (FBP), which has shown therapeutic effect in various pathological situations, having antioxidant and anti-inflammatory proprieties. The objective of the present study was to evaluate the activity of rapamycin in combination with the FBP in HepG2 cell proliferation and the mechanisms involved. HepG2 cells were analyzed after 72 h of treatment with both drugs. Cell proliferation, cytotoxicity, cytokines, apoptosis, senescence, autophagy and oxidative stress were accessed. Ιt was demonstrated that the combination is more efficient than the single use of substances, because subtherapeutic doses of rapamycin, when associated to FBP become effective, reducing cell proliferation, through a significant increase in the production of tiobarbituric acid reactive substances (TBARS), suggesting that this might be the cause of death by apoptosis. According to these results, we believe that the association of both drugs may be a promising choice for the treatment of hepatocarcinoma.

Effect of chemotherapy on dietary glycemic index and load in patients with breast cancer and their relationships to body fat and phase angle.

Dietary glycemic index (GI) and glycemic load (GL) are indicators of carbohydrate consumption and widely used in studies evaluating the risk for breast cancer. However, the effect of chemotherapy on these indices has been scarcely studied. The aim of this study was to evaluate dietary levels of GI and GL in women with breast cancer during chemotherapy treatment and their relationships to body fat and phase angle. Twenty-five patients were assessed according to demographic, clinical, anthropometric, and food consumption data. Dietary intake was assessed by 24-h dietary recalls applied on nonconsecutive days. Anthropometric measures and body composition were determined at all study timepoints: prior to the first chemotherapy cycle (T0), immediately after the last chemotherapy cycle (T1), and 2 months after T1 (T2). There was no difference in mean GI and GL among study timepoints. However, a high prevalence of inadequate GI and GL values was noted, independent of study timepoint. GI and GL were associated with phase angle at T1. GI was associated with percentage fat at T0 only. Dietary GI and GL were unchanged during chemotherapy, but were associated with indicators of clinical outcome, such as percentage fat and phase angle.

Sequencing and phylogenetic analysis of the infectious bursal disease virus isolates from outbreak in layer flocks in the state of Minas Gerais.

Sequencing and phylogenetic analysis based on the nucleotide sequence of the gene encoding VP2 protein was carried out in order to characterize the agent of two outbreaks of infectious bursal disease in layer flocks in the state of Minas Gerais in 2004. The results indicate the outbreaks could be related to the vaccinal virus.