Effect of Pethidine Injection on the Duration of Labor and Pregnancy Outcomes: A Retrospective Cohort Study.

Background and Objectives: Long and ineffective labor causes hardships for mothers and doctors and increases the rate of cesarean sections and medical comorbidities. Several factors contribute to effective and less painful labor, including maternal age, parity, fetal characteristics, and the medications or procedures that obstetricians use for labor. We aimed to study the factors that affect labor duration and identify those that make labor more effective. Materials and Methods: This retrospective study included 141 patients who underwent normal vaginal deliveries at the Daegu Catholic University Medical Center between April 2013 and April 2022. Among the 141 patients, 44 received pethidine intravenously, 88 received oxytocin intravenously, and 64 received epidural anesthesia. The duration of the active phase and second stage of labor were recorded according to the findings of a manual examination of the cervix and continuous external electronic monitoring. We analyzed maternal and neonatal medical records and performed binomial logistic regression to identify the factors associated with a shorter active phase of labor. The clinical outcomes in mothers and neonates were also evaluated. Results: Among the various clinical factors, multiparity (odds ratio of parity 0.325) and the use of pethidine (odds ratio 2.906) were significantly associated with shortening the active phase of labor to less than 60 min. The use of epidural anesthesia or oxytocin was not significantly associated with reducing the active phase of labor. When patients were divided into two groups based on whether a pethidine injection had been used during labor, the duration of the active phase was shorter in the pethidine injection group than in the control group for both nulliparas and multiparas. No significant differences in the duration of the second stage of labor were observed between the pethidine injection and control groups. There were no significant differences in pregnancy outcomes, including the need for mechanical ventilation of neonates, Apgar scores, neonatal intensive care unit admissions, number of precipitous deliveries, maternal adverse side effects of drugs, or duration of maternal hospitalization between the two groups. Conclusions: Pethidine can be safely administered to women during labor to help reduce the duration of the active phase by promoting dilatation of the cervix and preventing complications that may result from prolonged labor. Pethidine may be helpful, especially for those who cannot receive epidural anesthesia or who cannot afford it. However, large-scale randomized controlled studies are required to evaluate the efficacy and safety of this drug during labor. Furthermore, it would be helpful if various studies were conducted depending on the timing of administration and indications for delivery.

Association between Patients' Self-Judgement, Coagulated Menstrual Blood, and Menorrhagia: Results from a Questionnaire Survey and Blood Test Analysis.

Background and Objectives: Menorrhagia is defined as a blood loss of more than 80 mL, which is significant enough to cause anemia. Previously known methods for evaluating menorrhagia, such as the alkalin-hematin method, pictograms, and measuring the weight of sanitary products, were all impractical, complex, and time-consuming. Therefore, this study aimed to determine which item among menstrual history taking was most associated with menorrhagia and devised a simple evaluating method for menorrhagia through history taking that can be applied clinically. Materials and Methods: The study was conducted from June 2019 to December 2021. A survey was conducted on premenopausal women who underwent outpatient treatment or surgery and those who underwent a gynecologic screening test, and their blood tests were analyzed. The presence of iron deficiency anemia was identified with a Hb level of less than 10 g/dL with microcytic hypochromic anemia on a complete blood count performed within one month of the survey. A questionnaire survey was conducted on six items related to menorrhagia to investigate whether each item was related to "significant menorrhagia". Results: There were 301 participants in the survey during the period. In univariate analysis, the results revealed a statistically significant association between significant menorrhagia and the following items: self-judgement of menorrhagia; menstruation lasting over 7 days; total pad counts in a single menstrual period; Number of sanitary products changed per day; and leakaging of menstrual blood and presence of coagulated menstrual blood. In multivariate analysis, only the "self-judgement of menorrhagia" item showed a statistically significant result (p-value = 0.035; an odds ratio = 2.217). When the "self-judgement of menorrhagia" item was excluded, the "passage of clots larger than one inch in diameter" item showed a statistically significant result (p-value = 0.023; an odds ratio = 2.113). Conclusions: "Patient self-judgement of menorrhagia" is a reliable item for evaluating menorrhagia. Among several symptoms indicating menorrhagia, determining the presence of the "passage of clots larger than one inch in diameter" during the menstrual period is the most useful item for evaluating menorrhagia in clinical history taking. This study suggested using these simple menstrual history taking items to evaluate menorrhagia in real clinical practice.

Prognostic factors in women with cervical cancer stage IIIC1r treated with concurrent chemoradiotherapy.

AIM: The purpose of this study was to evaluate the prognostic factors of patients with stage IIIC1r cervical cancer who underwent concurrent chemoradiotherapy. METHODS: A total of 134 patients treated with chemoradiotherapy for cervical cancer with pelvic and/or paraaortic lymph node metastasis (PALNM) were enrolled in this study. Clinical variables were investigated through review of the patients' medical records. RESULTS: The 5-year overall survival (OS) rate in patients with stage IIICr cervical cancer was 70.5%. Age, PALNM, parametrial invasion, T stage, pelvic side wall invasion, differentiation, lymphovascular space involvement and high squamous cell carcinoma antigen level (>8 ng/mL) were prognostic factors for survival. The 5-year OS rate of patients with stage IIIC1r was 74.5%, and that of stage IIIC2r was 38.1% (P-value = 0.012). The 5-year OS rate of patients with stage IIIC1r with the presence of pelvic side wall invasion was 48.3% and that in its absence was 83.0% (P-value < 0.001). The 5-year OS rate of patients with stage IIIC1r with the presence of parametrial invasion was 68.9% and that in its absence was 82.4% (P-value = 0.031). In multivariable analysis via backward conditional modeling, age, PALNM and pelvic side wall invasion were independent prognostic factors for survival of stage IIICr. Age and pelvic side wall invasion were independent prognostic factors for survival of stage IIIC1r cervical cancer. CONCLUSION: In stage IIICr cervical cancer, patients with PALNM, and/or pelvic side wall invasion can expect to have a poor prognosis. Particularly, pelvic side wall invasion in stage IIIC1r is an independent prognosis factor.

Bleeding control using intrauterine continuous running suture during cesarean section in pregnant women with placenta previa.

PURPOSE: The purpose of this study was to evaluate the effectiveness of intrauterine continuous running suture during cesarean section in pregnant women with placenta previa. METHODS: We enrolled 277 women and medical records were retrospectively reviewed. Pregnant women were grouped according to uterine bleeding control methods as follows: Group A, using intrauterine continuous running suture and Group B (control group) using figure-of-eight suture. RESULTS: Intrauterine continuous running sutures were used in 104 pregnant women. Mean total blood loss in Group A was significantly less than that in Group B (1332.70 ± 152.92 mL vs 1861.56 ± 157.74 mL, P = 0.029). Mean total transfusion unit of Group A was significantly less than that in Group B (1.74 ± 0.41 vs 3.52 ± 0.75, P = 0.037). CONCLUSIONS: Intrauterine continuous running sutures can significantly reduce postpartum blood loss and transfusion units during cesarean section in pregnant women with placenta previa.

Purification of small molecule-induced cardiomyocytes from human induced pluripotent stem cells using a reporter system.

In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/ß-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research.

Fucoidan improves bioactivity and vasculogenic potential of mesenchymal stem cells in murine hind limb ischemia associated with chronic kidney disease.

Chronic kidney disease (CKD) is a significant risk factor for cardiovascular and peripheral vascular disease. Although mesenchymal stem cell (MSC)-based therapy is a promising strategy for treatment of ischemic diseases associated with CKD, the associated pathophysiological conditions lead to low survival and proliferation of transplanted MSCs. To address these limitations, we investigated the effects of fucoidan, a sulfated polysaccharide, on the bioactivity of adipose tissue-derived MSCs and the potential of fucoidan-treated MSCs to improve neovascularization in ischemic tissues of CKD mice. Treatment of MSCs with fucoidan increased their proliferative potential and the expression of cell cycle-associated proteins, such as cyclin E, cyclin dependent kinase (CDK) 2, cyclin D1, and CDK4, via focal adhesion kinase and the phosphatidylinositol-4,5-bisphosphate 3-kinase-Akt axis. Moreover, fucoidan enhanced the immunomodulatory activity of MSCs through the ERK-IDO-1 signal cascade. Fucoidan was found to augment the proliferation, incorporation, and endothelial differentiation of transplanted MSCs at ischemic sites in CKD mice hind limbs. In addition, transplantation of fucoidan-treated MSCs enhanced the ratio of blood flow and limb salvage in CKD mice with hind limb ischemia. To our knowledge, our findings are the first to reveal that fucoidan enhances the bioactivity of MSCs and improves their neovascularization in ischemic injured tissues of CKD. In conclusion, fucoidan-treated MSCs may provide an important pathway toward therapeutic neovascularization in patients with CKD.

Oleic acid enhances the motility of umbilical cord blood derived mesenchymal stem cells through EphB2-dependent F-actin formation.

The role of unsaturated fatty acids (UFAs) is essential for determining stem cell functions. Eph/Ephrin interactions are important for regulation of stem cell fate and localization within their niche, which is significant for a wide range of stem cell behavior. Although oleic acid (OA) and Ephrin receptors (Ephs) have critical roles in the maintenance of stem cell functions, interrelation between Ephs and OA has not been explored. Therefore, the present study investigated the effect of OA-pretreated UCB-MSCs in skin wound-healing and underlying mechanism of Eph expression. OA promoted the motility of UCB-MSCs via EphB2 expression. OA-mediated GPR40 activation leads to Gαq-dependent PKCα phosphorylation. In addition, OA-induced phosphorylation of GSK3ß was followed by ß-catenin nuclear translocation in UCB-MSCs. Activation of ß-catenin was blocked by PKC inhibitors, and OA-induced EphB2 expression was suppressed by ß-cateninsiRNA transfection. Of those Rho-GTPases, Rac1 was activated in an EphB2-dependent manner. Accordingly, knocking down EphB2 suppressed F-actin expression. In vivo skin wound-healing assay revealed that OA-treated UCB-MSCs enhanced skin wound repair compared to UCB-MSCs pretreated with EphB2siRNA and OA. In conclusion, we showed that OA enhances UCB-MSC motility through EphB2-dependent F-actin formation involving PKCα/GSK3ß/ß-catenin and Rac1 signaling pathways.

VvpE mediates the intestinal colonization of Vibrio vulnificus by the disruption of tight junctions.

The disruption of gastrointestinal tight junctions and their colonization evoked by enteric pathogens are hallmarks of the pathogenesis. Vibrio (V.) vulnificus, VvpE, is an elastase which is responsible for host surface adherence and vascular permeability; however, the functional roles of VvpE in the pathogenesis of V. vulnificus (WT) are poorly understood. In the present study, we have investigated the role of VvpE in regulation of intestinal tight junctions and the colonization of WT. We found that mutation of the vvpE gene from V. vulnificus (vvpE mutant) prevents intestinal tight/adherens junction dysregulation due to a WT infection and maintains the physiological level of the epithelial paracellular permeability. Interestingly, the vvpE mutant exhibited defective intestinal colonization abilities, whereas WT colonization was significantly elevated in the ileum in a time-dependent manner. Finally, the vvpE mutant negated the enterotoxicity, the breakdown of red blood cells, and pro-inflammatory responses, all of which are induced by the WT infection. In addition, the results of a LC-MS/MS analysis showed that VvpE contributes to WT pathogenesis in multiple ways by interacting with intestinal proteins, including ß-globin, Annexin A2, Annexin A4, F-actin, and intelectin-1b. These results demonstrate that VvpE plays important role in promoting the tight junction disruption and intestinal colonization of V. vulnificus and that it also has the ability to interact with the intestinal proteins responsible for microbial pathogenesis.

Autotaxin-LPA axis regulates hMSC migration by adherent junction disruption and cytoskeletal rearrangement via LPAR1/3-dependent PKC/GSK3ß/ß-catenin and PKC/Rho GTPase pathways.

Bioactive molecules and stem cell-based regenerative engineering is emerging a promising approach for regenerating tissues. Autotaxin (ATX) is a key enzyme that regulates lysophosphatidic acid (LPA) levels in biological fluids, which exerts a wide range of cellular functions. However, the biological role of ATX in human umbilical cord blood-derived mesenchymal stem cells (hMSCs) migration remains to be fully elucidated. In this study, we observed that hMSCs, which were stimulated with LPA, accelerated wound healing, and LPA increased the migration of hMSCs into a wound site in a mouse skin wound healing model. In an experiment to investigate the effect of LPA on hMSC migration, ATX and LPA increased hMSC migration in a dose-dependent manner, and LPA receptor 1/3 siRNA transfections inhibited the ATX-induced cell migration. Furthermore, LPA increased Ca(2+) influx and PKC phosphorylation, which were blocked by Gαi and Gαq knockdown as well as by Ptx pretreatment. LPA increased GSK3ß phosphorylation and ß-catenin activation. LPA induced the cytosol to nuclear translocation of ß-catenin, which was inhibited by PKC inhibitors. LPA stimulated the binding of ß-catenin on the E-box located in the promoter of the CDH-1 gene and decreased CDH-1 promoter activity. In addition, the ATX and LPA-induced increase in hMSC migration was blocked by ß-catenin siRNA transfection. LPA-induced PKC phosphorylation is also involved in Rac1 and CDC42 activation, and Rac1 and CDC42 knockdown abolished LPA-induced F-actin reorganization. In conclusion, ATX/LPA stimulates the migration of hMSCs through LPAR1/3-dependent E-cadherin reduction and cytoskeletal rearrangement via PKC/GSK3ß/ß-catenin and PKC/Rho GTPase pathways.

Novel Pathway for Hypoxia-Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF-1α, FASN, and mTORC1.

The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCB-hMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1α)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCB-hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1α/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. Stem Cells 2015;33:2182-2195.

C(16)-Ceramide-induced F-actin regulation stimulates mouse embryonic stem cell migration: involvement of N-WASP/Cdc42/Arp2/3 complex and cofilin-1/α-actinin.

Ceramide, a major structural element in the cellular membrane, is a key regulatory factor in various cellular behaviors that are dependent on ceramide-induced association of specific proteins. However, molecular mechanisms that regulate ceramide-induced embryonic stem cell (ESC) migration are still not well understood. Thus, we investigated the effect of ceramide on migration and its related signal pathways in mouse ESCs. Among ceramide species with different fatty acid chain lengths, C(16)-Cer increased migration of mouse ESCs in a dose- (≥1µM) and time-dependent (≥8h) manners, as determined by the cell migration assay. C(16)-Cer (10µM) increased protein-kinase C (PKC) phosphorylation. Subsequently, C(16)-Cer increased focal adhesion kinase (FAK) and Paxillin phosphorylation, which were inhibited by PKC inhibitor Bisindolylmaleimide I (1µM). When we examined for the downstream signaling molecules, C(16)-Cer activated small G protein (Cdc42) and increased the formation of complex with Neural Wiskott-Aldrich Syndrome Protein (N-WASP)/Cdc42/Actin-Related Protein 2/3 (Arp2/3). This complex formation was disrupted by FAK- and Paxillin-specific siRNAs. Furthermore, C(16)-Cer-induced increase of filamentous actin (F-actin) expression was inhibited by Cdc42-, N-WASP-, and Arp2/3-specific siRNAs, respectively. Indeed, C(16)-Cer increased cofilin-1/F-actin interaction or F-actin/α-actinin-1 and α-actinin-4 interactions in the cytoskeleton compartment, which was reversed by Cdc42-specific siRNA. Finally, C(16)-Cer-induced increase of cell migration was inhibited by knocking down each signal pathway-related molecules with siRNA or inhibitors. In conclusion, C(16)-Cer enhances mouse ESC migration through the regulation of PKC and FAK/Paxillin-dependent N-WASP/Cdc42/Arp2/3 complex formation as well as through promoting the interaction between cofilin-1 or α-actinin-1/-4 and F-actin.

Glucosamine-induced Sp1 O-GlcNAcylation ameliorates hypoxia-induced SGLT dysfunction in primary cultured renal proximal tubule cells.

The aim of this study is to determine whether GlcN could recover the endoplasmic reticulum (ER) stress-induced dysfunction of Na(+) /glucose cotransporter (SGLT) in renal proximal tubule cells (PTCs) under hypoxia. With the rabbit model, the renal ischemia induced tubulointerstitial abnormalities and decreased SGLTs expression in tubular brush-border, which were recovered by GlcN. Thus, the protective mechanism of GlcN against renal ischemia was being examined by using PTCs. Hypoxia decreased the level of protein O-GlcNAc and the expression of O-GlcNAc transferase (OGT) while increased O-GlcNAcase (OGA) and these were reversed by GlcN. Hypoxia also decreased the expression of SGLTs (SGLT1 and 2) and [(14) C]-α-methyl-D-glucopyranoside (α-MG) uptake which were recovered by GlcN and PUGNAc (OGA inhibitor). Hypoxia enhanced reactive oxygen species (ROS) and then ER stress proteins, glucose-regulated protein 78 (GRP78), and C/EBP-homologous protein (CHOP). However, the expression of GRP78 increased till 6 h and then decreased whereas CHOP increased gradually. Moreover, decreased GRP78 and increased CHOP were reversed by NAC (antioxidant) and GlcN. GlcN ameliorated hypoxia-induced decrease of O-GlcNAc modification of Sp1 but OGT or Sp1 siRNAs blocked the recovery effect of GlcN on SGLT expression and α-MG uptake. In addition, hypoxia-decreased GRP78 and HIF-1α expression was reversed by GlcN but OGT siRNA or Sp1 siRNA ameliorated the effect of GlcN. When PTCs were transfected with GRP78 siRNA or HIF-1α siRNA, SGLT expression and α-MG uptake was decreased. Taken together, these data suggest that GlcN-induced O-GlcNAc modified Sp1 with stimulating GRP78 and HIF-1α activity ameliorate hypoxia-induced SGLT dysfunction in renal PTCs. J. Cell. Physiol. 229: 1557-1568, 2014. © 2014 Wiley Periodicals, Inc.

Blood levels of cadmium and lead in residents near abandoned metal mine areas in Korea.

We analyzed national data on blood lead levels (BLL) and blood cadmium levels (BCL) in residents living near 38 abandoned metal mining areas (n = 5,682, 18-96 years old) in Korea that were collected by the first Health Effect Surveillance for Residents in Abandoned Metal mines (HESRAM) from 2008 to 2011. The geometric mean BCL and BLL were 1.60 µg/L (95 % CI = 1.57-1.62 µg/L) and 2.87 µg/dL (95 % CI = 2.84-2.90 µg/dL), respectively, notably higher than levels in the general population in Korea and other countries. We found significantly higher BLL and BCL levels in people living within 2 km of an abandoned metal mine (n = 3,165, BCL = 1.87 µg/L, BLL = 2.91 µg/dL) compared to people living more than 2 km away (n = 2,517, BCL = 1.31 µg/L, BLL = 2.82 µg/dL; P < 0.0001) and to the general population values reported in the literature.

Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-ß-cyclodextrin (MßCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: involvement of RhoA, Ca(2+)/CaM, and ERK.

BACKGROUND: Extracellular matrix (ECM) components and intracellular pH (pH(i)) may serve as regulators of cell migration in various cell types. METHODS: The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na(+)/H(+) exchanger (NHE)-1 activity was evaluated by measuring pH(i) and [(22)Na(+)] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed. RESULTS: ECM components (FN, laminin, fibrinogen, and collagen type I) increased [(22)Na(+)] uptake, pH(i), and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin ß1 and subsequently stimulates caveolin-1 phosphorylation and Ca(2+) influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca(2+)/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [(22)Na(+)] uptake and pH(i). Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration. CONCLUSIONS: FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca(2+)/CaM signaling pathways. GENERAL SIGNIFICANCE: The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.

PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways.

BACKGROUND: Regulation of glucose transporter (GLUT) expression and activity plays a vital role in the supply of glucose to embryonic stem (ES) cells. METHODS: To observe the effect of 6-phenyl cyclic monophosphate (cAMP) on glucose uptake and cell proliferation, 2-deoxyglucose (2-DG) uptake, immunohistochemistry, Western blotting, and immunoprecipitation were carried out. RESULTS: Among GLUT isoforms in mouse ES cells, GLUT1 was predominantly expressed and 6-phenyl cAMP increased GLUT mRNA levels. Among cAMP agonists, 6-phenyl cAMP increased 2-DG uptake more than that of 8-p-chlorophenylthio-2'-O-methyl-cAMP. 6-Phenyl cAMP increased GLUT1 expression and translocation from the cytosol to the plasma membrane. 6-Phenyl cAMP increased 2-DG uptake in a time- and concentration-dependent manner due to an increase in V(max) but not K(m). 6-Phenyl cAMP increased phosphorylation of nuclear factor-κB (NF-κB) and cAMP response element binding (CREB) and expression of the CREB protein (CBP) and transducer of regulated CREB activity 2 (TORC2) in sequence. 6-Phenyl cAMP induced complex formation of NF-κB/CREB/CBP/TORC2, which are involved in the increase of gluconeogenic enzyme expression. 6-Phenyl cAMP also increased cell cycle regulatory protein expression levels, the proportion of S-phase cells, and proto-oncogene expression via protein kinase A (PKA)-dependent NF-κB signaling. Finally, GLUT1 siRNA blocked the 6-phenyl cAMP-induced increase in ES cell proliferation. We conclude that PKA stimulated the complex formation of CREB/CBP/TORC2 via NF-κB, which induced effective coordination of glucose uptake as well as proliferation in ES cells. GENERAL SIGNIFICANCE: 6-Phenyl cAMP-induced PKA activation modified the proliferation, which may be beneficial for expanding ES cell use to cell therapy.

LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.

Quorum sensing is a cell-to-cell communication system known to control many bacterial processes. In the present study, the functions of quorum sensing in the pathogenesis of Vibrio vulnificus, a food-borne pathogen, were assessed by evaluating the virulence of a mutant deficient in SmcR, a quorum-sensing regulator and homologue of LuxR. When biofilms were used as an inoculum, the smcR mutant was impaired in virulence and colonization capacity in the infection of mice. The lack of SmcR also resulted in decreased histopathological damage in mouse jejunum tissue. These results indicated that SmcR is essential for V. vulnificus pathogenesis. Moreover, the smcR mutant exhibited significantly reduced biofilm detachment. Upon exposure to INT-407 host cells, the wild type, but not the smcR mutant, revealed accelerated biofilm detachment. The INT-407 cells increased smcR expression by activating the expression of LuxS, an autoinducer-2 synthase, indicating that host cells manipulate the cellular level of SmcR through the quorum-sensing signaling of V. vulnificus. A whole-genome microarray analysis revealed that the genes primarily involved in biofilm detachment and formation are up- and downregulated by SmcR, respectively. Among the SmcR-regulated genes, vvpE encoding an elastolytic protease was the most upregulated, and the purified VvpE appeared to dissolve established biofilms directly in a concentration-dependent manner in vitro. These results suggest that the host cell-induced SmcR enhances the detachment of V. vulnificus biofilms entering the host intestine and thereby may promote the dispersal of the pathogen to new colonization loci, which is crucial for pathogenesis.

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition.

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt(thr308) and (ser473); these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.

Prostaglandin E2 maintains mouse ESC undifferentiated state through regulation of connexin31, connexin43 and connexin45 expression: involvement of glycogen synthase kinase 3ß/ß-catenin.

BACKGROUND INFORMATION: Although previous reports have examined the function of prostaglandin E2 (PGE2) on gap junctions and undifferentiated stem cells, its effects on the reciprocal action of connexin (Cx) isoforms and undifferentiation in embryonic stem cells (ESCs) are unclear. Therefore, we investigated the role of PGE2 on Cx isoforms and maintenance of mouse ESC undifferentiated state. RESULTS: We have analysed 10 Cx genes, but found nine of them. PGE2 (50 µM) stimulated Cx31, Cx32, Cx40, Cx43 and Cx45 mRNA expression. Amongst them, PGE2 maximally stimulated the Cx43 mRNA expression and gap junction inter-cellular coupling. Therefore, we investigated the effect of PGE2 on Cx43 expression. PGE2 activated cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation. In addition, treatments of adenylate cyclase activators increased Cx43 expression, but not PI3K/Akt phosphorylation. PGE2 also inactivated GSK-3ß and stimulated active-ß-catenin. Furthermore, a ChiP assay demonstrated the association of ß-catenin with the Cx26 (as control) and Cx43 promoter. Finally, down-regulation of PGE2-induced Cx isoforms by AH 6809, Cx31-, Cx43-, Cx45 small interfering (si)RNA and 18α-glycyrrhetinic acid decreased levels of undifferentiated markers of ESCs, including Oct4, FoxD3, Sox2 and SSEA-1, but Nanog did not be down-regulated by Cx43 siRNA. CONCLUSIONS: PGE2 stimulates Cx isoforms via GSK-3ß/ß-catenin via EP2-receptor-dependent cAMP/PKA and PI3K/Akt in mouse ESCs, thereby partially contributing to the maintenance of their undifferentiated state.

Association between Preoperative 18-FDG PET-CT SUVmax and Next-Generation Sequencing Results in Postoperative Ovarian Malignant Tissue in Patients with Advanced Ovarian Cancer.

This study investigated the association between maximum standardized uptake values (SUVmax) on preoperative 18-FDG PET-CT and next-generation sequencing (NGS) results in post-surgical ovarian malignant tissue in patients with advanced ovarian cancer. Twenty-five patients with stage IIIC or IV ovarian cancer who underwent both preoperative 18-FDG PET-CT and postoperative NGS for ovarian malignancies were retrospectively enrolled. Two patients had no detected variants, 21 of the 23 patients with any somatic variant had at least one single nucleotide variant (SNV) or insertion/deletion (indel), 10 patients showed copy number variation (CNV), and two patients had a fusion variant. SUVmax differed according to the presence of SNVs/indels, with an SUVmax of 13.06 for patients with ≥ 1 SNV/indel and 6.28 for patients without (p = 0.003). Seventeen of 20 patients with Tier 2 variants had TP53 variants, and there was a statistically significant association between SUVmax and the presence of TP53 variants (13.21 vs. 9.35, p = 0.041). Analysis of the correlation between the sum of the Tier 1 and Tier 2 numbers and SUVmax showed a statistically significant correlation (p = 0.002; Pearson's r = 0.588). In conclusion, patients with advanced ovarian cancer with SNVs/indels on NGS, especially those with TP53 Tier 2 variants, showed a proportional association with tumor SUVmax on preoperative PET-CT.