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Hypoxic-ischemic encephalopathy (HIE) is a diffuse brain tissue injury caused by acute ischemia and hypoxia, and it is most commonly found in newborn infants but can also occur in adults. Mesenchymal stem cell (MSC) therapies have showed improved outcomes for treating HIE-induced neuronal defects. However, many key issues associated with poor cell viability and tolerance of grafted MSCs after HIE remain to be resolved. Genetic engineering could endow MSCs with more robust regenerative capacities. Our research, along with that of other scientists, has found that the expression of intracellular erythropoietin (EPO) in human umbilical cord MSCs (hUC-MSCs) increases proportionally with the duration of hypoxia exposure. Furthermore, we observed that EPO, when introduced into the EPO gene-modified hUC-MSCs, can be secreted into the extracellular space. However, the underlying mechanisms that support the neuroprotective effects of EPO-MSCs remain unclear. EPO-MSCs, hUC-MSCs, and NC-MSCs were identified by flow cytometry, osteogenic, and adipogenic differentiation assays. The oxygen-glucose deprivation (OGD)-induced SH-SY5Y cell-line was established, and five groups were set up: control, 24-h ischemia-hypoxia, co-cultured with hUC-MSCs, NC-MSCs, and EPO-MSCs after hypoxia. LEGENDplex™ multi-factor flow cytometry was used to detect the secretion of inflammatory factors in cell supernatants and cerebrospinal fluid. Chromosome-targeted excision and tagging (CUT&Tag) sequencing was applied to detect genomic H3K4me2 modifications, and conjoint analysis with transcriptome sequencing (RNA-seq) was performed. Lentiviral vector infection was used to construct SH-SY5Y cells with stable knockdown of RE1-silencing transcription factor (REST), and flow cytometry was used to detect alterations in apoptosis. Finally, the molecular mechanism underlying the neuroprotective and anti-apoptotic effects of EPO-MSCs was investigated using RNA sequencing, qRT-PCR, and western blot assays. Our results suggest that EPO-MSCs are genetically engineered to secrete significantly more EPO. EPO-MSCs treatment has anti-apoptotic properties and offers neuronal protection during ischemic-hypoxic injury. Furthermore, RNA-seq results suggest that multiple inflammation-related genes were down-regulated after EPO-MSCs treatment. Application of RNA-seq and CUT&Tag combined analysis found that the expressions of REST were significantly up-regulated. Lentiviral vector infection to construct REST knockdown SH-SY5Y failed to rescue apoptosis after hypoxia and co-culture with EPO-MSCs, and SETD2-mediated H3K36me3 protein level expression was reduced. EPO-MSCs may promote neuronal survival by affecting H3K4me2 and thus activating the expression of REST and TET3. EPO-MSCs also upregulated the modification level of SETD2-mediated H3K36me3 and regulated the expression of inflammation-related genes such as PLCG2, as well as apoptosis genes BCL2A1. To investigate the neuroprotective effects of EPO-modified hUC-MSCs and the underlying epigenetic regulatory mechanisms, this study aims to provide a theoretical foundation for the potential application of EPO gene-modified hUC-MSCs in the treatment of HIE. [ABSTRACT FROM AUTHOR]

Pre-hospital potentially preventable trauma related deaths are mainly due to hypoperfusion-induced tissue hypoxia leading to irreversible organ dysfunction at or near the point of injury or during transportation prior to receiving definitive therapy. The prolyl hydroxylase domain (PHD) is an oxygen sensor that regulates tissue adaptation to hypoxia by stabilizing hypoxia inducible factor (HIF). The benefit of PHD inhibitors (PHDi) in the treatment of anemia and lactatemia arises from HIF stabilization, which stimulates endogenous production of erythropoietin and activates lactate recycling through gluconeogenesis. The results of this study provide insight into the therapeutic roles of MK-8617, a pan-inhibitor of PHD-1, 2, and 3, in the mitigation of lactatemia in anesthetized rats with polytrauma and hemorrhagic shock. Additionally, in an anesthetized rat model of lethal decompensated hemorrhagic shock, acute administration of MK-8617 significantly improves one-hour survival and maintains survival at least until 4 h following limited resuscitation with whole blood (20% EBV) at one hour after hemorrhage. This study suggests that pharmaceutical interventions to inhibit prolyl hydroxylase activity can be used as a potential pre-hospital countermeasure for trauma and hemorrhage at or near the point of injury. [ABSTRACT FROM AUTHOR]

With the aging of the population, an increasing number of elderly patients are opting for artificial joint replacement, leading to the exploration of various rapid rehabilitation programs in the perioperative period. In this study, we aimed to investigate the effectiveness of combining recombinant human erythropoietin and iron sucrose in altering the range and trend of postoperative hemoglobin in patients undergoing arthroplasty. Specifically, we will examine whether this combination can effectively alter the rise and fall of postoperative haemoglobin, identify the inflection point of haemoglobin change or recovery after arthroplasty, and assess the effect of treatment on serum iron in postoperative blood. We conducted a retrospective study of 138 patients who underwent unilateral total joint arthroplasty by the same surgeon in the same hospital before July 2022. The results of this study may provide valuable insights for the development of effective rehabilitation programs for patients undergoing arthroplasty. [ABSTRACT FROM AUTHOR]

MIS-C is a systemic inflammation disorder with poorly characterised immunopathological mechanisms. We compared changes in the systemic immune response in children with MIS-C (n = 12, 5–13 years) to healthy controls (n = 14, 5–15 years). Analysis was done in whole blood treated with LPS. Expression of CD11b and Toll-like receptor-4 (TLR4) in neutrophils and monocytes were analysed by flow cytometry. Serum cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-Ira, TNF-α, TNF-β, IFN-Υ, VEGF, EPO and GM-CSF) and mRNA levels of inflammasome molecules (NLRP3, ASC and IL-1β) were evaluated. Subpopulations of lymphocytes (CD3+, CD19+, CD56+, CD4+, CD8+, TCR Vδ1+, TCR Vδ2+) were assessed at basal levels. Absolute counts of neutrophils and NLR were high in children with MIS-C while absolute counts of lymphocytes were low. Children with MIS-C had increased levels of IL-6, IL-10, TNF-β and VEGF serum cytokines at the basal level, and significantly increased TNF-β post-LPS, compared to controls. IL-1RA and EPO decreased at baseline and post-LPS in MIS-C patients compared to controls. The percentage of CD3+ cells, NK cells and Vδ1 was lower while B cells were higher in children with MIS-C than in controls. Dysregulated immune response in children with MIS-C was evident and may be amenable to immunomodulation. [ABSTRACT FROM AUTHOR]

Myelination is a process tightly regulated by a variety of neurotrophic factors. Here, we show—by analyzing two transgenic mouse lines, one overexpressing EPO selectively in the brain Tg21(PDGFB-rhEPO) and another with targeted removal of EPO receptors (EPORs) from oligodendrocyte progenitor cells (OPC)s (Sox10-cre;EpoRfx/fx mice)—a key function for EPO in regulating developmental brain myelination. Overexpression of EPO resulted in faster postnatal brain growth and myelination, an increased number of myelinating oligodendrocytes, faster axonal myelin ensheathment, and improved motor coordination. Conversely, targeted ablation of EPORs from OPCs reduced the number of mature oligodendrocytes and impaired motor coordination during the second postnatal week. Furthermore, we found that EPORs are transiently expressed in the subventricular zone (SVZ) during the second postnatal week and EPO increases the postnatal expression of essential oligodendrocyte pro-differentiation and pro-maturation (Nkx6.2 and Myrf) transcripts, and the Nfatc2/calcineurin pathway. In contrast, ablation of EPORs from OPCs inactivated the Erk1/2 pathway and reduced the postnatal expression of the transcripts. Our results reveal developmental time windows in which EPO therapies could be highly effective for stimulating oligodendrocyte maturation and myelination. [ABSTRACT FROM AUTHOR]

With the aging of the population, an increasing number of elderly patients are opting for artificial joint replacement, leading to the exploration of various rapid rehabilitation programs in the perioperative period. In this study, we aimed to investigate the effectiveness of combining recombinant human erythropoietin and iron sucrose in altering the range and trend of postoperative hemoglobin in patients undergoing arthroplasty. Specifically, we will examine whether this combination can effectively alter the rise and fall of postoperative haemoglobin, identify the inflection point of haemoglobin change or recovery after arthroplasty, and assess the effect of treatment on serum iron in postoperative blood. We conducted a retrospective study of 138 patients who underwent unilateral total joint arthroplasty by the same surgeon in the same hospital before July 2022. The results of this study may provide valuable insights for the development of effective rehabilitation programs for patients undergoing arthroplasty. [ABSTRACT FROM AUTHOR]

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation. [ABSTRACT FROM AUTHOR]

Radiation injury from medical, accidental, or intentional sources can induce acute and long-term hepatic dysregulation, fibrosis, and cancer. This long-term hepatic dysregulation decreases quality of life and may lead to death. Our goal in this study is to determine acute changes in biological pathways and discover potential RNA biomarkers predictive of radiation injury. We performed whole transcriptome microarray analysis of mouse liver tissue (C57BL/6 J) 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray to identify significant expression changes in mRNAs, lncRNAs, and miRNAs, We also validated changes in specific RNAs through qRT-PCR. We used Ingenuity Pathway Analysis (IPA) to identify pathways associated with gene expression changes. We observed significant dysregulation of multiple mRNAs across all doses. In contrast, miRNA dysregulation was observed upwards of 2 Gray. The most significantly upregulated mRNAs function as tumor suppressors: Cdkn1a, Phlda3, and Eda2r. The most significantly downregulated mRNAs were involved in hemoglobin synthesis, inflammation, and mitochondrial function including multiple members of Hbb and Hba. The most significantly upregulated miRNA included: miR-34a-5p, miR-3102-5p, and miR-3960, while miR-342-3p, miR-142a-3p, and miR-223-3p were most significantly downregulated. IPA predicted activation of cell cycle checkpoint control pathways and inhibition of pathways relevant to inflammation and erythropoietin. Clarifying expression of mRNA, miRNA and lncRNA at a short time point (48 h) offers insight into potential biomarkers, including radiation markers shared across organs and animal models. This information, once validated in human models, can aid in development of bio-dosimetry biomarkers, and furthers our understanding of acute pathway dysregulation. [ABSTRACT FROM AUTHOR]

The objective of this study was to explore intracellular molecular changes during the differentiation of human-induced pluripotent stem cells (iPSCs) into erythropoietin (EPO)-producing cells using Raman spectroscopy and imaging. Raman imaging data of fixed cells at four stages of cell differentiation were analyzed by a partial least squares (PLS) regression model, and the variations in the intracellular molecular compositions with cell differentiation were investigated. As a result, three biomarkers characterizing the cell phases were identified: dimethyl sulfoxide (DMSO), fatty acids with a low grade of unsaturation, and glycoproteins. The uptake of DMSO by EPO-producing cells, which was added into a culture medium as an inducer for cell differentiation, was detected, and the increase in unsaturated fatty acid concentrations was revealed that lipid metabolism changed over the course of cell differentiation. The decrease in the glycoprotein concentration after the cell phase during which iPSCs differentiated into EPO-producing cells was also made clear. Raman imaging successfully visualized chemical images of these three biomarkers in two dimensions, where the biomarker concentrations independently varied during cell differentiation. These results demonstrated the application potential of the proposed method to regenerative medicine for monitoring cell differentiation and discriminating cell maturation in situ at the molecular level. [ABSTRACT FROM AUTHOR]

Human metallothionein-2A (MT2A) protein participates in metal homeostasis, detoxification, oxidative stress reduction, and immune defense. It decreases heavy metal ions and reactive oxygen species (ROS) during injury of cells and tissues. The single nucleotide polymorphisms at the MT2A gene have been associated in various human diseases including cancer. The current study aimed to elucidate associations between MT2A genotypes with the clinical, biochemical, and molecular characteristics that potentially related to lowered MT2A ex-pression. One hundred and forty-one healthy Taiwanese subjects were enrolled from Changhua Show-Chwan Memorial Hospital. Clinical, biochemical and molecular characteristics including the frequent minor allele SNPs, rs28366003 and rs10636, within the MT2A gene were determined. The genotype distribution of MT2A rs10636 fits the Hardy–Weinberg equilibrium. The significant associations with gradually decline of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were identified with MT2A rs10636 and rs28366003 using analysis of variance (ANOVA) with Tukey's analysis as a post hoc test. We further validated the correlations between the expressions of genes in erythropoiesis, cholesterol synthesis, platelet synthesis, insulin with MT2A using the web-based Gene Expression Profiling Interactive Analysis (GEPIA) databases. The results revealed that hypoxia-inducible factor 1α (HIF-1α), erythropoietin (EPO), lipoprotein lipase (LPL), and lecithin-cholesterol acyltransferase (LCAT) mRNA ex-pression are significantly correlated with MT2A mRNA expression. In conclusion, these results suggested that genetic variations of MT2A rs10636 and rs28366003 might be an important risk factor for erythropoiesis in the Taiwanese general population. [ABSTRACT FROM AUTHOR]

Akt and nuclear factor kappa B (NF-κB) signaling pathways are involved in germ cell apoptosis and inflammation after testicular heat stress (THS). We observed that after THS induced by the exposure of rat testes to 43 °C for 20 min, their weight decreased, the fraction of apoptotic testicular germ cells significantly increased, and the proliferation of germ cells was inhibited. In addition, THS lowered serum testosterone (T) level, whereas the levels of follicle stimulating hormone and luteinizing hormone were not significantly changed. The ultrastructure of the seminiferous tubules became abnormal after THS, the structure of the blood-testis barrier (BTB) became loose, and the Sertoli cells showed a trend of differentiation. The level of phosphorylated Akt was reduced, whereas the amount of phosphorylated NF-κB p65 was augmented by THS. Wuzi-Yanzong (WZYZ), a classic Chinese medicine prescription for the treatment of male reproductive dysfunctions, alleviated the changes induced by THS. In order to determine the mechanism of action of WZYZ, we investigated how this preparation modulated the levels of T, androgen receptor (AR), erythropoietin (EPO), EPO receptor, and Tyro-3, Axl, and Mer (TAM) family of tyrosine kinase receptors. We found that WZYZ activated the Akt pathway, inhibited the Toll-like receptor/MyD88/NF-κB pathway, and repaired the structure of BTB by regulating the levels of T, AR, TAM receptors, and EPO. In conclusion, these results suggest that WZYZ activates the Akt pathway and inhibits the NF-κB pathway by acting on the upstream regulators, thereby improving spermatogenesis deficit induced by THS. [ABSTRACT FROM AUTHOR]