Identification of phthalate mixture exposure targets in the human and mouse ovary in vitro.

Chemical health risk assessment is based on single chemicals, but humans and wildlife are exposed to extensive mixtures of industrial substances and pharmaceuticals. Such exposures are life-long and correlate with multiple morbidities, including infertility. How combinatorial effects of chemicals should be handled in hazard characterization and risk assessment are open questions. Further, test systems are missing for several relevant health outcomes including reproductive health and fertility in women. Here, our aim was to screen multiple ovarian cell models for phthalate induced effects to identify biomarkers of exposure. We used an epidemiological cohort study to define different phthalate mixtures for in vitro testing. The mixtures were then tested in five cell models representing ovarian granulosa or stromal cells, namely COV434, KGN, primary human granulosa cells, primary mouse granulosa cells, and primary human ovarian stromal cells. Exposures at epidemiologically relevant levels did not markedly elicit cytotoxicity or affect steroidogenesis in short 24-hour exposure. However, significant effects on gene expression were identified by RNA-sequencing. Altogether, the exposures changed the expression of 124 genes on the average (9-479 genes per exposure) in human cell models, without obvious concentration or mixture-dependent effects on gene numbers. The mixtures stimulated distinct changes in different cell models. Despite differences, our analyses suggest commonalities in responses towards phthalates, which forms a starting point for follow-up studies on identification and validation of candidate biomarkers that could be developed to novel assays for regulatory testing or even into clinical tests.

Derivation of Bovine Primed Embryonic Stem Cells from Somatic Cell Nuclear Transfer Embryos.

Derivation of bovine embryonic stem cells from somatic cell nuclear transfer embryos enables the derivation of genetically matched pluripotent stem cell lines to valuable and well-characterized animals. In this chapter, we describe a step-by-step procedure for deriving bovine embryonic stem cells from whole blastocysts produced by somatic cell nuclear transfer. This simple method requires minimal manipulation of blastocyst-stage embryos, relies on commercially available reagents, supports trypsin passaging, and allows the generation of stable primed pluripotent stem cell lines in 3-4 weeks.

Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells.

Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

Importance of WNT-dependent signaling for derivation and maintenance of primed pluripotent bovine embryonic stem cells†.

The WNT signaling system plays an important but paradoxical role in the regulation of pluripotency. In the cow, IWR-1, which inhibits canonical WNT activation and has WNT-independent actions, promotes the derivation of primed pluripotent embryonic stem cells from the blastocyst. Here, we describe a series of experiments to determine whether derivation of embryonic stem cells could be generated by replacing IWR-1 with other inhibitors of WNT signaling. Results confirm the importance of inhibition of canonical WNT signaling for the establishment of pluripotent embryonic stem cells in cattle and indicate that the actions of IWR-1 can be mimicked by the WNT secretion inhibitor IWP2 but not by the tankyrase inhibitor XAV939 or WNT inhibitory protein dickkopf 1. The role of Janus kinase-mediated signaling pathways for the maintenance of pluripotency of embryonic stem cells was also evaluated. Maintenance of pluripotency of embryonic stem cells lines was blocked by a broad inhibitor of Janus kinase, even though the cells did not express phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Further studies with blastocysts indicated that IWR-1 blocks the activation of pSTAT3. A likely explanation is that IWR-1 blocks differentiation of embryonic stem cells into a pSTAT3+ lineage. In conclusion, results presented here indicate the importance of inhibition of WNT signaling for the derivation of pluripotent bovine embryonic stem cells, the role of Janus kinase signaling for maintenance of pluripotency, and the participation of IWR-1 in the inhibition of activation of STAT3.

Similarities between bovine and human germline development revealed by single-cell RNA sequencing.

The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes and exist transiently during development until they differentiate into oogonia in females, or spermatogonia in males. Little is known about the molecular characteristics of primordial germ cells in cattle. By performing single-cell RNA-sequencing, quantitative real-time PCR, and immunofluorescence analyses of fetal gonads between 40 and 90 days of fetal age, we evaluated the molecular signatures of bovine germ cells at the initial stages of gonadal development. Our results indicate that at 50 days of fetal age, bovine primordial germ cells were in the early stages of development, expressing genes of early primordial germ cells, including transcriptional regulators of human germline specification (e.g. SOX17, TFAP2C, and PRDM1). Bovine and human primordial germ cells also share expression of KIT, EPCAM, ITGA6, and PDPN genes coding for membrane-bound proteins, and an asynchronous pattern of differentiation. Additionally, the expression of members of Notch, Nodal/Activin, and BMP signaling cascades in the bovine fetal ovary, suggests that these pathways are involved in the interaction between germ cells and their niche. Results of this study provide insights into the mechanisms involved in the development of bovine primordial germ cells and put in evidence similarities between the bovine and human germline.

Changes in testicular gene expression following reduced estradiol synthesis: A complex pathway to increased porcine Sertoli cell proliferation.

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Derivation of sheep embryonic stem cells under optimized conditions.

Until recently, it has been difficult to derive and maintain stable embryonic stem cells lines from livestock species. Sheep ESCs with characteristics similar to those described for rodents and primates have not been produced. We report the derivation of sheep ESCs under a chemically defined culture system containing fibroblast growth factor 2 (FGF2) and a tankyrase/Wnt inhibitor (IWR1). We also show that several culture conditions used for stabilizing naïve and intermediate pluripotency states in humans and mice were unsuitable to maintain ovine pluripotency in vitro. Sheep ESCs display a smooth dome-shaped colony morphology, and maintain an euploid karyotype and stable expression of pluripotency markers after more than 40 passages. We further demonstrate that IWR1 and FGF2 are essential for the maintenance of an undifferentiated state in de novo derived sheep ESCs. The derivation of stable pluripotent cell lines from sheep blastocysts represents a step forward toward understanding pluripotency regulation in livestock species and developing novel biomedical and agricultural applications.

Livestock pluripotency is finally captured in vitro.

Pluripotent stem cells (PSCs) have demonstrated great utility in improving our understanding of mammalian development and continue to revolutionise regenerative medicine. Thanks to the improved understanding of pluripotency in mice and humans, it has recently become feasible to generate stable livestock PSCs. Although it is unlikely that livestock PSCs will be used for similar applications as their murine and human counterparts, new exciting applications that could greatly advance animal agriculture are being developed, including the use of PSCs for complex genome editing, cellular agriculture, gamete generation and invitro breeding schemes.

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Pluripotent stem cells and livestock genetic engineering.

The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.

Coordination modes and different hapticities for fullerene organometallic complexes.

The different coordination modes in fullerene organometallic complexes are reviewed. The main modes are η2 and η5, but there are some interesting studies about the other four, all of them are revised in order to show which is the state of art of this kind of compounds with the respect of the hapticity.

[Chemical and sensory characterization of cakes enriched with dietary fiber and micronutrient for the elderly]. / Caracterización química y sensorial de biscochuelos enriquecidos con fibra dietética y micronutrientes para el anciano.

This study presents the development of individual cakes enriched with dietary fiber (lupin and oat fiber), vitamins and minerals; as recent research has shown that the average daily dietary fibre intake of the elderly population in Chile is only 12 g. Each cake contains 4.8 g of dietary fibre and a 30% of the RDA of vitamins A, B1, B2, B6, B12, E, nicotinamide and folic acid, 40% of the RDA of vitamin D3, 15% of the RDA of calcium, 12% of the RDA of magnesium and 3% the RDA of zinc. Polydextrose and sorbitol were added to improve flavor and texture. Response Surface Methodology (RSM) was used for optimization, based on a two-variable composite design. Thirteen experimental runs were carried out, with polydextrose (1 to 40% based on flour content) and sorbitol (1 to 30% based on flour content) as independent variables. The response variable was sensory quality obtained by the Karlsruhe test. Sensory attributes of texture and overall quality showed a good fitting with high determination coefficients and were used for optimization. The optimized cake contained 11.5% polydextrose and 4.4% sorbitol (both based on flour content). Quality of the optimized product was controlled by means of physical, chemical, microbiological and sensory analyses. Overall sensory quality was 8.18 ("very good") and good quality and nutritive value were achieved. In an acceptance test carried out with 150 adults, 100% acceptability was obtained in the hedonic scale categories "like it" and "like it very much". A shelf life study performed with cakes packaged in polypropylene bags of 30 microns thickness indicated a shelf life of 13 days stored at room conditions (25 degrees C and 55-60% R.H.).

Determinacion de la variacion de materia organiza segun el uso de la tierra en la comunidad Obrajes-Tarija con apoyo de SIG / sss

Segun el uso actual de la tierra en la comunidad e Obrajes, se evaluaron 139 Has., el resto no evaluado comprende tierras micelaneas, complejo de colinas montañosas, llanura lacustre donde estan los bad lands y el area ocupada por el rio Guadalquivir. Al realizar el analisis de valoracion de la materia organiza de los perfiles representativos de la capa arable se concluye que los niveles de materia organiza del suelo y su variacion, se considera como indicadores de la interaccion entre el uso actual de la tierra y segun a la aplicacion del sistema de cultivo empleado frente al medio ambiente. Es evidente que la perdida directa mas importante de la fraccion organiza de los suelos agricolas va unida a la perdida de suelo (erosion) ya que precisamente es el horizonte superficial donde encontramos mayor proporcion de materia organiza de mayor calidad (por el tipo de descomposicion) y una textura favorable para el desarrollo de la vegetacion..

[Optimization of a spaghetti formula enriched with dietary fiber and micronutrients for elderly people]. / Optimización de una formulacion de espaguetis enriquecidos con fibra dietética y micronutrientes para el adulto mayor.

Several studies have demonstrated low dietary fiber intake in elderly people, which increases the risk of diseases such as constipation, colon cancer and diverticulosis. A spaghetti formula enriched with lupin fibre was developed to increase the dietary fibre intake in elderly people, as spaghetti are frequently consumed in this age group. Sweet lupin bran (Vitafiber) was used as fibre source and gluten was used as improving additive. Response surface methodology with a two variable composite rotatable design was applied to optimize the formulations. The independent variables were lupin bran (7.14-14.29%) and gluten Vital (0.1-2.0%). The dependent variables were the responses of a trained 10-member sensory panel who evaluated the sensory quality parameters color, shape, aroma, flavor and texture by the Karlsruhe 9-point test. The optimized formula was prepared with 66.7% semoline, 7.14% lupin bran, 1.05% gluten and 24.7% water, enriched with 0.019% of a vitamin premix (A. E, D, B2, B12 and folic acid) and with 0.41% of a mineral premix (Ca, Fe, Zn), in order to meet 30% of the RDA for the elderly per 100 g dry spaghetti. The dietary fibre content of the optimized product was 11.05 g/100 g. The study showed that fibre-enriched spaghetti formula is a good way to increase dietary fibre intake in elderly people, as it is a common food, simple to prepare and easy to eat.

Optimización de ua formulación de espaguetis enriquecidos con fibra dietética y micronutrientes para el adulto mayor / Optimization od a spaghetti formula enriched with dietary fibre and micronutrients for elderly people

Numerosos estudios han demostrado que debido a diferentes causas, existe un déficit en la ingesta de fibra en la población adulta mayor, la cual puede causar diferentes patologías tales como constipación, cáncer de colon, diverticulosis. Para ofrecer alternativas de solución a esta realidad se desarrollaron formulaciones de espaguetis enriquecidos con fibra dietética, por ser un alimento de consumo habitual. La fibra usada fue harina de salvado de lupino dulce (Vitafiber) y se empleó gluten (Vital de trigo 75 por ciento) como aditivo mejorador. Se usó el método de superficie de respuesta para optimizar la formulación, con un diseño rotacional compuesto, con dos variables, ensayando concentraciones entre 7,14 y 14,29 por ciento de harina de salvado de lupino dulce y 0,1 a 2,0 por ciento de gluten como variables independientes. Las variables dependientes fueron las respuestas de 10 jueces entrenados que evaluaron los diferentes parámetros de calidad sensorial (color, forma, olor, sabor y textura) con el test de Karlsruhe de 9 puntos. La formulación optimizada correspondió a la eleborada con 66,7 por ciento de sémola, 7,14 por ciento de harina de salvado de lupino dulce, 24,7 por ciento de agua y 1,05 por ciento de gluten. Este producto optimizado fue enriquecido con 0,019 por ciento de un premix de vitaminas (A;E;B2,D3 y ácido fólico) y 0,41por ciento de minerales (Ca,Fe,Zn) de forma tal que 100 g de fideos secos cumplieran con aportar alrededor del 30 por ciento de las recomendaciones de la ODR para adultos mayores. El producto final contiene 11,05 g/100g de fibra dietética total, 984 UI de vitamina A, 4,52 UI de vitamina E y 0,38 mg de vitamina B, por 100g y 208 mg de Calcio 3,16 mg de Hierro y 4,8 mg de Zinc, por 100g, respectivamente. Se concluye que el producto elegido es buen vehículo para aumentar el consumo de fibra dietética, por ser un alimento de uso habitual, de preparación simple y de fácil consumo

Elaboración y optimización de un producto de repostería para el adulto mayor / Manufacture and optimization of a pastry product for the elderly

Los adultos mayores presentan por lo general una gran tendencia a la malnutrición, ya sea porque sus requerimientos son diferentes a los del resto de la población o porque no tienen la capacidad de cubrirlos satisfactoriamente. Con el fin de ayudar a reestablecer y matener la salud de este grupo etáreo, se decidió elaborar y optimizar un producto de pastelería hipocalórico relleno con crema, que fuera liviano y fácil de ingerir incluso para adultos mayores con problemas dentales y también para aquellos con problemas de obesidad o diabetes. La crema fue suplementada con vitaminas y sales minerales en forma de premix, aportando cada uno el 10 por ciento de la ingesta diaria recomendada. El premix de sales minerales se compone de sulfato de zinc y hierro en forma de óxido sacaratado; y el premix de vitaminas, de vitaminas D,E,B2,B6,B12, Niacina y ácido fólico. El producto optimizado fue controlado a través de análisis físicos, químicos, microbiológicos y sensoriales, determinándose que su calidad corresponde a Grado 1, con una vida útil de 5 días en almacenamiento refrigerado 3-5ªC y 65-70 por ciento de humedad relativa. Se realizaron pruebas de aceptabilidad y tolerancia con grupos de adultos mayores, encontrándose un 98 por ciento de aceptación y ausencia de efectos secundarios

Pérfil profesional del nutricionista en Chile / Professional profile of the nutritionist in Chile

Se estudió el perfil y las características del ejercicio profesional del nutricionista en Chile, a través de una encuesta que se aplicó a una muestra del 20 por ciento de los profesionales (335) en 10 regiones del país, incluyendo la Región Metropolitana. Se usó la metodología propuesta por OPS/INCAP y ACODIN adaptada a la realidad nacional. La información se agrupó en 4 campos ocupacionales: nutricionistas clínicas (25,1 por ciento ); atención primaria (18,2 por ciento ); alimentación institucional (13,3 por ciento ); nutricionistas académicos (80,0 por ciento ). Se observó que un 17,0 por ciento de los profesionales trabajan en instituciones gubernamentales, hospitales, como también en instituciones responsables de la alimentación institucional. Los empleadores privados ocupan más del 50 por ciento de sus profesionales en atención primaria e industria. Los salarios más bajos es encontraron en hospitales y atención primaria. La necesidad de capacitación para la labor administrativa fue enfatizada por un 47 por ciento de la muestra. Autoestima y autonomía en las decisiones fue señalado como importantes elementos que es necesario mejorar. Los conocimientos en planificación en los campos institucionales deben ser mejorados si estos resultados se aplican a la enseñanza formal