ß-Carotene Supplementation Improves Pancreas Function during Moderate Ethanol Consumption: Initial Characterization from a Morphological Overview.

Alcohol is believed to harm acinar cells, pancreatic ductal epithelium, and pancreatic stellate cells. After giving ethanol and/or ß-carotene to C57BL/6 mice, our goal was to evaluate their biochemistry, histology, and morpho-quantitative features. There were six groups of C57BL/6 mice: 1. Group C (control), 2. Group LA (low-dose alcohol), 3. Group MA (moderate-dose alcohol), 4. Group B (ß-carotene), 5. Group LA + B (low-dose alcohol combined with ß-carotene), and 6. Group MA + B (moderate-dose alcohol combined with ß-carotene). After the animals were euthanized on day 28, each specimen's pancreatic tissue was taken. Lipase, uric acid, and amylase were assessed using biochemical assessment. Furthermore, the examination of the pancreatic structure was conducted using Ammann's fibrosis scoring system. Finally, the morpho-quantitative characteristics of the pancreatic islets and acinar cells were determined. In the serum of the MA + B group, there were higher amounts of total amylase (825.953 ± 193.412 U/L) and lower amounts of lipase (47.139 ± 6.099 U/L) (p < 0.05). Furthermore, Ammann's fibrosis punctuation in the pancreas revealed significant variations between the groups (p < 0.001). Finally, the stereological analysis of pancreatic islets showed that the groups were different (p < 0.001). These findings suggest that antioxidant treatments might help decrease the negative effects of ethanol exposure in animal models.

Genotype analysis to clarify RhD variants in discrepant samples of Chilean population.

Introduction: The D antigen variants are classified as weak, partial, and extremely weak (DEL) and can be differentiated using molecular tests. In Chile, the laboratories of local blood centers do not identify variants of the D antigen, referring them for study to the Reference Laboratory of the Public Health Institute of Chile. So, our aim was to talk about the results of the molecular analysis of variants of the D antigen in samples that had different results in the serological classification. Methods: In the D antigen classification of the Rh system, 479 samples with serological discrepant results were sent for molecular analysis. The Rh phenotype was performed with monoclonal anti-C, anti-c, anti-E, and anti-e antisera by direct agglutination. To find the D antigen, researchers used direct agglutination with monoclonal antisera and indirect antiglobulin testing with the column (gel) agglutination method. Molecular analysis was performed with a polymerase chain reaction with sequence-specific primers (SSP-PCR) and sequencing. Results and discussion: The presence of D antigen variants was confirmed in 332 samples (69.3%), with an initial discrepancy in serological classification. In this group of discrepant samples, the frequency of weak RhD variants was 66% (219/332), that of extremely weak RhD was 28% (93/332), and that of partial RhD was 6% (20/332). The weak variants type 2 (27.4%), type 3 (8.4%), type 48 (8.4%), and type 1 (8.1%) were the next most prevalent variants after RHD*DEL43 (28%). The ccEe (R2r) phenotype was the most frequently detected (38.4%) and is present in 87% of the RHD*DEL43 samples. The E antigen is associated with the presence of this variant. Our analyses give the first description of D antigen variants in Chile. The most common variants are DEL type (RHD*DEL43) and weak (weak type 2), which are linked to the ccDEe (R2r) phenotype. These findings allow us to characterize the variants of the D antigen in Chile and, according to the obtained data, to design strategies for the management of donors, patients, and pregnant women.

Characterization of the Germplasm Bank for the Spanish Autochthonous Bull Breed "Asturiana de la Montaña".

Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm's viability. This study reports an investigation of the "Asturiana de la Montaña" breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4-6 mL, 5-10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26-43%, progressive 14-28%), with higher values for viability (47-62%). Insemination results showed a good performance for this breed (NRR: 47-56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results.

Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation.

BACKGROUND: The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. OBJECTIVES: In this study, we analyzed the response of capercaillie sperm to the freezing-thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. METHODS: We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl-acetamide (DMA) v:v] of cryoprotectants were evaluated using in-straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. RESULTS: In chicken, we found that the LR84-4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. CONCLUSIONS: Our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.

Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta.

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8-71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field fertility.

Semen banking is critical to preserving rare and autochthonous breeds. However, protocols can change with time, leaving heterogeneous semen batches. The objective of this study was to assess differences in sperm quality and field fertility. We report differences between batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were analysed by CASA and flow cytometry. The 85-days non-return rates from AI records were used to assess the fertility of 23,853 AI. BIOXCell showed higher quality post-thawing. Differences increased after a 5-hr incubation at 37°C, and Biociphos yielded doses with lower resilience. Field fertility did not differ between extenders (Biociphos: 57.4% ± 1.2; BIOXCell: 56.6% ± 3.0), possibly because of AI protocols compensating for differences in quality. However, this needs to be confirmed by controlled intervention studies. In conclusion, batches frozen with Biociphos may require specific strategies for compensating for the lower sperm quality. Regular surveys and evaluation of cryobank procedures may be useful to characterizing stored batches and defining strategies to guaranteeing success in their future use.

Síndrome de Lambert - Eaton: Características clínicas y neurofisiológicas / Lambert Syndrome - Eaton: Clinical and neurophysiological characteristics

Resumen: El síndrome miasténico de Lambert Eaton (LEMS) es una enfermedad de la transmisión neuromuscular pre sináptica poco frecuente e infra diagnosticada, con síntomas clínicos característicos aunque poco específicos pero hallazgos neurofisiológicos muy típicos. En la mayor parte de los casos se presenta como un síndrome paraneoplásico aunque existen formas idiopáticas. Describimos el caso de un paciente que se presentó con un cuadro clínico compatible y hallazgos neurofisiológicos contundentes que permitieron realizar el diagnóstico. Si bien no se encontró una neoplasia oculta en el tamizaje inicial es fundamental el seguimiento clínico-paraclínico por su elevada asociación con la misma. La evolución fue satisfactoria con tratamiento sintomático. Discutimos la fisiopatología del trastorno, estableciendo, como hecho fundamental la presencia de anticuerpos contra los canales de calcio voltaje dependientes pre sinápticos, siendo este hecho fundamental para la comprensión del tratamiento sintomático planteado. Repasamos las características que se asocian a mayor riesgo de neoplasia oculta y planteamos la estrategia de seguimiento para despistarla precozmente.

Abstract: Lambert Eaton myasthenic syndrome (LEMS) is an uncommon neuromuscular joint disease, usually infra diagnosed, with characteristic but nonspecific clinical symptoms but very typical neurophysiological findings. In most cases it is a paraneoplastic syndrome although idiopathic forms also exist. We describe the case of a patient with typical clinical presentation and neurophysiological findings that allowed the diagnosis. The initial screening for hidden malignancy was negative, but the follow up is essential to rule it out given its strong association. The evolution was satisfactory with symptomatic treatment. We discuss the pathophysiology of the disorder, establishing as fundamental fact, the presence of antibodies against presynaptic voltage-dependent calcium channels, which is essential to understand the symptomatic treatment. We review the features that are associated with increased risk of hidden malignancy and propose a follow-up strategy to throw it off early.

Expression and localization of interleukin 1 beta and interleukin 1 receptor (type I) in the bovine endometrium and embryo.

The interleukin-1 (IL1) system likely mediates mammalian embryo-maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium.

Frequency and specificity of red blood cell alloimmunization in chilean transfused patients.

BACKGROUND: Alloimmunization is an adverse effect of blood transfusions. In Chile, alloimmunization frequency is not established, and for this reason the aim of this study was to investigate the prevalence and specificity of red blood cell (RBC) alloantibodies in Chilean transfused subjects. METHODS: Records from 4,716 multi-transfused patients were analyzed. In these patients, antibody screening was carried out prior to cross-matching with a commercially available two-cell panel by the microcolum gel test, and samples with a positive screen were analyzed for the specificity of the alloantibody with a 16-cell identification panel. RESULTS: The incidence of RBC alloimmunization in transfused patients was 1.02% (48/4,716) with a higher prevalence in women (40/48). We detected 52 antibodies, the most frequent specificities identified were anti-E (30.8%), anti-K (26.9%), anti-D (7.7%), and anti-Fy(a) (5.8%). The highest incidence of alloantibodies was observed in cancer and gastroenterology patients. CONCLUSION: The data demonstrated a low alloimmunization frequency in Chilean transfused patients, principally associated with antibodies anti-E, anti-K, anti-D, and anti-Fy(a).

Efficacy of ezetimibe is not related to NPC1L1 gene polymorphisms in a pilot study of Chilean hypercholesterolemic subjects.

BACKGROUND AND OBJECTIVE: Niemann-Pick C1 Like 1 (NPC1L1) is a multi-transmembrane transport protein highly expressed in the small intestine. It mediates sterol transfer throughout the brush border membrane of enterocytes, becoming essential for intestinal cholesterol absorption and ensuing whole-body cholesterol homeostasis. This protein is targeted by ezetimibe, a potent cholesterol absorption inhibitor. Single nucleotide polymorphisms (SNPs) in NPC1L1 have been associated to variation in both plasma low-density lipoprotein (LDL) cholesterol levels and lipid-lowering medication with ezetimibe. However, there are no data evaluating the impact of NPC1L1 variants on Chilean subjects medicated with ezetimibe monotherapy. Therefore, we assessed the contribution of two unexplored NPC1L1 variants on plasma lipids and response to ezetimibe in Chilean hypercholesterolemic individuals. METHODS: Using PCR-restriction fragment length polymorphism (RFLP), we analyzed the SNP distribution of two common variants; -133A>G (rs17655652) and 1679C>G (rs2072183), and their relation with plasma lipids and lipid-lowering response to ezetimibe in 60 hypercholesterolemic Chilean subjects. RESULTS: Genotype distribution for the rs17655652 variant was AA 57 %, 40 % AG and 3 % GG, whereas for the rs2072183 SNP was 57 % CC, 35 % CG and 8 % GG. Minor allele frequencies (MAFs) were 0.23 and 0.26, respectively. No association was observed between NPC1L1 SNPs and baseline cholesterol. After therapy, none of the polymorphisms affected ezetimibe response in the studied cohort (P > 0.05). CONCLUSION: Data obtained indicates that polymorphisms rs17655652 and rs2072183 were not related to cholesterol variability. Also, lipid-lowering response to ezetimibe is not impacted by the NPC1L1 polymorphisms studied in Chilean hypercholesterolemic subjects.

Metabolomic prediction of pregnancy viability in superovulated cattle embryos and recipients with fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

Embryonic sex induces differential expression of proteins in bovine uterine fluid.

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.

Prevalence and correlates of cognitive impairment in adults with subjective memory complaints in primary care centres.

AIMS: To examine the prevalence and correlates of cognitive impairment (CI) in adults over 50 years old attending primary care centres with complaints of memory failure. METHODS: A sample of 580 individuals aged 50+ years with no previous diagnosis of dementia was assessed by use of the Mini Mental State Examination, the Cambridge Cognitive Assessment-Revised and the California Verbal Learning Test - to evaluate CI-dependent variables - and administration of a questionnaire on memory complaints and other instruments - to measure correlates. RESULTS: The prevalence of CI was 46.20% and positive associations were found for age, gender, level of education, subjective memory complaints, instrumental activities of daily living, reading habits and frequency of leisure activities. In the logistic regression, modelled CI was associated with older age, gender (49.12% women, 39.66% men), instrumental activities of daily living, and reading habits. CONCLUSION: Almost half of the adults aged 50+ years attending primary care centres with subjective memory complaints were affected by CI. Early evaluation of cognitive functioning is essential to establish adequate preventive and intervention strategies.

La respuesta terapéutica a ezetimiba en ratones C57BL/6 es mediada por cambios en la expresión de NPC1L1, ABCG5 y ABCG8 en el enterocito / Therapeutic response to ezetimibe in C57BL/6 mice is mediated by changes in NPC1L1, ABCG5 and ABCG8 expression in the enterocyte

Las proteínas NPC1L1, ABCG5 y ABCG8 participan en la absorción intestinal de colesterol. Ezetimiba inhibe este proceso bloqueando a NPC1L1, sin embargo, su efecto sobre ABCG5 y ABCG8 aún no está claro. Así, el objetivo del presente trabajo fue evaluar en ratones C57BL/6 con hipercolesterolemia inducida por dieta y tratados con ezetimiba, la expresión de NPC1L1, ABCG5 y ABCG8 mediante PCR en tiempo real y Western blot, en 3 grupos de animales: 1, dieta hipercolesterolémica D12336; 2, dieta D12336 más 5 mg/kg/día de ezetimiba; 3, dieta control. El nivel sérico de colesterol total fue significativamente diferente entre los grupos estudiados (control: 1,85 +/- 0,49 mmol/L; dieta D12336: 3,11 +/- 0,73 mmol/L; ezetimiba: 2,11 +/- 0,50 mmol/L, P = 0,001). La expresión génica de NPC1L1 aumentó 5,4 veces en el grupo que recibió la dieta D12336 (P = 0,003). Por otro lado, la expresión génica de ABCG5 y ABCG8 no fue diferente en el grupo con hipercolesterolemia (P = 0,239 y P = 0,201, respectivamente). Después del tratamiento con ezetimiba, la expresión génica de ABCG5 se incrementó 15,6 veces (P = 0.038). No hubo diferencias significativas en la expresión génica de NPC1L1 (P = 0,134) y ABCG8 (P = 0,067). En relación a la expresión proteica, la dieta D12336 incrementó los niveles de expresión de NPC1L1 (P = 0,022) y ABCG5 (P = 0,008); el tratamiento con ezetimiba incrementó los niveles de NPC1L1 (P = 0,048) y redujo los niveles de ABCG5 (P = 0,036) y ABCG8 (P = 0,016). En conclusión, nuestros resultados sugieren que tanto la dieta hipercolesterolémica como el tratamiento con ezetimiba, en un modelo experimental, afectan los niveles de expresión de NPC1L1, ABCG5 y ABCG8, sugiriendo que ABCG5 y ABCG8 están involucrados en la respuesta hipolipemiante a este fármaco. No obstante, el mecanismo mediante el cual se explica esta interacción requiere de un futuro estudio.

Proteins NPC1L1, ABCG5 and ABCG8 are involved in the intestinal absorption of cholesterol. Ezetimibe inhibits this process by blocking NPC1L1, however, its effect on ABCG5 and ABCG8 is not yet clear. Thus, the objective of this study was to evaluate in C57BL / 6 mice with diet-induced hypercholesterolemia treated with ezetimibe, the expression of NPC1L1, ABCG5 and ABCG8 by real time PCR and Western blot, in 3 groups of animals: 1, diet hypercholesterolemic D12336, 2, D12336 diet plus 5 mg/kg/ day of ezetimibe, 3, diet control. The serum level of total cholesterol was significantly different between groups (control: 1.85 +/- 0.49 mmol / L; diet D12336: 3.11 +/- 0.73 mmol / L; ezetimibe: 2.11 +/- 0.50 mmol / L, P = 0.001). NPC1L1 gene expression increased 5.4-fold in the group receiving the diet D12336 (P = 0.003). Furthermore, the gene expression of ABCG5 and ABCG8 was not different in the group with hypercholesterolemia (P = 0.239 and P = 0.201, respectively). After treatment with ezetimibe, ABCG5 gene expression was increased 15.6 times (P = 0.038). No significant differences in gene expression of NPC1L1 (P = 0.134) and ABCG8 (P = 0.067). Regarding protein expression, the D12336 diet increased the levels of expression of NPC1L1 (P = 0.022) and ABCG5 (P = 0.008), treatment with ezetimibe increased the levels of NPC1L1 (P = 0.048) and reduced levels of ABCG5 (P = 0.036) and ABCG8 (P = 0.016). In conclusion, our results suggest that both hypercholesterolemic diet as treatment with ezetimibe, in an experimental model, affect the expression levels of NPC1L1, ABCG5 and ABCG8, suggesting that ABCG5 and ABCG8 are involved in lipid-lowering response to this drug. However, the mechanism by which this interaction is explained requires further study.

Effect of five single nucleotide polymorphisms of ABCG5 and ABCG8 genes on eLipid-lowering response / Efecto de cinco polimorfismos de los genes ABCG5 y ABCG8 sobre la respuesta hipolipemiante a ezetimiba

In this study we evaluated the possible association between five single nucleotide polymorphisms in ABCG5 (rs6720173) and ABCG8 (rs11887534, rs4148211, rs4148217 and rs6544718) genes and ezetimibe response in Chilean hypercholesterolemic subjects. A total of 60 non-related hypercholesterolemic subjects, aged 18 to 65 years old were included in this study. These subjects were treated with ezetimibe (10mg/day) during one month. The ABCG5 and ABCG8 genotypes were assessed by PCR-RFLP. The genotype distribution of the ABCG5/ABCG8 polymorphisms was in Hardy-Weinberg equilibrium. Our results showed that the investigated polymorphisms were not associated with the response to ezetimibe. Nevertheless, the T allele of rs6544718 polymorphism was related to higher baseline levels of LDL-cholesterol (p<0.001). In addition, the G allele for the rs4148211 polymorphism was associated with greater baseline concentrations of triglycerides (P=0.019). This allele was also associated with lower concentrations of HDL-cholesterol (P=0.027), after ezetimibe treatment. Our results suggest that the studied polymorphisms do not affect the therapeutic response to ezetimibe in the evaluated subjects.

En este estudio se evaluó la posible asociación entre cinco polimorfismos de nucleótido único en los genes ABCG5 (rs6720173) y ABCG8 (rs11887534, rs4148211, rs4148217 y rs6544718) y la respuesta a ezetimiba en pacientes hipercolesterolémicos chilenos. Un total de 60 individuos hipercolesterolemicos, no relacionados, con edades entre 18 y 65 años fueron incluidos. Estos sujetos fueron tratados con ezetimiba (10mg/día) durante un mes. Los genotipos de ABCG5 y ABCG8 fueron evaluados por PCR-RFLP. La distribución de genotipos de los polimorfismos de ABCG5/ABCG8 se encontraba en equilibrio de Hardy-Weinberg. Nuestros resultados mostraron que los polimorfismos estudiados no se asociaron con la respuesta a la ezetimiba. Sin embargo, el alelo T del polimorfismo rs6544718 fue relacionado con niveles basales elevados de LDL-colesterol (p <0,001). Además, el alelo G para el polimorfismo rs4148211 se asoció con una mayor concentración basal de triglicéridos (p = 0,019). Este alelo también se asoció con concentraciones más bajas de HDL-colesterol (p = 0,027), después del tratamiento con ezetimiba. Nuestros resultados sugieren que los polimorfismos estudiados no afectan a la respuesta terapéutica a la ezetimiba en los sujetos evaluados.

Efficient derivation of bovine embryonic stem cells needs more than active core pluripotency factors.

Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.

Proteome of the early embryo-maternal dialogue in the cattle uterus.

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

TP53 codon 72 polymorphism is associated with coronary artery disease in Chilean subjects.

OBJECTIVE: To investigate the possible association between the codon 72 polymorphism (Pro72Arg, rs1042522) of the tumor suppressor gene (TP53) and the presence of coronary artery disease (CAD) in Chilean subjects. SUBJECTS AND METHODS: A total of 209 unrelated patients with a diagnosis of CAD confirmed by angiography (33-74 years old) and 216 healthy controls (30-68 years old) were included in this study. The Pro72Arg polymorphism of the TP53 gene was evaluated by PCR-RFLP. RESULTS: The genotype distribution for the Pro72Arg variant of the TP53 gene in CAD patients (PP: n = 13, 6.2%; PR: n = 61, 29.4%; RR: n = 135, 64.6%) and controls (PP: n = 18, 8.3%; PR: n = 94, 43.5%; RR: n = 104, 48.1%) was significantly different (p = 0.003). Similarly, the allelic frequency was also different (p = 0.003). The odds ratio for CAD related to the 72Arg allele was 2.0 (95% CI = 1.33-2.90), confirming the presence of an association. CONCLUSION: These findings suggest that the Pro72Arg polymorphism of the TP53 gene is associated with CAD in Chilean individuals.