NRF2 attenuation aggravates detrimental consequences of metabolic stress on cultured porcine parthenote embryos.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

Biotechnological insights into extracellular enzyme production by thermotolerant fungi from hot springs and caves: Morphology, pellets formation, and protease production.

This study investigates the thermotolerant fungal biodiversity in caves and hot springs, focusing on their potential for extracellular enzyme production, specifically proteases. Samples were collected from the Cardonal region in Hidalgo, Mexico, using three different isolation methods. The study characterizes the morphological diversity of the isolated fungi and identifies various genera, including Aspergillus, Penicillium, Trichoderma, Cladosporium, and Fusarium, based on morphology. The isolated fungi were screened for their ability to produce extracellular enzymes on solid media, with a particular emphasis on proteases due to their industrial significance. Among the 35 isolated fungi, 20 exhibited proteolytic activity, and 12 strains were identified as good protease producers based on enzymatic index values. The study also evaluated the formation of fungal pellets by proteolytic fungi and found certain strains to display significant pellet formation. Additionally, protease production was examined by fungal pellets in submerged cultures, with isolate 6 demonstrating the highest protease activity. The findings highlight the diverse thermotolerant fungal biodiversity in extreme environments, and emphasize their potential for enzymatic production. This research contributes to our understanding of fungal ecology and provides insights into the biotechnological applications of these enzymes. The study recommends further molecular investigations to enhance biodiversity studies in such extreme environments.

DCAF13 and RNF114 participate in the regulation of early porcine embryo development.

In brief: Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development. Abstract: Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.

Optimization of gonadotropin stimulation protocols for in vitro embryo production in prepubertal Mediterranean water buffalo.

Embryos can be produced from prepubertal donor animals using laparoscopic ovum pickup and in vitro embryo production technologies (LOPU-IVEP). Together, these tools can shorten the interval between generations, rapidly accelerating the rate of genetic gain. Here, we assessed the impact of different gonadotropin stimulation protocols in Mediterranean water buffalo heifer calves aged between 2 and 6 months old. Following gonadotropin stimulation, LOPU was performed at two-week intervals, with animals receiving different protocols on subsequent LOPUs. After collection, the cumulus-oocyte complexes (COCs) were matured and fertilized in vitro, and embryos were cultured to the blastocyst stage followed by transfer into synchronized adult recipients. The number and size of follicles aspirated during LOPU, the number and quality of COCs recovered, as well as cleavage, embryo development and pregnancy rates were assessed. First, we evaluated the impact of using FSH with and without eCG (administered 24-h prior to LOPU) and found that a combination of FSH and eCG was able to significantly improve embryo development rates (20.6 ± 2.0% vs. 9.0 ± 3.6%; P < 0.05). Second, we compared this protocol to a slow-release formulation of FSH reconstituted in hyaluronan. In addition to requiring less work to prepare the animals for LOPU, this slow-release formulation yielded numerically higher, but not statistically different, average number of recovered COCs (14.4 ± 2.1 vs. 10.3 ± 2.0; P > 0.05) and embryo development rates (22.9 ± 4.7% vs. 14.1 ± 5.2%; P > 0.05) compared to FSH given every 12 h. Next, we compared the length of gonadotropin treatment over 3-, 4- and 5-days prior to LOPU and found that as the length of gonadotropin treatment increased, although the number of COCs recovered steadily decreased (14.1 ± 2.4 vs. 8.7 ± 1.0 vs. 6.9 ± 0.7; P < 0.05), the embryo development rates steadily increased (14.4 ± 3.9 vs. 27.3 ± 4.4 vs. 35.9 ± 7.0; P < 0.05), presumably due to an increase in the proportion of large follicles at the time of LOPU. Numerically, the 4-day treatment yielded more transferrable embryos per donor per LOPU (2.70 ± 0.5) than 3-day (1.94 ± 0.6) and 5-day (2.25 ± 0.5) treatments. Finally, following embryo transfer, 26 of 90 recipient females became pregnant (28.9%). Pregnancies were established from all treatments, which suggests that post-implantation development was not affected among the gonadotropin treatments assessed.

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo.

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation.

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos.

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

Enhancement of Chromatin and Epigenetic Reprogramming in Porcine SCNT Embryos-Progresses and Perspectives.

Over the last 25 years, cloned animals have been produced by transferring somatic cell nuclei into enucleated oocytes (SCNT) in more than 20 mammalian species. Among domestic animals, pigs are likely the leading species in the number of clones produced by SCNT. The greater interest in pig cloning has two main reasons, its relevance for food production and as its use as a suitable model in biomedical applications. Recognized progress in animal cloning has been attained over time, but the overall efficiency of SCNT in pigs remains very low, based on the rate of healthy, live born piglets following embryo transfer. Accumulating evidence from studies in mice and other species indicate that new strategies for promoting chromatin and epigenetic reprogramming may represent the beginning of a new era for pig cloning.

The Search for Cryptic L-Rhamnosyltransferases on the Sporothrix schenckii Genome.

The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied of the Sporothrix genus. The cell wall of this organism has been previously analyzed, and rhamnoconjugates are signature molecules found on the surface of both mycelia and yeast-like cells. Similar to other reactions where sugars are covalently linked to other sugars, lipids, or proteins, the rhamnosylation process in this organism is expected to involve glycosyltransferases with the ability to transfer rhamnose from a sugar donor to the acceptor molecule, i.e., rhamnosyltransferases. However, no obvious rhamnosyltransferase has thus far been identified within the S. schenckii proteome or genome. Here, using a Hidden Markov Model profile strategy, we found within the S. schenckii genome five putative genes encoding for rhamnosyltransferases. Expression analyses indicated that only two of them, named RHT1 and RHT2, were significantly expressed in yeast-like cells and during interaction with the host. These two genes were heterologously expressed in Escherichia coli, and the purified recombinant proteins showed rhamnosyltransferase activity, dependent on the presence of UDP-rhamnose as a sugar donor. To the best of our knowledge, this is the first report about rhamnosyltransferases in S. schenckii.

Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.

CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.

Supplementation of oleic acid, stearic acid, palmitic acid and ß-hydroxybutyrate increase H3K9me3 in endometrial epithelial cells of cattle cultured in vitro.

There is growing evidence that greater than homeostatic blood concentrations of nonesterified fatty acids (NEFAs) and ß-hydroxybutyrate (BHBA) have negative consequences on dairy cow's fertility, but effects on cell homeostasis in the reproductive system is not completely understood. In this study, lipids accumulation, reactive oxygen species (ROS) concentrations, abundance of gene transcripts, and immunofluorescence signal of H3K4me3 and H3K9me3 were evaluated in endometrial epithelial cells of cattle cultured with NEFAs (Oleic (OA), Stearic (SA) and Palmitic (PA) acids), BHBA, NEFAs + BHBA or each of the three NEFAs alone. The cellular lipids were in greater concentrations as a result of NEFAs + BHBA, NEFAs, SA or OA supplementation, but not by BHBA or PA. The ROS concentrations were greater when there were treatments with NEFAs + BHBA, NEFAs or BHBA. The relative mRNA abundance for genes involved in the regulation of apoptosis (XIAP), glucose transport (GLUT3), and DNA methylation (DNMT1) were greater when there were NEFAs + BHBA, but not NEFAs, BHBA, OA, SA or PA treatments. The immunofluorescence signal for H3K9me3 was greater when there were NEFAs + BHBA, NEFAs or PA, but not by BHBA, OA or SA treatments. These findings indicate that NEFAs and BHBA have an additive effect on endometrial cells of cattle by altering epigenetic markers and the expression of genes controlling important cellular pathways. Furthermore, there was cellular lipid accumulation and increased H3K9me3 in cultured bovine endometrial cells that was mainly induced by OA and PA treatments, respectively.

Tauroursodeoxycholic acid/TGR5 signaling promotes survival and early development of glucose-stressed porcine embryos†.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

All-Fiber Measurement of Surface Tension Using a Two-Hole Fiber.

An all-fiber approach is presented to measure surface tension. The experimental realization relies on the use of a specialty fiber, a so-called two-hole fiber (THF), which serves a two-fold purpose: providing a capillary channel to produce bubbles while having the means to measure the power reflected at the end facet of the fiber core. We demonstrate that provided a controlled injection of gas into the hollow channels of the THF, surface tension measurements are possible by simply tracking the Fresnel reflection at the distal end of the THF. Our results show that the characteristic times involved in the bubble formation process, from where the surface tension of the liquids under test is retrieved, can be measured from the train of pulses generated by the continuous formation and detachment of bubbles.

Histone Lysine Demethylases KDM5B and KDM5C Modulate Genome Activation and Stability in Porcine Embryos.

The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos around the genome activation stage. Attenuation of KDM5B and KDM5C mRNA hampered embryo development to the blastocyst stage in fertilized, parthenogenetically activated and nuclear transfer embryos. While KDM5B attenuation increased H3K4me2-3 levels on D3 embryos and H3K4me1-2-3 on D5 embryos, KDM5C attenuation increased H3K9me1 on D3 embryos, and H3K9me1 and H3K4me1 on D5 embryos. The relative mRNA abundance of EIF1AX and EIF2A on D3 embryos, and the proportion of D4 embryos presenting a fluorescent signal for uridine incorporation were severely reduced in both KDM5B- and KDM5C-attenuated compared to control embryos, which indicate a delay in the initiation of the embryo transcriptional activity. Moreover, KDM5B and KDM5C attenuation affected DNA damage response and increased DNA double-strand breaks (DSBs), and decreased development of UV-irradiated embryos. Findings from this study revealed that both KDM5B and KDM5C are important regulators of early development in porcine embryos as their attenuation altered H3K4 and H3K9 methylation patterns, perturbed embryo genome activation, and decreased DNA damage repair capacity.

Tauroursodeoxycholic acid acts via TGR5 receptor to facilitate DNA damage repair and improve early porcine embryo development.

DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNA-mediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.

Burnout Syndrome in nursing professionals in Punta Arenas, Chile / Síndrome de Burnout em profissionais de enfermagem da cidade de Punta Arenas, Chile / Síndrome de Burnout en profesionales de enfermería de la ciudad de Punta Arenas, Chile

ABSTRACT Objective: to identify the prevalence of burnout syndrome in nursing professionals in Punta Arenas, Chile. Method: quantitative, descriptive, cross-sectional research in a sample of nursing professionals from Punta Arenas, Chile. Data were collected between January and March 2019, using the Maslach Burnout Inventory and a sociodemographic questionnaire. Descriptive statistics were applied in the data analysis. Results: 163 individuals participated in the study. 18% of the sample suffered from burnout syndrome, who were female young adults, i.e. between 20 and 40 years of age, and who worked in direct user care. Conclusion: the prevalence of burnout in the as-yet-unexplored place of study and in different work scenarios, such as education, management, and hospital-based and primary health care, confirms the implications of this study for occupational health in this population. This knowledge offers recent evidence to ground the implementation of occupational health promotion policies and strategies in Chilean nursing professionals.

RESUMO Objetivo: identificar a prevalência da síndrome de burnout entre os profissionais de enfermagem da cidade de Punta Arenas, Chile. Método: investigaçāo quantitativa, descritiva, de corte transversal, com uma amostra de profissionais de enfermagem da cidade de Punta Arenas, Chile. Para a coleta dos dados, realizada entre janeiro e março de 2019, utilizou-se a escala Maslach Burnout Inventory e o questionário sociodemográfico. Para a análise dos dados aplicou-se a estatística descritiva. Resultados: 163 indivíduos participaram do estudo. 18% da amostra sofría da síndrome de burnout, correspondendo a mulheres no grupo etário de jovens adultos, isto é, entre 20 e 40 anos de idade e trabalhando na área assistencial ou de atençāo direta aos usuários. Conclusāo: a prevalência do burnout no local ainda nāo investigado e em diferentes cenários ocupacionais, tais como a educaçāo, a gestāo e a atençāo, hospitalária e primária de saúde, confirma as implicações deste trabalho para a saúde ocupacional desta populaçāo. Este conhecimento proporciona evidências atualizadas para fundamentar a implementaçāo de políticas e estratégias de promoçāo da saúde ocupacional em profissionais de enfermagem chilenos.

RESUMEN Objetivo: identificar la prevalencia de síndrome de burnout en los profesionales de enfermería de la ciudad de Punta Arenas, Chile. Método: investigación de enfoque cuantitativo, descriptivo, de corte transversal, con una muestra de profesionales de enfermería de la ciudad de Punta Arenas, Chile. Para la recolección de datos, realizada entre enero y marzo de 2019, se utilizó la escala Maslach Burnout Inventory y el cuestionario sociodemográfico. Se aplicó estadística descriptiva para el análisis de datos. Resultados: participaron 163 individuos en el estudo. El 18% de la muestra tenía síndrome de burnout, correspondiente a personas de sexo femenino, del grupo etario adulto joven, es decir entre 20 y 40 años de edad y que se desempeña en el área asistencial o de atención directa de usuarios. Conclusión: la prevalencia del burnout en el local aún no explorado y en diferentes escenarios de trabajo, como educación, gestión y atención, hospitalaria y primaria de salud, confirma las implicaciones de este trabajo em la salud ocupacional en esta población. Este conocimiento proporciona evidencia actualizada para fundamentar la implementación de políticas y estrategias de promoción de la salud laboral en profesionales de enfermería chilenos.

The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos.

There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of KDM7A mRNA reduced blastocyst formation by 69.2% in in vitro fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in KDM7A knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, KDM7A knockdown altered mRNA expression, including the downregulation of KDM3C on D3, NANOG on D5 and D7, and OCT4 on D7 embryos, and the upregulation of CDX2, KDM4B and KDM6B on D5 embryos. On D3 and D5 embryos, total cell number and mRNA expression of embryo genome activation (EGA) markers (EIF1AX and PPP1R15B) were not affected by KDM7A knockdown. However, the ratio of inner cell mass (ICM)/total number of cells in D7 blastocysts was reduced by 45.5% in KDM7A knockdown compared to control embryos. These findings support a critical role for KDM7A in the regulation of early development and cell lineage specification in porcine embryos, which is likely mediated through the modulation of H3K9me1/me2 and H3K27me1/me2 levels, and changes in the expression of other KDMs and pluripotency genes.

A fast and reliable protocol for activation of porcine oocytes.

Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca2+ and Zn2+ levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different time intervals. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with PKCa + CDK1i for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different times to a Zn2+ chelator (TPEN) following Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximise oocyte activation and embryo development. Next, we observed that treatment with PKCa + CDK1i for 4 h after TPEN for 15 min decreased embryo development compared to TPEN alone. Lastly, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs 49.2%) and blastocyst from cleaved embryos (65.5% vs 46.2%) compared to CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol compared with CT-245 and the non-activated (N-A) group. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a fast and effective protocol for activation of porcine oocytes.