Bone marrow cells are differentiated into MDSCs by BCC-Ex through down-regulating the expression of CXCR4 and activating STAT3 signalling pathway.

Studies showed that the increase of myeloid-derived suppressor cells (MDSCs) in tumour microenvironment is closely related to the resistant treatment and poor prognosis of metastatic breast cancer. However, the effect of tumour-derived exosomes on MDSCs and its mechanism are not clear. Here, we reported that breast cancer cells (4T1)-secreted exosomes (BCC-Ex) were able to differentiate bone marrow cells into MDSCs and significantly inhibited the proliferation of T lymphocytes to provide an immunosuppressive microenvironment for cancer cells in vivo and in vitro. The number of MDSCs in bone marrow and spleen of 4T1 tumour-bearing mice and BCC-Ex infused mice was significantly higher than that of normal mice, whereas the number of T lymphocytes in spleen was significantly decreased. In addition, BCC-Ex markedly promoted the differentiation of MDSCs from bone marrow cells or bone marrow cells derived macrophages, seen as the increased expressions of MDSCs-related functional proteins Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS). Furthermore, BCC-Ex significantly down-regulated the expressions of chemokine receptor CXCR4 and markedly up-regulated the levels of inflammatory cytokines IL-6 and IL-10 in bone marrow cells and macrophages and remarkably inhibited the division and proliferation of T cells. Importantly, CXCR4 agonist, CXCL12, could reverse the function of BCC-Ex, indicating that BCC-Ex-induced MDSCs might be dependent on the down-regulation of CXCR4. Western blot showed that BCC-Ex significantly promoted the phosphorylation of STAT3 in bone marrow cells, resulting in the inhibitions of the proliferation and apoptosis of bone marrow cells, and the aggravation of the differentiation of bone marrow cells into MDSCs.

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acetaminophen-induced acute liver failure through activating ERK and IGF-1R/PI3K/AKT signaling pathway.

This study aimed to investigate the therapeutic potential of human umbilical cord mesenchymal stem cells derived exosomes (hUCMSC-Exo) in acute liver failure (ALF) in mice as well as its underlying mechanism. We found that a single tail vein administration of hucMSC-Exo effectively enhanced the survival rate, inhibited apoptosis in hepatocytes, and improved liver function in APAP-induced mouse model of ALF. Furthermore, the deletion of glutathione (GSH) and superoxide dismutase (SOD), generation of malondialdehyde (MDA), and the over production of cytochrome P450 E1 (CYP2E1) and 4-hydroxynonenal (4-HNE) caused by APAP were also inhibited by hucMSC-Exo, indicating that hucMSC-Exo inhibited APAP-induced apoptosis of hepatocytes by reducing oxidative stress. Moreover, hucMSC-Exo significantly down-regulated the levels of inflammatory cytokines IL-6, IL-1ß, and TNF-α in APAP-treated livers. Western blot showed that hucMSC-Exo significantly promoted the activation of ERK1/2 and IGF-1R/PI3K/AKT signaling pathways in APAP-injured LO2 cells, resulting in the inhibition of apoptosis of LO2 cells. Importantly, PI3K inhibitor LY294002 and ERK1/2 inhibitor PD98059 could reverse the function of hucMSC-Exo on APAP-injured LO2 cells in some extent. Our results suggest that hucMSC-Exo offer antioxidant hepatoprotection against APAP in vitro and in vivo by inhibitiing oxidative stress-induced apoptosis via upregulation of ERK1/2 and PI3K/AKT signaling pathways.

Human amniotic mesenchymal stem cells inhibit hepatocellular carcinoma in tumour-bearing mice.

Hepatocellular carcinoma (HCC) is the third leading cause of the cancer-related death in the world. Human amniotic mesenchymal stem cells (hAMSCs) have been characterized with a pluripotency, low immunogenicity and no tumorigenicity. Especially, the immunosuppressive and anti-inflammatory effects of hAMSCs make them suitable for treating HCC. Here, we reported that hAMSCs administrated by intravenous injection significantly inhibited HCC through suppressing cell proliferation and inducing cell apoptosis in tumour-bearing mice with Hepg2 cells. Cell tracking experiments with GFP-labelled hAMSCs showed that the stem cells possessed the ability of migrating to the tumorigenic sites for suppressing tumour growth. Importantly, both hAMSCs and the conditional media (hAMSC-CM) have the similar antitumour effects in vitro, suggesting that hAMSCs-derived cytokines might be involved in their antitumour effects. Antibody array assay showed that hAMSCs highly expressed dickkopf-3 (DKK-3), dickkopf-1 (DKK-1) and insulin-like growth factor-binding protein 3 (IGFBP-3). Furthermore, the antitumour effects of hAMSCs were further confirmed by applications of the antibodies or the specific siRNAs of DKK-3, DKK-1 and IGFBP-3 in vitro. Mechanically, hAMSCs-derived DKK-3, DKK-1 and IGFBP-3 markedly inhibited cell proliferation and promoted apoptosis of Hepg2 cells through suppressing the Wnt/ß-catenin signalling pathway and IGF-1R-mediated PI3K/AKT signalling pathway, respectively. Taken together, our study demonstrated that hAMSCs possess significant antitumour effects in vivo and in vitro and might provide a novel strategy for HCC treatment clinically.

Enhancement of protein mechanical stability: Correlated deformations are handcuffed by ligand binding.

As revealed by previous experiments, protein mechanical stability can be effectively regulated by ligand binding with the binding site distant from the force-bearing region. However, the mechanism for such long-range allosteric control of protein mechanics is still largely unknown. In this work, we use protein topology-based elastic network model (ENM) and all-atomic steered molecular dynamics (SMD) simulations to study the impact of ligand binding on protein mechanical stability in two systems, i.e., GB1 and CheY-binding P2-domain of CheA (CBDCheA). Both ENM and SMD results show that the ligand binding has considerable and negligible effects on the mechanical stability of these two proteins, respectively. These results are consistent with the experimental observations. A physical mechanism for the enhancement of protein mechanical stability was then proposed: the correlated deformations of the force-bearing region and the binding site are handcuffed by the binding of ligand. The handcuff effect suppresses the propagation of internal force in the force-bearing region, thus improving the resistance to the loading force. Our study indicates that ENM method can effectively identify the structure motifs allosterically related to the deformation in the force bearing region, as well as the force propagation pathway within the structure of the studied proteins. Hence, it should be helpful to understand the molecular origin of the different mechanical properties in response to ligand binding for GB1 and CBDCheA.

Catalytic Conversion of Carbon Dioxide through C-N Bond Formation.

From the viewpoint of green chemistry and sustainable development, it is of great significance to synthesize chemicals from CO2 as C1 source through C-N bond formation. During the past several decade years, many studies on C-N bond formation reaction were involved, and many efforts have been made on the theory. Nevertheless, several great challenges such as thermodynamic limitation, low catalytic efficiency and selectivity, and high pressure etc. are still suffered. Herein, recent advances are highlighted on the development of catalytic methods for chemical fixation of CO2 to various chemicals through C-N bond formation. Meanwhile, the catalytic systems (metal and metal-free catalysis), strategies and catalytic mechanism are summarized and discussed in detail. Besides, this review also covers some novel synthetic strategies to urethanes based on amines and CO2. Finally, the regulatory strategies on functionalization of CO2 for N-methylation/N-formylation of amines with phenylsilane and heterogeneous catalysis N-methylation of amines with CO2 and H2 are emphasized.

Does the Surgery-First Approach Produce Better Outcomes in Orthognathic Surgery? A Systematic Review and Meta-Analysis.

PURPOSE: The surgery-first approach (SFA) in orthognathic surgery, performed without presurgical orthodontic treatment, has gained attention, but the results remain controversial. The purpose of this study was to assess the current evidence on stability, efficacy, and surgical results of SFA versus conventional 3-stage method (CTM) orthognathic surgery. MATERIALS AND METHODS: A comprehensive search in PubMed and Web of Science was conducted. A systematic review and cumulative meta-analysis of all comparative studies were performed to assess the 2 strategies (SFA and CTM) using a random- or a fixed-effects model. Outcomes included treatment duration, postoperative stability, surgical movement, and postoperative occlusion. RESULTS: Ten nonrandomized controlled studies including 513 patients were identified. Compared with CTM, patients in the SFA group benefited from shorter total treatment duration (weighted mean difference [WMD], -5.25; 95% confidence interval [CI], -8.21 to -2.29; P = .0005), similar postoperative stability of the mandible (WMD, 0.35 mm; 95% CI, -0.24 to 0.94; P = .55) and maxilla (WMD, 0.13 mm; 95% CI, -0.35 to 0.60; P = .60), similar surgical movements, and other surgical results. CONCLUSIONS: SFA offers an efficient alternative to CTM with shorter total treatment duration, similar postoperative stability, and other surgical results but longer postoperative orthodontic time.

Amygdalin and exercise training exert a synergistic effect in improving cardiac performance and ameliorating cardiac inflammation and fibrosis in a rat model of myocardial infarction.

This study investigated the effects of amygdalin (AMY, a cyanogenic glycoside widely distributed in the fruits and seeds of Rosaceae plants) on cardiac performance and ventricular remodeling in a rat model of myocardial infarction (MI). We also investigated whether the combination of AMY with exercise training (ExT) has a beneficial synergistic effect in treating MI rats. MI was induced by the ligation of the left anterior descending coronary artery in male SD rats. ExT or AMY treatment was started 1 week after MI and continued for 1 week (short-term) or 8 weeks (long-term). Cardiac function was evaluated by echocardiographic and hemodynamic parameters. Heart tissues were harvested and subjected to 2,3,5-triphenyl-tetrazolium chloride, Masson's trichrome, hematoxylin-eosin, and immunohistochemical staining. Gene expression was determined by quantitative polymerase chain reaction. Western blot gave a qualitative assessment of protein levels. AMY or ExT improved cardiac function and reduced infarct size in MI rats. AMY or ExT also suppressed myocardial fibrosis and attenuated inflammation in the infarct border zone of hearts from MI rats, as evidenced by inhibition of collagen deposition, inflammatory cell infiltration, and pro-inflammatory markers (interleukin 1ß, interleukin 6, tumor necrosis factor-α, and cyclooxygenase 2). Notably, the effects of AMY combined with ExT were superior to those of AMY alone or ExT alone. Mechanistically, these beneficial functions were correlated with the inhibition of MI-induced activation of the transforming growth factor-ß/Smad pathway. Collectively, AMY and ExT exert a synergistic effect on improving cardiac performance and ameliorating cardiac inflammation and fibrosis after MI, and the effects of long-term intervention were better than short-term intervention.

Human amniotic mesenchymal stem cell-islet organoids enhance the efficiency of islet engraftment in a mouse diabetes model.

AIMS: Despite islet transplantation has proved a great potential to become the standard therapy for type 1 diabetes mellitus (T1DM), this approach remains limited by ischemia, hypoxia, and poor revascularization in early post-transplant period as well as inflammation and life-long host immune rejection. Here, we investigate the potential and mechanism of human amniotic mesenchymal stem cells (hAMSCs)-islet organoid to improve the efficiency of islet engraftment in immunocompetent T1DM mice. MAIN METHODS: We generated the hAMSC-islet organoid structure through culturing the mixture of hAMSCs and islets on 3-dimensional-agarose microwells. Flow cytometry, whole-body fluorescent imaging, immunofluorescence, Calcein-AM/PI staining, ELISA, and qPCR were used to assess the potential and mechanism of shielding hAMSCs to improve the efficiency of islet transplantation. KEY FINDINGS: Transplant of hAMSC-islet organoids results in remarkably better glycemic control, an enhanced glucose tolerance, and a higher ß cell mass in vivo compared with control islets. Our results show that hAMSCs shielding provides an immune privileged microenvironment for islets and promotes graft revascularization in vivo. In addition, hAMSC-islet organoids show higher viability and reduced dysfunction after exposure to hypoxia and inflammatory cytokines in vitro. Finally, our results show that shielding with hAMSCs leads to the activation of PKA-CREB-IRS2-PI3K and PKA-PDX1 signaling pathways, up-regulation of SIL1 mRNA levels, and down-regulation of MT1 mRNA levels in ß cells, which ultimately promotes the synthesis, folding and secretion of insulin, respectively. SIGNIFICANCE: hAMSC-islet organoids can evidently increase the efficiency of islet engraftment and might develop into a promising alternative for the clinical treatment of T1DM.

MiR-30c-1-3p targets matrix metalloproteinase 9 involved in the rupture of abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) can be fatal if ruptured, but there is no predictive biomarker. Our aim was to evaluate the prognostic potential of microRNAs (miRNAs/miRs) in an AAA mouse model and patients with unruptured AAA (URAAA) and ruptured AAA (RAAA). Among the 64 miRNAs differentially expressed in mice with AAA compared to control, miR-30c-1-3p, miR-432-3p, miR-3154, and miR-379-5p had high homology with human miRNAs. MiR-30c-1-3p plasma levels were significantly lower in patients with RAAA than in those with URAAA or control and tended to negatively correlate with the maximum aortic diameter (r = -0.3153, P = 0.06109). MiR-30c-1-3p targeted matrix metalloproteinase (MMP)-9 mRNA through the coding region and downregulated its expression in vitro. MMP-9 plasma concentrations were significantly higher in the RAAA group than in the URAAA group (P < 0.001) and were negatively associated with miR-30c-1-3p levels (r = -0.3671, P = 0.01981) and positively-with the maximal aortic diameter (r = 0.6251, P < 0.0001). The optimal cutoff values for MMP-9 expression and the maximal aortic diameter were 461.08 ng/ml and 55.95 mm, with areas under the curve of 0.816 and 0.844, respectively. Our results indicate that plasma levels of miR-30c-1-3p and MMP-9 may be candidate biomarkers of AAA progression. KEY MESSAGES: Downregulation of miR-30c-1-3p expression and upregulation of its potential target MMP-9 are predictors of the devastation of AAA. Downregulation of miR-30c-1-3p expression and its downstream impact on MMP-9 have a potential on predicting the development and rupture of AAA.

C2-C3 Anterior Cervical Diskectomy and Fusion for Hangman's Fractures with C2 Posterior Dislocation: Technical Notes.

BACKGROUND: The overwhelming majority of hangman's fractures cause anterior dislocation of C2. Hangman's fracture with C2 posterior dislocation is extremely rare; only 1 pediatric case was reported in 2018 to date. This kind of injury cannot be cataloged using current classification schemes, and no established treatment recommendations exist. The purpose of this article is to report a rare case of a hangman's fracture with C2 posterior dislocation, which does not fit into existing classification systems and discuss management technical notes to avoid pitfalls. METHODS: We describe this case, review relevant literature, and share our experience. RESULTS: A 31-year-old male sustained a hangman's fracture with C2 posterior dislocation after he fell into a 50-cm deep roadside ditch when riding a motorcycle. Radiograph and computed tomography on admission showed fractures through both pars of C2 and C2 posterior dislocation. Magnetic resonance imaging on admission showed high T2-weighted signal intensity of cervical spinal cord and compression of the cervical spinal cord by posterior dislocation of the C2 vertebral body. A C2-3 anterior cervical diskectomy and fusion was performed. At 6 months after operation, bony fusion was achieved and magnetic resonance imaging showed the T2-weighted signal hyperintensity of cervical spinal cord before surgery disappeared. CONCLUSIONS: C2-C3 anterior cervical diskectomy and fusion is recommended for hangman's fractures with C2 posterior dislocation. Traction before surgery is not recommended.

Influence of fluvastatin on cardiac function and baroreflex sensitivity in diabetic rats.

AIM: To investigate whether fluvastatin is able to ameliorate the impaired cardiac function or baroreflex sensitivity (BRS) in rats with type 1 diabetes. METHODS: Type 1 diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) and then administered fluvastatin (1.5, 3.0, and 6.0 mg·kg(-1)·d(-1)) for 30 d. Food and drink intake was recorded every day. Fasting blood glucose (FBG) level, blood lipid level, cardiac function and BRS were measured in diabetic rats after fluvastatin treatment for 30 d. RESULTS: The polydipsia, polyphagia and abnormal biochemical indexes of blood were significantly ameliorated by the the 3.0- and 6.0-mg doses of fluvastatin in STZ-induced diabetic rats. FBG was decreased in diabetic rats after fluvastatin treatment for 30 d. The left ventricular systolic pressure (LVSP) and the maximum rate of change of left ventricular pressure in the isovolumic contraction and relaxation period (±dp/dt(max)) were elevated, and left ventricular diastolic pressure (LVEDP) was decreased by fluvastatin. The attenuated heart rate responses to arterial blood pressure (ABP) increase induced by phenylephrine (PE) and ABP decrease induced by sodium nitroprusside (SNP) were reversed by the 3.0-mg dose of fluvastatin. CONCLUSION: Fluvastatin regulates blood lipid levels and decreases the FBG level in diabetic rats. These responses can protect the diabetic heart from complications by improving cardiac function and BRS.

(E)-N'-(2-Chloro-benzyl-idene)-3,5-di-hydroxy-benzohydrazide dihydrate.

In the Schiff base mol-ecule of the title compound, C(14)H(11)ClN(2)O(3)·2H(2)O, the benzene rings form a dihedral angle of 20.6 (1)°. The water molecules of crystallization are involved in the formation of a three-dimensional hydrogen-bonding network via O-H⋯O and N-H⋯O hydrogen bonds.

Role of craniofacial surgery in oral and maxillofacial tumors involving the skull base: A retrospective analysis of 126 patients.

OBJECTIVES: Oral and maxillofacial tumors involving the skull base (SB) are rare and complex, making treatment difficult and controversial. The purpose of the present study was to evaluate the treatment efficacy of craniofacial surgery (CFS). STUDY DESIGN: Patients who underwent CFS for these tumors between May 2000 and November 2017 were retrospectively analyzed. Clinicopathologic and treatment modality data were collected and follow-up was recorded. Kaplan-Meier and log-rank tests and Cox-regression model were used for survival analysis. RESULTS: In total, 126 patients were enrolled (70 males and 56 females; 97 malignant tumors). Squamous cell carcinoma accounted for the majority of tumors. The lip-submandibular-neck approach was most frequently applied. Through-and-through SB bone or partial dura resection was performed in 42 cases. A pathologic positive margin was found in 18 cases. Of the included patients, 80 underwent simultaneous craniofacial reconstruction. The postoperative complications rate was 11.1%. Estimated 1-year, 3-year, and 5-year overall survival rates were 78.8%, 68.2%, and 54.4% respectively; and the 1-year, 3-year, and 5-year recurrence-free survival rates were 77.4%, 66.8%, and 63.8%, respectively. Multivariate analysis indicated postoperative complications, radiotherapy, recurrence, and metastasis status had a negative impact on survival (P < .05). CONCLUSIONS: Although tumors involving the SB had various clinicopathologic characteristics, with interdisciplinary cooperation, CFS is an optimal option.

Vesicles from the self-assembly of the ultra-small fatty acids with amino acids under aqueous conditions.

The properties of vesicles formed from the self-assembly of amphiphilic molecules can mimic the functionality of the natural lipid membranes. In this study, the self-assembly process of the amphiphilic structures formed by the interaction between ultra-small fatty acids [FAs, Cn (n = 4-8)] and amino acids (AAs) to generate vesicles under aqueous conditions were investigated in detail, along with the corresponding dynamic vesiculation mechanisms. Our results showed that the molar ratio of FAs/AAs and the chain length of FAs largely affected the structural characteristics and dispersion of vesicles. The detailed information about the entire size distributions and morphology of obtained vesicles were explored by the cryogenic transmission electron microscopy (Cryo-EM). Fourier transform infrared (FT-IR) spectra and quantum calculations suggested that the intermolecular hydrogen bond and electrostatic interactions between ultra-small molecules (FAs and AAs) during the aggregation processes were responsible for the formation of vesicles, where the hydrogen-bonding effect was dominant. Our findings shed new light on the effective and simple preparation of biological vesicles via ultra-small molecules self-assembly in aqueous solutions, which may have potential applications in vesicle physiology and drug delivery systems, and also get a mature understanding of the fundamental intermolecular interactions in life process.

Human Menstrual Blood-Derived Stem Cells Inhibit the Proliferation of HeLa Cells via TGF-ß1-Mediated JNK/P21 Signaling Pathways.

Human menstrual blood-derived stem cells (hMBSCs) are a novel type of mesenchymal stem cells (MSCs) that have a high proliferative rate, multilineage differentiation potential, low immunogenicity, and low oncogenicity, making them suitable candidates for regenerative medicine. The therapeutic efficacy of hMBSCs has been demonstrated in some diseases; however, their effects on cervical cancer remain unclear. In the present study, we investigated whether hMBSCs have anticancer properties on cervical cancer cells in vivo and in vitro, which has not yet been reported. In vitro, transwell coculturing experiments revealed that hMBSCs suppress the proliferation and invasion of HeLa cervical cancer cells by inducing G0/G1 cell cycle arrest. In vivo, we established a xenografted BALB/c nude mouse model by subcutaneously coinjecting HeLa cells with hMBSCs for 21 days. We found that hMBSCs significantly decrease the average volume and average weight of xenografted tumors. ELISA, TGF-ß1 antibody, and recombinant human TGF-ß1 (rhTGF-ß1) were used to analyze whether TGF-ß1 contributed to cell cycle arrest. We found that hMBSC-secreted TGF-ß1 and rhTGF-ß1 induced cell cycle arrest and increased the expression of phospho-JNK and phospho-P21 in HeLa cells, which was mostly reversed by TGF-ß1 antibody. These results indicate that hMBSCs have antitumor properties on cervical cancer in vitro and in vivo, mediated by the TGF-ß1/JNK/p21 signaling pathway. In conclusion, this study suggests that hMBSC-based therapy is promising for the treatment of cervical cancer.

Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway.

BACKGROUND: Increasing evidence has shown that mesenchymal stem cells (MSCs) yield a favorable therapeutic benefit for thermal burn skin wounds. Human amniotic MSCs (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating skin wounds. However, the exact effects of hAMSCs on the healing of thermal burn skin wounds and their potential mechanisms are not explored. METHODS: hAMSCs were isolated from amniotic membrane and characterized by RT-PCR, flow cytometry, immunofluorescence, and tumorigenicity test. We assessed the effects of hAMSCs and hAMSC conditional medium (CM) on wound healing in a deep second-degree burn injury model of mice. We then investigated the biological effects of hAMSCs and hAMSC-CM on the apoptosis and proliferation of heat stress-injured human keratinocytes HaCAT and dermal fibroblasts (DFL) both in vivo and in vitro. Next, we explored the underlying mechanisms by assessing PI3K/AKT and GSK3ß/ß-catenin signaling pathways in heat injured HaCAT and DFL cells after hAMSCs and hAMSC-CM treatments using PI3K inhibitor LY294002 and ß-catenin inhibitor ICG001. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may activate PI3K/AKT signaling pathway. RESULTS: Our results showed that hAMSCs expressed various markers of embryonic stem cells and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAMSC and hAMSC-CM transplantation significantly promoted thermal burn wound healing by accelerating re-epithelialization with increased expression of CK19 and PCNA in vivo. hAMSCs and hAMSC-CM markedly inhibited heat stress-induced apoptosis in HaCAT and DFL cells in vitro through activation of PI3K/AKT signaling and promoted their proliferation by activating GSK3ß/ß-catenin signaling. Furthermore, we demonstrated that hAMSC-mediated activation of GSK3ß/ß-catenin signaling was dependent on PI3K/AKT signaling pathway. Antibody array assay showed that a panel of cytokines including PAI-1, C-GSF, periostin, and TIMP-1 delivered from hAMSCs may contribute to the improvement of the wound healing through activating PI3K/AKT signaling pathway. CONCLUSION: Our results demonstrated that hAMSCs and hAMSC-CM efficiently cure heat stress-induced skin injury by inhibiting apoptosis of skin cells and promoting their proliferation through activating PI3K/AKT signaling pathway, suggesting that hAMSCs and hAMSC-CM may provide an alternative therapeutic approach for the treatment of skin injury.

Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hMSCs) are a promising source for clinical stem cell transplantation. However, telomere regulation mechanisms, as one of the possible major mechanisms by which hMSCs sustain their stem cell characteristics, remain unknown. We isolated hMSCs by plastic adhesion and characterized these cells by morphology, immune phenotype and differentiation capacity. Telomerase was found negative in hMSCs, but slightly up-regulated in hMSC-derived adipocytes by the Telomeric Repeat Amplification Protocol (TRAP) assay. Moreover, hMSCs lack the alternative lengthening of telomeres (ALT) mechanism, because the hallmarks of ALT, such as very long and heterogeneous telomeres, extra-chromosome telomere repeat DNA (ECTR), and ALT-associated promyelocytic leukemia bodies (APBs), were not evident. However, when hMSCs were arrested in S phase with a combination of serum deprivation and aphidicolin, previously undetectable telomerase activity became predominantly positive. Meanwhile, the expression level of hTERT protein and mRNA increased, paralleled with the appearance of a large cohort of synchronized hMSCs at S phase. These findings provide a profile of telomere regulation by cell cycle dependent expression of telomerase in hMSCs and may lead to a better understanding of the stem cell nature of these cells.

Synthesis and positive inotropic evaluation of 2-(4-(4-substituted benzyloxy)-3-methoxybenzyl)-1,4-diazepan-1-yl)-N-(4,5-dihydro-1-methyl[1,2,4]triazolo[4,3-a]quinolin-7-yl)-acetamides.

In an attempt to search for more potent positive inotropic agents, a series of 2-(4-(4-substituted benzyloxy)-3-methoxybenzyl)-1,4-diazepan-1-yl)-N-(4,5-dihydro-1-methyl[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides was synthesized and their positive inotropic activities were evaluated by measuring left atrium stroke volume on isolated rabbit-heart preparations. Several compounds showed favorable activity compared with the standard drug Milrinone among which 2-(4-(4-(2-chlorobenzyloxy)-3-methoxybenzyl)-1,4-diazepan-1-yl)-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamide 6e was found to have the most desirable potency with the 6.79 +/- 0.18% increased stroke volume (Milrinone: 1.67 +/- 0.64%) at a concentration of 1 x 10(-5) M in our in-vitro study. The chronotropic effects of those compounds having inotropic effects were also evaluated in this work.

Therapeutic efficiency of human amniotic epithelial stem cell-derived functional hepatocyte-like cells in mice with acute hepatic failure.

BACKGROUND: Hepatocyte transplantation has been proposed as an effective treatment for patients with acute liver failure (ALF), but its application is limited by a severe shortage of donor livers. Human pluripotent stem cells (hPSCs) have emerged as a potential cell source for regenerative medicine. Human amniotic epithelial stem cells (hAESCs) derived from amniotic membrane have multilineage differentiation potential which makes them suitable for possible application in hepatocyte regeneration and ALF treatment. METHODS: The pluripotent characteristics, immunogenicity, and tumorigenicity of hAESCs were studied by various methods. hAESCs were differentiated to hepatocyte-like cells (HLCs) using a non-transgenic and three-step induction protocol. ALB secretion, urea production, periodic acid-Schiff staining, and ICG uptake were performed to investigate the function of HLCs. The HLCs were transplanted into ALF NOD-SCID (nonobese diabetic severe combined immunodeficient) mouse, and the therapeutic effects were determined via liver function test, histopathology, and survival rate analysis. The ability of HLCs to engraft the damaged liver was evaluated by detecting the presence of GFP-positive cells. RESULTS: hAESCs expressed various markers of embryonic stem cells, epithelial stem cells, and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAESC-derived hepatocytes possess the similar functions of human primary hepatocytes (hPH) such as producing urea, secreting ALB, uptaking ICG, storing glycogen, and expressing CYP enzymes. HLC transplantation via the tail vein could engraft in live parenchymal, improve the liver function, and protect hepatic injury from CCl4-induced ALF in mice. More importantly, HLC transplantation was able to significantly prolong the survival of ALF mouse. CONCLUSION: We have established a rapid and efficient differentiation protocol that is able to successfully generate ample functional HLCs from hAESCs, in which the liver injuries and death rate of CCl4-induced ALF mouse can be significantly rescued by HLC transplantation. Therefore, our results may offer a superior approach for treating ALF.

[Peripheral benzodiazepine receptor agonist Ro5-4864 inhibits mitochondrial permeability transition in rat heart].

Opening of mitochondrial permeability transition (MPT) pores leads to mitochondrial injury during oxidative stress. The peripheral benzodiazepine receptor (PBR) located at mitochondrial outer-membrane has been shown to be involved in several mitochondrial functions. In the present study, we used Ro5-4864, a PBR agonist, to test if activation of PBR could prevent MPT pore opening during Ca(2+) overloading. Cardiac mitochondria isolated from Sprague-Dawley rats were treated by 150 mmol/L Ca(2+) to induce MPT. Ro5-4864 (50, 100 and 200 micromol/L) was added into incubation buffer before adding 150 micromol/L Ca(2+). In additional group, atractyloside (ATR, 20 micromol/L), an opener of MPT pores was added 5 min before the addition of 100 micromol/L Ro5-4864. The change of absorbance at 520 nm was monitored with a spectrophotometer at 30 degrees C for 10 min. Western blot was used to detect cytochrome C loss. The mitochondrial membrane potential was monitored with the fluorescence dye JC-1. Ro5-4864 inhibited the decrease of absorbance at 520 nm compared to that in the untreated Ca(2+) group (P<0.01, P<0.05). In the presence of ATR, Ro5-4864 was not able to prevent MPT anymore. Opening of MPT pores by Ca(2+) decreased the content of cytochrome C in mitochondria, but increased cytochrome C content in cytosol. Ro5-4864 preserved cytochrome C content in mitochondria and led to less cytochrome C release to cytosol. ATR treatment reversed the protective effect of Ro5-4864 on cytochrome C content. Opening of MPT pores led to mitochondrial depolarization, whereas Ro5-4864 treatment maintained mitochondrial membrane potential. Thus, prevention of MPT by activation of PBR during calcium overloading maintains mitochondrial cytochrome C content and membrane potential. Activation of PBR during cardiac ischemia and reperfusion may be an alternative way for cardioprotection.