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BACKGROUND: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium. METHODS: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions. RESULTS: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions. CONCLUSIONS: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.
Subject(s)
Embryonic Stem Cells , Animals , Culture Media, Serum-Free/pharmacology , Swine , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Cell Differentiation , Feeder Cells/cytology , Feeder Cells/metabolism , Cell Culture Techniques/methods , Cell Proliferation , Blastocyst/cytology , Blastocyst/metabolism , Cells, CulturedABSTRACT
Abnormal Wnt5a expression is associated with dysregulated inflammation and organ dysfunction. However, the effect of Wnt5a activation on the duration of organ dysfunction remains unclear. This prospective study investigated the association between Wnt5a levels and persistent acute kidney injury (AKI) in patients with urosepsis. Serum creatinine and Wnt5a levels were measured on days 1 and 5 and at discharge in 87 patients diagnosed with urosepsis. Patients with urosepsis were classified into an improving acute kidney injury (AKI) group and a persistent or worsening AKI group according to the AKI stage on days 1 and 5. AKI recovery was defined as a discharge-to-baseline serum creatinine ratio of <1.5. Twenty-eight patients with urosepsis (32.2%) had persistent or worsening AKI, and their Wnt5a levels were higher on days 1 and 5 and at discharge than those with improving AKI. The association between Wnt5a levels and persistent or worsening AKI was maintained after adjusting for age, sex, baseline serum creatinine levels, and disease severity. Moreover, elevated Wnt5a levels were associated with an increased risk of major adverse kidney events. High Wnt5a levels at discharge were associated with unrecovered AKI and participants with AKI recovery had a steeper Wnt5a slope over time than those without recovery, irrespective of age, sex, baseline serum creatinine level, or disease severity. Assessment of Wnt5a expression was helpful in predicting AKI persistence and adverse outcomes in patients with urosepsis. Therefore, Wnt5a may serve as a valuable bio-marker for identifying the risk of persistence of AKI.
Subject(s)
Acute Kidney Injury , Creatinine , Sepsis , Wnt-5a Protein , Humans , Wnt-5a Protein/metabolism , Wnt-5a Protein/blood , Acute Kidney Injury/etiology , Acute Kidney Injury/blood , Acute Kidney Injury/metabolism , Acute Kidney Injury/diagnosis , Male , Female , Sepsis/complications , Sepsis/blood , Middle Aged , Aged , Prospective Studies , Creatinine/blood , Urinary Tract Infections/complications , Urinary Tract Infections/blood , Biomarkers/blood , Severity of Illness IndexABSTRACT
The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.
Subject(s)
4-Butyrolactone , Angiopoietin-2 , Homoserine , Human Umbilical Vein Endothelial Cells , Pseudomonas aeruginosa , Humans , Pseudomonas aeruginosa/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Angiopoietin-2/metabolism , Angiopoietin-2/genetics , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Homoserine/analogs & derivatives , Homoserine/pharmacology , Homoserine/metabolism , Cadherins/metabolism , Cadherins/genetics , rac1 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/genetics , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , ras GTPase-Activating Proteins/metabolism , ras GTPase-Activating Proteins/genetics , Signal Transduction/drug effects , Antigens, CD/metabolism , Antigens, CD/genetics , Angiopoietin-1/metabolism , Angiopoietin-1/geneticsABSTRACT
Introduction: Embryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals. Methods: Oocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze-thawing. Results: The survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05). Conclusion: Our study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze-thawing.
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Electronic flat-band materials host quantum states characterized by a quenched kinetic energy. These flat bands are often conducive to enhanced electron correlation effects and emergent quantum phases of matter1. Long studied in theoretical models2-4, these systems have received renewed interest after their experimental realization in van der Waals heterostructures5,6 and quasi-two-dimensional (2D) crystalline materials7,8. An outstanding experimental question is if such flat bands can be realized in three-dimensional (3D) networks, potentially enabling new materials platforms9,10 and phenomena11-13. Here we investigate the C15 Laves phase metal CaNi2, which contains a nickel pyrochlore lattice predicted at a model network level to host a doubly-degenerate, topological flat band arising from 3D destructive interference of electronic hopping14,15. Using angle-resolved photoemission spectroscopy, we observe a band with vanishing dispersion across the full 3D Brillouin zone that we identify with the pyrochlore flat band as well as two additional flat bands that we show arise from multi-orbital interference of Ni d-electrons. Furthermore, we demonstrate chemical tuning of the flat-band manifold to the Fermi level that coincides with enhanced electronic correlations and the appearance of superconductivity. Extending the notion of intrinsic band flatness from 2D to 3D, this provides a potential pathway to correlated behaviour predicted for higher-dimensional flat-band systems ranging from tunable topological15 to fractionalized phases16.
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Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.
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In vitro generation of porcine embryos is an indispensable method in the realms of both agriculture and biomedicine. Nonetheless, the extant procedures encounter substantial obstacles pertaining to both the caliber and efficacy of the produced embryos, necessitating extensive research to in vitro maturation (IVM), the seminal commencement phase. One potentially fruitful approach may lie in refining the media and supplements composition utilized for oocyte maturation. Fibroblast growth factor-7 (FGF7), alternatively termed keratinocyte growth factor, is a theca-derived cytokine integral to folliculogenesis. This study aimed to examine the ramifications of supplementing FGF7 during the IVM phase. To determine the FGF7 location and its receptor in porcine ovaries, immunohistochemistry was executed based on follicle size categories (1-2, 3-6, and 7-9 mm). Regardless of follicle size, it was determined that FGF7 was expressed in theca and granulosa cells (GCs), whereas the FGF7 receptor was only expressed in the GCs of the larger follicles. During the IVM process, the maturation medium was supplied with various concentrations of FGF7, aiming to mature porcine cumulus-oocyte complexes (COCs). The data indicated a significant augmentation in the nuclear maturation rate only within the group treated with 10 ng/mL of FGF7 (p < 0.05). Post-IVM, the oocytes diameter exhibited a significant expansion in all groups that received FGF7 supplementation (p < 0.05). Additionally, all FGF7-supplemented groups exhibited a substantial elevation in intracellular glutathione levels, coupled with a noticeable reduction in reactive oxygen species levels (p < 0.05). With respect to gene expressions related to apoptosis, FGF7 treatment elicited a downregulation of pro-apoptotic genes and an upregulation of anti-apoptotic genes. The expression of genes associated with antioxidants underwent a significant enhancement (p < 0.05). In terms of the FGF7 signaling pathway-associated genes, there was a significant elevation in the mRNA expression of ERK1, ERK2, c-kit, and KITLG (p < 0.05). Remarkably, the group of 10 ng/mL of FGF7 demonstrated an appreciable uptick in the blastocyst formation rate during embryonic development post-parthenogenetic activation (p < 0.05). In conclusion, the FGF7 supplementation during IVM substantially augments the quality of matured oocytes and facilitates the subsequent development of parthenogenetically activated embryos. These results offer fresh perspectives on improved maturation and following in vitro evolution of porcine oocytes.
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Introduction: Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Methods: Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Results: Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. Conclusion: The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.
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Finding d-electron heavy fermion states has been an important topic as the diversity in d-electron materials can lead to many exotic Kondo effect-related phenomena or new states of matter such as correlation-driven topological Kondo insulator. Yet, obtaining direct spectroscopic evidence for a d-electron heavy fermion system has been elusive to date. Here, we report the observation of Kondo lattice behavior in an antiferromagnetic metal, FeTe, via angle-resolved photoemission spectroscopy, scanning tunneling spectroscopy and transport property measurements. The Kondo lattice behavior is represented by the emergence of a sharp quasiparticle and Fano-type tunneling spectra at low temperatures. The transport property measurements confirm the low-temperature Fermi liquid behavior and reveal successive coherent-incoherent crossover upon increasing temperature. We interpret the Kondo lattice behavior as a result of hybridization between localized Fe 3dxy and itinerant Te 5pz orbitals. Our observations strongly suggest unusual cooperation between Kondo lattice behavior and long-range magnetic order.
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Neurotrophin-4 (NT-4), a neurotrophic factor, appears to affect early embryonic development because it is secreted not only by neurons but also by oviductal and uterine epithelial cells. However, no studies have characterized the effects of NT-4 on early embryonic development in pigs. In this study, we applied the experimental model of parthenogenetic-activation (PA)-derived embryos. Herein, we investigated the effect of NT-4 supplementation during the in vitro culture (IVC) of embryos, analyzed the transcription levels of specific genes, and outlined the first cell lineage specification for porcine PA-derived blastocysts. We confirmed that NT-4 and its receptor proteins were localized in both the inner cell mass (ICM) and trophectoderm (TE) in porcine blastocysts. Across different concentrations (0, 1, 10, and 100 ng/mL) of NT-4 supplementation, the optimal concentration of NT-4 to improve the developmental competence of porcine parthenotes was 10 ng/mL. NT-4 supplementation during porcine IVC significantly (p < 0.05) increased the proportion of TE cells by inducing the transcription of TE lineage markers (CDX2, PPAG3, and GATA3 transcripts). NT-4 also reduced blastocyst apoptosis by regulating the transcription of apoptosis-related genes (BAX and BCL2L1 transcripts) and improved blastocyst quality via the interaction of neurotrophin-, Hippo-yes-associated protein (Hippo-YAP) and mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway. Additionally, NT-4 supplementation during IVC significantly (p < 0.05) increased YAP1 transcript levels and significantly (p < 0.01) decreased LATS2 transcript levels, respectively, in the porcine PA-derived blastocysts. We also confirmed through fluorescence intensity that the YAP1 protein was significantly (p < 0.001) increased in the NT-4-treated blastocysts compared with that in the control. NT-4 also promoted differentiation into the TE lineage rather than into the ICM lineage during porcine early embryonic development. In conclusion, 10 ng/mL NT-4 supplementation enhanced blastocyst quality by regulating the apoptosis- and TE lineage specification-related genes and interacting with neurotrophin-, Hippo-YAP-, and MAPK/ERK signaling pathway during porcine in vitro embryo development.
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BACKGROUND: Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. OBJECTIVES: This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). METHODS: Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. RESULTS: After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. CONCLUSIONS: Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.
Subject(s)
Antioxidants , Ergothioneine , Pregnancy , Female , Swine , Animals , Antioxidants/pharmacology , Antioxidants/metabolism , Ergothioneine/pharmacology , Ergothioneine/analysis , Ergothioneine/metabolism , In Vitro Oocyte Maturation Techniques/veterinary , Reactive Oxygen Species/metabolism , Oocytes , Embryonic Development , Glutathione/analysis , Glutathione/metabolism , Glutathione/pharmacology , Fertilization in Vitro/veterinary , Blastocyst/metabolismABSTRACT
Porcine embryos are used for a variety of applications. However, the maturation rate in vitro remains low, and novel in vitro maturation (IVM) techniques that facilitate the collection of mature oocytes are necessary. C-C motif chemokine ligand 2 (CCL2) is a key periovulatory chemokine present in cumulus-oocyte complexes (COCs). We aimed to examine the effects of CCL2 supplementation during IVM on oocyte maturation and embryonic development. The CCL2 concentration was significantly higher in porcine follicular fluid (pFF) derived from follicles >8 mm in size than in pFF derived from smaller follicles. There was a significant increase in CCL2 mRNA levels in all follicular cells after IVM compared with that before IVM. We analyzed the localization of CCL2 and its receptor, the CCL2 receptor, in follicular cells. During IVM, different concentrations of CCL2 were added to COCs cultured in a maturation medium. After IVM, the group treated with 100 ng/mL CCL2 showed significantly higher metaphase II rates than the control group. All CCL2-treatment groups showed a significant increase in intracellular glutathione levels and a significant decrease in reactive oxygen species levels, compared to the control. In CCs treated with 100 ng/mL CCL2, the mRNA levels of BAX, CASP3, and NPR2 were significantly decreased. Furthermore, the mRNA levels of SOD1, SOD2, and CD44 were significantly increased. In oocytes treated with 10 ng/mL CCL2, mRNA levels of BAX and CASP3 were significantly decreased, whereas, NRF2 and NPM2 were significantly increased. ERK1 exhibited significantly increased mRNA expression in both CCs and oocytes treated with 10 ng/mL CCL2. The protein expression ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly increased in CCs treated with 10 ng/mL CCL2. After parthenogenetic activation, cleavage rates were significantly improved in the 100 ng/mL CCL2 treatment group, and blastocyst formation rates were significantly enhanced in the 10 ng/mL CCL2 treatment group. Overall, our results suggest that IVM medium along with CCL2 improves porcine oocyte maturation and the development of parthenogenetically-activated embryos.
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BACKGROUND: Sarcopenia is a major component of geriatric syndrome and associated with poor clinical outcomes and mortality. However, diagnosing sarcopenia in the very elderly is difficult, and data on its epidemiology and devastating effects in this group are scarce. Phase angle (PA) is measured using bioimpedance spectroscopy and known to reflect cellular integrity and health. This study aimed to clarify the impact of sarcopenia and PA on mortality risk in very elderly people living in long-term care facilities. METHODS: This prospective cohort study enrolled elderly residents living in nine long-term care facilities. We collected the participants' data, such as body mass index (BMI), comorbidities and laboratory data, from September to October 2017 and mortality data until October 2019. Nutritional status was evaluated using the Mini Nutritional Assessment (MNA) score, and multifrequency bioimpedance spectroscopy was used to assess body composition including PA. Appendicular skeletal muscle mass was calculated using the body composition monitor-derived equation of Taiwan's researchers. Sarcopenia was diagnosed using the European Working Group on Sarcopenia in Older People 2 (EWGSOP2) definition (sarcopenia vs. normal group). We divided the participants into two groups according to the median PA value of 3.65° (high vs. low group) and performed multivariate regression analyses to verify the association with mortality risk according to sarcopenia diagnosis or PA group. RESULTS: A total of 279 elderly participants were enrolled; of them, 238 (85.3%) were diagnosed with sarcopenia according to EWGSOP2 guidelines. The median patient age was 83 years, 211 (75.6%) were female and the median BMI was 20.4 kg/m2 . The sarcopenia group was older than the normal group (84 vs. 81 years; P = 0.002), had a lower mean BMI (19.8 vs. 26.6 kg/m2 , P < 0.001) and had a lower MNA score (9 vs. 12 points, P < 0.001). Sarcopenia was associated with a higher mortality risk after the adjustment for age, sex and diabetes mellitus (hazard ratio [HR], 3.744; 95% confidence interval [CI], 1.155-12.134; P = 0.028). A low PA was associated with sarcopenia, older age, female sex, low MNA score and overhydration volume; it was also a significant predictor of mortality after the adjustment for age, sex, diabetes mellitus and MNA score (HR, 0.593; 95% CI, 0.420-0.837; P = 0.003). CONCLUSIONS: Sarcopenia is prevalent among the very elderly patients in long-term care facilities. Sarcopenia and low PA are significantly associated with higher mortality risk.
Subject(s)
Sarcopenia , Humans , Female , Aged , Aged, 80 and over , Male , Sarcopenia/diagnosis , Sarcopenia/epidemiology , Sarcopenia/complications , Prospective Studies , Nutritional Status , Comorbidity , Body Mass IndexABSTRACT
Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.
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The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.
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Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 µg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly (p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.
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Objective: Previously developed Intradialytic hypotension (IDH) prediction models utilize clinical variables with potential privacy protection issues. We developed an IDH prediction model using minimal variables, without the risk of privacy infringement. Methods: Unidentifiable data from 63,640 hemodialysis sessions (26,746 of 79 patients for internal validation, 36,894 of 255 patients for external validation) from two Korean hospital hemodialysis databases were finally analyzed, using three IDH definitions: (1) systolic blood pressure (SBP) nadir <90 mmHg (Nadir90); (2) SBP decrease ≥20 mmHg from baseline (Fall20); and (3) SBP decrease ≥20 mmHg and/or mean arterial pressure decrease ≥10 mmHg (Fall20/MAP10). The developed models use 30 min information to predict an IDH event in the following 10 min window. Area under the receiver operating characteristic curves (AUROCs) and precision-recall curves were used to compare machine learning and deep learning models by logistic regression, XGBoost, and convolutional neural networks. Results: Among 344,714 segments, 9,154 (2.7%), 134,988 (39.2%), and 149,674 (43.4%) IDH events occurred according to three different IDH definitions (Nadir90, Fall20, and Fall20/MAP10, respectively). Compared with models including logistic regression, random forest, and XGBoost, the deep learning model achieved the best performance in predicting IDH (AUROCs: Nadir90, 0.905; Fall20, 0.864; Fall20/MAP10, 0.863) only using measurements from hemodialysis machine during dialysis session. Conclusions: The deep learning model performed well only using monitoring measurement of hemodialysis machine in predicting IDH without any personal information that could risk privacy infringement.
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Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15, sperm-oocyte interaction regulator CD9, and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.
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The Veterans Health Administration (VHA), responsible for providing 9 million veterans with quality healthcare, is not insulated from concerns about efficiency. In the aftermath of the Veterans Affairs (VA) hospital scandal in 2014, Congress passed the Veterans Choice Act of 2014, which allows eligible veterans to use non-VA hospitals instead of VA hospitals. After analyzing 118 or 119 VA hospitals each year from 2012 through 2017 in the U.S, this paper evaluates the efficiency scores of VA hospitals and examines how the 2014 Act has influenced their technical efficiency over time. Slack analysis shows that inefficient VA hospitals can improve efficiency by reallocating input resources, and regression analysis demonstrates that the overall technical efficiency of VA hospitals decreased by 0.164 after the implementation of the Act. This means that as more veterans used non-VA hospitals under the 2014 Act, the technical efficiency of VA hospitals decreased considerably. Given that a substantial portion of veterans' demands for healthcare transferred out to non-VA hospitals, the VHA should evaluate whether the current capacity of VA hospitals is appropriate and try to reduce wasted input resources to improve efficiency.