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Se presentan antecedentes de manejo de regeneración en 2 bosques de pino radiata afectados por los incendios del año 2017 en la comuna de Florida, región del Biobío, donde se realizó manejo utilizando desbrozadora, herbicidas y herramientas manuales. Los resultados indican que a los 4 años de aplicados los tratamientos de manejo, el control manual sigue presentando los menores valores de crecimiento en diámetro y altura de las plantas, mientras que la utilización del control químico continúa siendo el mejor tratamiento, con diferencias estadísticamente significativas en relación a los tratamientos restantes, tanto para el DAP como para la altura. La utilización de desbrozadora o el método manual, no siempre permiten eliminar la totalidad de las especies que no son de interés, por lo que generan mayor competencia por nutrientes, luz y agua; estos métodos tampoco limitan el desarrollo de regeneración natural a través del tiempo, de modo que es necesario repetirlos en el tiempo para mantener el rendimiento deseado. Los resultados iniciales confirman la potencialidad del manejo de la regeneración natural post incendio de bosques de pino radiata como herramienta de gestión forestal para el repoblamiento de bosques productivos con esta especie. Se requiere de evaluaciones permanentes que permitan determinar el efecto de los diferentes tratamientos utilizados sobre el crecimiento y productividad del rodal en distintas etapas de la rotación, información necesaria para la toma de decisiones en relación a la recuperación de plantaciones afectadas por incendios forestales, especialmente en el sector de pequeños y medianos propietarios.

En Chile la producción, demanda y consumo del pellet presenta un permanente crecimiento, utilizando en este proceso subproductos derivados principalmente de la industria del aserrío y remanufactura de pino radiata. El crecimiento de la industria del pellet se deriva de problemas de contaminación ambiental atribuibles al mal uso de la leña, a programas de descontaminación, comodidad de su uso y por cambios culturales de los usuarios. En la producción de pellet, la calidad y cantidad de biomasa forestal es fundamental, permitiendo reducir emisiones de CO2 (mitigación) y con ello, garantizar el rendimiento y eficiencia. Una gran contrariedad de la industria del pellet es la incertidumbre respecto al abastecimiento de biomasa en cantidad y calidad adecuada, para responder a la demanda, por cuanto la variación en la actividad del aserrío afecta directamente a la disponibilidad de subproductos que son la materia prima para el pellet. En apoyo al fortalecimiento de la industria del pellet en Chile, se presentan antecedentes respecto a disponibilidad y características de los subproductos que son potencialmente utilizables como materia prima, sus perspectivas, y opciones para suplir los mayores requerimientos de biomasa. Lo anterior con el objetivo de favorecer el desarrollo de las energías renovables y contribuir a la diversificación de la matriz energética, a partir de las potencialidades del territorio forestal del país. Se concluye que la estrecha relación entre la industria maderera y la productora de pellet, representa un escenario donde esta última actualmente tiene un límite que está dado por la oferta de biomasa de subproductos proveniente de la industria del aserrío.

La búsqueda de opciones eficientes de manejo forestal para especies de rápido crecimiento es una de las líneas de trabajo del Instituto Forestal, investigando opciones en especial para rodales de pequeños y medianos propietarios forestales. La capacidad de retoñación y crecimiento de los retoños de Eucalyptus globulus generan una opción en este sentido, por lo que se presentan antecedentes de rodales de Eucalyptus globulus manejados como monte bajo en la Región del Biobío, en las comunas de Cañete y Copiulemu. A los 6 años, los rodales logran entre 8 y 19 metros de altura y diámetros de 5,9 a 12,5 cm, cifras similares a lo reportado para Eucalyptus globulus originados por semillas creciendo en las mismas condiciones. Se observan valores de 1.320 a 2.500 árboles por hectárea, en una situación de uno o dos retoños por tocón. Las parcelas con un retoño presentan DAP, altura y volumen mayores con respecto a las parcelas con manejo de dos varas. En cuanto a volumen, se proyecta que este puede alcanzar de 140 a 270 m3/ha a los 13 años. Ello es un indicar del comportamiento de este tipo de rodales, dependiendo de las condiciones de sitio, características del rodal y manejo realizado. Se recomienda la mantención de las unidades experimentales para aumentar la base de datos de trabajo, abarcando, además, otras áreas geográficas.

Se detalla la normativa nacional que regula la producción de pellet en Chile y se contrasta con las normativas análogas usadas en Europa. Las normas nacionales son voluntarias y similares en sus alcances a las europeas, coincidiendo en gran medida en cuanto a la definición de parámetros de calidad y clasificación del pellet. Sin embargo, existen diferencias importantes en cuanto a la implementación de procesos de certificación que acrediten el cumplimiento de la normativa.

Introduction:An accident at work is defined as “an event that occurs during working hours or in connection with it that causes an injury”. Therefore, the objective of the study is to characterize occupational accidents in health workers at a hospital during a 3-year follow-up.Methodology:A continuation and expansion of the study "Characterization of occupational accidents in workers of a highly complex hospital in Bogotá D.C. Colombia in the period between January 2020 – June 2022”. Through a descriptive analysis of occupational accidents based on information obtained from a database of occupational accidents in the period between January 2020 and December 2022. A proposal was made for a device to reduce accidents with the support of the department of industrial design.Results:The accident rate was higher in the female population (78.42%). The age range in which the most reports of occupational accidents were presented is 30–39 years (37.48%). The year with the highest number of accidents was 2020 with 268 cases. The position with the highest accident rate was that of health personnel representing (76.21%). The most frequent type of accident was the non-biological accident, with 399 reported cases (55.19%). The wound was the type of injury that occurred the most (24.76%). In injury mechanisms, in the first place, there is overexertion, excessive effort, or false movement (24.48%). The part of the body most affected in occupational accidents is the hands (40.11%). Just over half of the people who reported work accidents required medical leave.Conclusions:The accident rate of the workers of a hospital was described, and followed up for 3 years, to identify areas of intervention in reducing risks for institutional personnel, but also in optimizing the costs generated to the institution due to related absenteeism.

SUMMARY: Progesterone receptors are expressed in the reproductive organs of adult sheep, where they regulate morphofunctional and reproductive development. However, various studies have shown the presence of these receptors in the uterus of prepubertal females. It is not clear what role these receptors have at this level of development in uterine tissue. Therefore, it is relevant as a first step in the investigation, to determine the expression and histological distribution of the progesterone receptor in prepubertal sheep in order to determine possible functions at this level of reproductive development. Immunohistochemical analysis allows visualizing the specific presence of a protein in the cellular and histological context, however, the results displayed through digital images are qualitative data and subject to the observer's criteria. In this work, a quantitative analysis method of immunohistochemical expression of the progesterone receptor in ovine endometrium is presented, using digital analysis of images, by means of integrated optical density of digital photographs of histological sections processed with immunohistochemical methods. The results show the possibility of quantitatively evaluating the expression of progesterone receptors in the endometrial stroma and prepubertal endometrial glands by applying the integrated optical density analysis of digital images.

La estimación de la densidad básica de la madera se realiza mediante el método tradicional regido por la Norma Tappi T 258 om-94, o bien, por la norma chilena NCh 176/2, las cuales son métodos destructivos, que demandan extensos tiempos, altos gastos en mano de obra y gran utilización de material. Buscando métodos que permitan reducir esos inconvenientes se testeó la metodología Seis Sigmas, utilizando el método DMAMC (Definir, Medir, Analizar, Mejorar y Controlar), para mejorar este proceso, y estimar este parámetro en tiempos y costos más reducidos. Para esto se probó la espectroscopía NIR (Near Infrarred) calibrada para predecir la densidad de la madera en Acacia mearnsii, utilizando polvo de astillado en estado seco obtenido de tarugos. El uso de esta tecnología fue apoyado con modelos estadísticos de regresión multivariantes de mínimos cuadrados parciales (PLS, Partial Least Squares), y se generó un modelo de calibración y un modelo de validación para el instrumento. El objetivo de este estudio fue mejorar los tiempos del cálculo de densidad básica, parámetro físico importante para la obtención de información de la biomasa, que se emplea como un estimador del material leñoso de una especie y del rendimiento que alcanzará en el pulpaje, así como para determinar la facilidad con la que esta se puede trabajar. Los niveles de predicción del modelo PLS fueron sobre el 80%, por lo que se valida el uso de la espectroscopía NIR como herramienta para predecir la densidad básica. Además, se aprecia un aumento en los indicadores de calidad, el nivel sigma aumentó de 0,18 a 3, el Cp de 0,26 a 1.08, el Cpk de 0,23 a 1 y los defectos por millón disminuyeron en un 99.6%. Además, utilizando la espectroscopía NIR disminuyen los costos en comparación con el método tradicional.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

El Instituto Forestal desde sus inicios ha desarrollado investigaciones sobre especies del género Acacia. A comienzos de los años 60 del siglo pasado se iniciaron los programas de introducción de especie forestales al país en los cuales fueron incluidas numerosas de ellas traídas de Australia. Con el transcurso de los años han destacado especialmente, por su adaptación y buen desarrollo, especies del género como Acacia dealbata Link., Acacia melanoxylon R. Br. y Acacia mearnsii D. Wild. En años más recientes INFOR ha realizado diferentes investigaciones orientadas a su silvicultura y manejo y a definir las propiedades físicas y mecánicas de sus maderas y sus aptitudes de uso, para posteriormente abordar un fuerte programa de difusión y transferencia tecnológica orientado a dar a conoces las experiencias y resultados de las investigaciones, enfocado en especial en pequeños y medianos propietarios forestales y pequeños y medianos industriales de la madera.

Introducción: La eutanasia fue incluida en la Constitución Colombiana en el año 2014 como medida que garantiza el derecho a la muerte digna. Actualmente este sigue siendo un tema controvertido y que genera diversas opiniones y actitudes. Objetivo: Explorar los conocimientos y actitudes que tienen estudiantes universitarios sobre la eutanasia en la Pontificia Universidad Javeriana de Bogotá y en la Universidad del Cauca en Popayán. Métodos: Estudio observacional de corte transversal con datos obtenidos por medio de una encuesta virtual voluntaria sobre eutanasia en estudiantes universitarios de dos universidades. El cuestionario se enfocaba en: datos generales, conocimientos sobre eutanasia y casos hipotéticos relacionados. Resultados: Participaron 758 estudiantes. Un 60% de sexo femenino y con media de edad de 22 años. El 53% de los estudiantes identificó la definición apropiada de eutanasia y el 77% consideraría recurriría a ella en un caso hipotético de padecer una enfermedad en fase terminal. Una proporción importante confundió la definición de eutanasia con la de cuidados paliativos. Conclusiones: Se observó un alto grado de desconocimiento respecto a la eutanasia y a pesar de su amplia aceptación por la población estudiada los resultados indican la necesidad de abrir espacios de información y diálogo sobre el tema.

Equine endometrial and adipose mesenchymal stem cells (eMSCs and aMSCs, respectively) were isolated from the same donors of thoroughbred mares. The cells displayed characteristic features of MSCs, including trilineage mesodermal and also neurogenic differentiation. We evaluated the influence of cellular origin on their transcriptome profile. Cellular RNA was isolated and sequenced and extracellular vesicles (EVs) were obtained from conditioned medium of cells cultured in medium depleted of EVs, and their microRNA (miRNA) cargo analyzed by sequencing. Differential expression of mRNAs and EV-miRNA was analyzed, as well as pathways and processes most represented in each cell origin. mRNA reads from all expressed genes clustered according to the cellular origin. A total of 125 up- and 51 downregulated genes were identified and 31 differentially expressed miRNAs. Based on mRNA sequencing, endometrial MSCs strongly upregulated genes involved in the Hippo, transforming growth factor beta, and pluripotency signaling pathways. Alongside with this, pathways involved in extracellular matrix reorganization were the most represented in the miRNA cargo of EVs secreted by eMSCs. The niche from which MSCs originated defined the transcriptomic signature of the cells, including the secretion of lineage-specific loaded EV to ensure proper communication and homeostasis. Identification and testing their biological functions can provide new tools for the therapeutic use of horse MSC.

Simple Summary Prostaglandin E2 plays a regulatory role in MSC self-renewal, and PGE2 receptor-mediated signalling is involved in cell proliferation and migration. The aim of this work was to establish a genetically modified equine cellular model in which receptor genes EP2 and EP4 were edited by the CRISPR/Cas9 system. We successfully targeted EP2 and EP4 receptors in horse adipose MSCs using the CRISPR/Cas9 system. This targeting did not affect the surface marker phenotype of the adipose MSCs generated. Gene edition significantly lowered the expression of each of the targeted receptors and affected the early migration ability of the edited MSCs. This opens the possibility of using these mutant cell lines as a model system to elucidate the role of EP2 and EP4 in MSCs and, particularly, as therapeutic tools in equine regenerative medicine. Abstract In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNFα (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1α, 6, 8, 10, TNFα and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 × 107 adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNFα expression in the ELISA (p

En el presente trabajo de describe la elaboración de un primer modelo de volumen de árbol individual para la especie acacia negra (Acacia mearnsii), los principales aspectos de la metodología utilizada y los resultados obtenidos, a partir de la información obtenida de un rodal en crecimiento en la comuna de Florida, Región del Bio Bio.

Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca(2+)](i)). Recently, we demonstrated that the application of high concentrations (10–20 μM) of the Ca(2+) ionophore A23187 promotes sperm immobilization after 10 min, and its removal thereafter allows motility recovery, hyperactivation, and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1(−/−), Slo3(−/−), and Adcy10(−/−), but not PMCA4(−/−), which strongly suggest that regulation of [Ca(2+)](i) is mandatory for sperm motility and hyperactivation. In this study, we found that prior to inducing sperm immobilization, high A23187 concentrations (10 μM) increase flagellar beat. While 5–10 μM A23187 substantially elevates [Ca(2+)](i) and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 μM) provoke smaller [Ca(2+)](i) increases and sperm hyperactivation, confirming that [Ca(2+)](i) increases act as a motility switch. Until now, the [Ca(2+)](i) thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca(2+)](i) and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187, which is then washed to evaluate [Ca(2+)](i) and flagellar beat recovery using the implemented method. We observe that [Ca(2+)](i) must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.

Con el propósito de aportar en el conocimiento y utilización del pellet, además de entregar antecedentes para el desarrollo de políticas públicas en el país, se presentan en este trabajo algunos de los principales antecedentes relacionados con los actores y el mercado del pellet en Chile. Se analizan distintos aspectos de la producción, mercados y usos asociados a la biomasa forestal para generación de energía. Este documento se preparó en el marco del proyecto CORFO “Mejoramiento del Entorno y Reducción de Brechas Tecnológicas para la Producción y Desarrollo del Mercado del Pellet en Chile”, solicitado por el Misterio de Energía y adjudicado a INFOR por CORFO.

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

Eclipsing time variations have been observed for a wide range of binary systems, including post-common-envelope binaries. A frequently proposed explanation, apart from the possibility of having a third body, is the effect of magnetic activity, which may alter the internal structure of the secondary star, particularly its quadrupole moment, and thereby cause quasi-periodic oscillations. Here we present two compressible non-ideal magneto-hydrodynamical (MHD) simulations of the magnetic dynamo in a solar mass star, one of them with three times the solar rotation rate ("slow rotator"), the other one with twenty times the solar rotation rate ("rapid rotator"), to account for the high rotational velocities in close binary systems. For the slow rotator, we find that both the magnetic field and the stellar quadrupole moment change in a quasi-periodic manner, leading to O-C (observed - corrected times of the eclipse) variations of ~0.025 s. For the rapid rotator, the behavior of the magnetic field as well as the quadrupole moment changes become considerably more complex, due to the less coherent dynamo solution. The resulting O-C variations are of the order 0.13 s. The observed system V471~Tau shows two modes of eclipsing time variations, with amplitudes of 151 s and 20 s, respectively. However, the current simulations may not capture all relevant effects due to the neglect of the centrifugal force and self-gravity. Considering the model limitations and that the rotation of V471 Tau is still a factor of 2.5 faster than our rapid rotator, it may be conceivable to reach the observed magnitudes., Comment: 16 pages, 24 figures, accepted for publication with MNRAS

This study used a comparative approach to gather clinical information to assess the effect of bovine somatotropin (bST) on follicular dynamics and ovulation in sheep and goats during an interovulatory cycle. The performance of general markers of ovarian function and specific features of follicular dynamics obtained by daily ultrasonography (US) were used to assess the hypothesis that bST, associated with supraphysiological levels of IGF-I, was able to disrupt the follicular dynamics and ovulation in Highlander ewes and Saanen goats. In Exp 1, 15 ewes and 14 goats were estrous-synchronized (P4-6 days + PGFα d-6) and then allocated to a bST-treated group (50 and 100 mg, Lactotropin®; n = 5 females each) and to an untreated control group (5 ewes and 4 goats) to assess the activity of bST through plasma IGF-I (RIA). In Exp 2, 12 animals from each species were synchronized. At day 6, they were divided into a bST-group (100 mg in sheep and 50 mg in goats, n = 6 each) and an untreated control group (n = 6 each). Starting at day 6 and up to 22 days after ovulation in sheep and 25 days in goats, each female was subjected to daily US (10 mHz probe) to assess follicular and luteal (CL) dynamics and ovulation. This included assessments of both general ovarian features and specific follicular wave features. Our results showed that bST increased plasma IGF-I by day 3 (p 0.10). Results from Exp.2 indicate that in sheep, bST administration had a subtle inhibitory effect on follicular function. However, bST in goats had a stronger influence, extending the interovulatory cycle (P = 0,034), increasing the number of follicular waves during the period (P = 0.003), and reducing the functional potential of large follicles as measured by their lower follicular diameter (P = 0.02), duration of the follicle waves (P = 0.02), and persistence of follicles after reaching their maximum diameters (P = 0.04). In addition, untreated sheep and goats shared common patterns of terminal follicular development and ovulations characterized by overlapping between follicular waves and ovulations of follicles from different waves, features not seen in cattle.

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+ and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, grew on plastic, had doubling population times of 46.4 ± 3.38 h, underwent tri-lineage (osteo, chondro and adipogenic) differentiation after appropriate inductions, migrated toward the attraction of fetal calf serum and displayed a pattern of surface markers commonly accepted for horse MSCs. All these are properties of MSCs. Some of these attributes were shared with equine adipose-derived MSCs, but the migration pattern of eMSC at 12 and 24 h after stimulation was reduced in comparison with adipose MSCs. Also, expression of CD44, CD90 and MHCI surface markers were dramatically down-regulated in eMSCs. In conclusion, equine-derived endometrial MSC share biological attributes with adipose MSC of this species, but displayed a different surface marker phenotype and an impaired migration ability. Conceivably, this phenotype is distinctive for MSC of this origin.

Post-mating induced endometritis (PMIE) is an acute inflammatory response of the endometrium to spermatozoa, linked to an incapability of some mares to drain out the fluids associated with inflammation. This is of pivotal importance for reproductive success in mares. Mesenchymal stem cells (MSCs) are potential candidates for anti-inflammatory uterine therapies. Here, we aimed to study inflammatory markers in the endometrium of healthy mares and of those with induced endometritis, before and after intrauterine inoculation of MSCs, and to characterise their homing potential invivo in an induced endometritis horse model. Nine mares during their ovulatory season were selected after gynaecologic examination (absence of free liquid in the uterus, no polymorphonuclear leucocytes (PMNs) at cytology, negative bacteriology, and grade I in Kenney's scale on uterine biopsies). Mares were infused in the uterine body with 2mL of 500×106 spermmL−1 previously killed by repeated frozen-thawing cycles. At 3h, uteri were flushed with 250mL of sterile saline and the inflammatory response was monitored in the lavages and biopsies. Parameters measured included cytology, protein expression of inflammatory markers (supernatant) after lavage centrifugation (800×g, 10min), ELISA, and immunostaining for interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). The mares were divided into three groups (3 mares each). Then, 24h after dead sperm challenge, group 1 received intrauterine infusion of 2×107 adipose MSC in 0.9% sterile saline; group 2, received the same amount of endometrial MSCs in the same vehicle; and group 3 received only saline. The volume of infusion in the uterine body was 20mL for all groups. Cells (passage 4) were previously labelled with 10μM Vybrant CFDA SE Cell Tracer Kit (ThermoFisher Scientific). After 48h, the same lavages, biopsies, and measurements as described above were performed. Additional biopsies were taken at Days 10 and 30 after intrauterine infusions. Biopsies were split in two, one for confocal microscopy and the other for quantitative PCR. Endometritis was induced in all mares, as judged by cytology and expression of protein markers of inflammation. After 48h, reduction in IL-6 and TNFα was detected by immunostaining of biopsies and confirmed by ELISA in the lavages, as well as by PCR. Homing was detected in all mares infused with MSC and it persisted at Days 10 and 30 after infusion. No homing was found in the control mares. As a result of these experiments, we conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells (adipose or endometrial). Both types of cells were nested in the endometrium at low quantities, although the number of cells actually detected at fixed time points was not quantified. Overall, we can propose that, given the number of homed cells detected and the marked decrease in inflammatory markers after inoculation of cells, MSCs exert their anti-inflammatory function preferentially by a paracrine mechanism and not necessarily by nesting and proliferation, although both events occur. Funding for this study was provided by Fondecyt 1150757.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. While the HCO3--dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this manuscript, we used Western blotting and cholera toxin-dependent ADP ribosylation to show Gs presence in the sperm head. Also, we showed that forskolin, a tmAC specific activator, induces cAMP accumulation in sperm from both WT and Adcy10 null mice. This increase is blocked by the tmAC inhibitor SQ-22536 but not by the Adcy10 inhibitor KH7. While Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome reacted sperm, and forskolin is able to increase intracellular Ca2+ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum. Fil: Wertheimer Hermitte, Eva Victoria. University Of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Krapf, Dario. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: de la Vega Beltran, José L.. Universidad Nacional Autónoma de México; México Fil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; México Fil: Navarrete, Felipe. University Of Massachussets; Estados Unidos Fil: Haddad, Douglas. University Of Massachussets; Estados Unidos Fil: Escoffier, Jessica. University Of Massachussets; Estados Unidos Fil: Salicioni, Ana M.. University Of Massachussets; Estados Unidos Fil: Levin, Lonny R.. Cornell University; Estados Unidos Fil: Buck, Jochen. Cornell University; Estados Unidos Fil: Mager, Jesse. University Of Massachussets; Estados Unidos Fil: Darszon, Alberto. Universidad Nacional Autónoma de México; México Fil: Visconti, Pablo E.. University Of Massachussets; Estados Unidos

We carried out targeted ALMA observations of 129 fields in the COSMOS region at 1.25 mm, detecting 152 galaxies at S/N$\geq$5 with an average continuum RMS of 150 $��$Jy. These fields represent a S/N-limited sample of AzTEC / ASTE sources with 1.1 mm S/N$\geq$4 over an area of 0.72 square degrees. Given ALMA's fine resolution and the exceptional spectroscopic and multiwavelength photometric data available in COSMOS, this survey allows us unprecedented power in identifying submillimeter galaxy counterparts and determining their redshifts through spectroscopic or photometric means. In addition to 30 sources with prior spectroscopic redshifts, we identified redshifts for 113 galaxies through photometric methods and an additional nine sources with lower limits, which allowed a statistically robust determination of the redshift distribution. We have resolved 33 AzTEC sources into multi-component systems and our redshifts suggest that nine are likely to be physically associated. Our overall redshift distribution peaks at $z\sim$2.0 with a high redshift tail skewing the median redshift to $\tilde{z}$=2.48$\pm$0.05. We find that brighter millimeter sources are preferentially found at higher redshifts. Our faintest sources, with S$_{1.25 \rm mm}$1.8 mJy, have a median redshift of $\tilde{z}$=3.08$\pm$0.17. After accounting for spectral energy distribution shape and selection effects these results are consistent with several previous submillimeter galaxy surveys, and moreover, support the conclusion that the submillimeter galaxy redshift distribution is sensitive to survey depth., Accepted for publication in Astronomy & Astrophysics for review 17 pages in body + 26 in appendices

We determine the radio size distribution of a large sample of 152 SMGs in COSMOS that were detected with ALMA at 1.3 mm. For this purpose, we used the observations taken by the VLA-COSMOS 3 GHz Large Project. One hundred and fifteen of the 152 target SMGs were found to have a 3 GHz counterpart. The median value of the major axis FWHM at 3 GHz is derived to be $4.6\pm0.4$ kpc. The radio sizes show no evolutionary trend with redshift, or difference between different galaxy morphologies. We also derived the spectral indices between 1.4 and 3 GHz, and 3 GHz brightness temperatures for the sources, and the median values were found to be $\alpha=-0.67$ and $T_{\rm B}=12.6\pm2$ K. Three of the target SMGs, which are also detected with the VLBA, show clearly higher brightness temperatures than the typical values. Although the observed radio emission appears to be predominantly powered by star formation and supernova activity, our results provide a strong indication of the presence of an AGN in the VLBA and X-ray-detected SMG AzTEC/C61. The median radio-emitting size we have derived is 1.5-3 times larger than the typical FIR dust-emitting sizes of SMGs, but similar to that of the SMGs' molecular gas component traced through mid-$J$ line emission of CO. The physical conditions of SMGs probably render the diffusion of cosmic-ray electrons inefficient, and hence an unlikely process to lead to the observed extended radio sizes. Instead, our results point towards a scenario where SMGs are driven by galaxy interactions and mergers. Besides triggering vigorous starbursts, galaxy collisions can also pull out the magnetised fluids from the interacting disks, and give rise to a taffy-like synchrotron-emitting bridge. This provides an explanation for the spatially extended radio emission of SMGs, and can also cause a deviation from the well-known IR-radio correlation., Comment: 32 pages (incl. 5 appendices), 17 figures, 7 tables; accepted for publication in A&A; abstract abridged for arXiv

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca 2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca 2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca 2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility. Fil: Navarrete, Felipe A.. University of Massachusetts; Estados Unidos Fil: Alvau, Antonio. University of Massachusetts; Estados Unidos Fil: Lee, Hoi Chang. University of Massachusetts; Estados Unidos Fil: Levin, Lonny R.. Weill Cornell Medical College; Estados Unidos Fil: Buck, Jochen. Weill Cornell Medical College; Estados Unidos Fil: Leon, Patricia Martin De. University of Delaware; Estados Unidos Fil: Santi, Celia M.. University of Washington; Estados Unidos Fil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Mager, Jesse. University of Massachusetts; Estados Unidos Fil: Fissore, Rafael A.. University of Massachusetts; Estados Unidos Fil: Salicioni, Ana M.. University of Massachusetts; Estados Unidos Fil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Visconti, Pablo E.. University of Massachusetts; Estados Unidos

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. Fil: Alvau, Antonio. University of Massachussets; Estados Unidos Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gervasi, Maria Gracia. University of Massachussets; Estados Unidos Fil: Navarrete, Felipe A.. University of Massachussets; Estados Unidos Fil: Xu, Xinran. State University of Colorado - Fort Collins; Estados Unidos Fil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: De la Vega Beltran, José Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Greer, Peter. Queens University; Canadá Fil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Krapf, Diego. State University of Colorado - Fort Collins; Estados Unidos Fil: Salicioni, Ana María. University of Massachussets; Estados Unidos Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Visconti, Pablo E.. University of Massachussets; Estados Unidos

Earlier we successfully isolated and characterised endometrial (eMSC) and adipose (aMSC) mesenchymal stem cells from the same donors. Mesenchymal stem cells share biological traits but display different surface marker phenotype and migration ability. Here we extended our research to their mRNA signature using next-generation sequencing. The RNA from cells (3 biological replicates from each cell type and 3 technical replicates) at 90% confluence was extracted using a total RNA extraction kit and sent for mRNA-Seq (Norgen, Ontario, Canada; Illumina Sequencing Platform NextSEqn 500). Raw 76-bp single-end reads were aligned against the EquCab3 genome using RNA-STAR aligner. Counts were filtrated at a minimum of 5. Pairwise comparisons between the cell types were the input for gene ontology enrichment analysis. Only genes differentially expressed (DE) with 5 folds change (FC; P50×: 8 and 13; >20×, 10×, 5×, 2×

SummaryElongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.

To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 ...

We used the PdBI to map a sample of 15 SMGs in the COSMOS field at the wavelength of 1.3 mm. The target SMGs were originally discovered in the JCMT/AzTEC 1.1 mm continuum survey at S/N=4-4.5. This paper presents, for the first time, interferometric millimetre-wavelength observations of these sources. The angular resolution of our observations, 1.8", allowed us to accurately determine the positions of the target SMGs. Using a detection threshold of S/N>4.5 regardless of multiwavelength counterpart association, and 4, Comment: 31 pages (incl. 5 appendices with 3 figures and 1 table), 6 figures and 4 tables in the main text. Accepted for publication in A&A

We present deep 3 GHz VLA observations of the potentially recoiling black hole CID-42 in the COSMOS field. This galaxy shows two optical nuclei in the HST/ACS image and a large velocity offset of ~ 1300 km/s between the broad and narrow H beta emission line although the spectrum is not spacially resolved (Civano et al. 2010). The new 3 GHz VLA data has a bandwidth of 2 GHz and to correctly interpret the flux densities imaging was done with two different methods: multi-scale multi-frequency synthesis and spectral windows stacking. The final resolutions and sensitivities of these maps are 0.7" with rms = 4.6 muJy/beam and 0.9" with rms = 4.8 muJy/beam respectively. With a 7 sigma detection we find that the entire observed 3 GHz radio emission can be associated with the South-Eastern component of CID-42, coincident with the detected X-ray emission. We use our 3 GHz data combined with other radio data from the literature ranging from 320 MHz to 9 GHz, which include the VLA, VLBA and GMRT data, to construct a radio synchrotron spectrum of CID-42. The radio spectrum suggests a type I unobscured radio-quiet flat-spectrum AGN in the South-Eastern component which may be surrounded by a more extended region of old synchrotron electron population or shocks generated by the outflow from the supermassive black hole. Our data are consistent with the recoiling black hole picture but cannot rule out the presence of an obscured and radio-quiet SMBH in the North-Western component., Accepted for publication in MNRAS; 7 pages, 4 figures, 2 tables