Bioinspired Fluorine Labeling for 19F NMR-Based Plasma Amine Profiling.

Metabolite profiling serves as a powerful tool that advances our understanding of biological systems, disease mechanisms, and environmental interactions. In this study, we present an approach employing 19F-nuclear magnetic resonance (19F NMR) spectroscopy for plasma amine profiling. This method utilizes a highly efficient and reliable fluorine-labeling reagent, 3,5-difluorosalicylaldehyde, which effectively emulates pyridoxal phosphate, facilitating the formation of Schiff base compounds with primary amines. The fluorine labeling allows for distinct resolution of 19F NMR signals from amine mixtures, leading to precise identification and quantification of amine metabolites in human plasma. This advancement offers valuable tools for furthering metabolomics research.

Chiral Discrimination of Monosaccharides Derivatized with 2-Fluorophenyl Hydrazine Using 19F NMR Spectroscopy.

Chiral discrimination of monosaccharides holds significant importance, especially given the growing interest of the pharmaceutical industry in their utilization. However, the majority of existing methods has predominantly centered around chromatographic techniques. In this study, we introduce a 19F NMR-based comprehensive approach for chiral analysis specifically tailored for 15 pairs of aldoses. This technique involves employing sugar hydrazones containing fluorine in combination with chiral octahedral gallium and scandium complexes. By utilizing highly sensitive 19F NMR spectroscopy, the fluorine label in the sugar hydrazone enables accurate differentiation between d and l enantiomers. The efficiency of the newly developed method was demonstrated through its successful application in both quantitative and qualitative analyses of mixtures containing various monosaccharides.

An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases.

Sirtuin 2 (Sirt2), an NAD+-dependent protein deacetylase, deacetylates tubulin, AKT, and other proteins. Previously, we showed that Sirt2 isoform 1 (Sirt2.1) increased replication of hepatitis B virus (HBV). Here, we show that HBV replication upregulates the expression of Sirt2 primary and alternatively spliced transcripts and their respective isoforms, 1, 2, and 5. Since Sirt2 isoform 5 (Sirt2.5) is a catalytically inactive nuclear protein with a spliced-out nuclear export signal (NES), we speculated that its different localization affects its activity. The overexpression of Sirt2.5 reduced expression of HBV mRNAs, replicative intermediate DNAs, and covalently closed circular DNA (cccDNA), an activity opposite that of Sirt2.1 and Sirt2.2. Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction was weakened by HBV replication. Unlike Sirt2.1, Sirt2.5 activated the AKT/GSK-3ß/ß-catenin signaling pathway very weakly and independently of HBV replication. When the NES and an N-terminal truncated catalytic domain were added to the Sirt2.5 construct, it localized in the cytoplasm and increased HBV replication (like Sirt2.1 and Sirt2.2). Chromatin immunoprecipitation assays revealed that more Sirt2.5 was recruited to cccDNA than Sirt2.1. The recruitment of histone lysine methyltransferases (HKMTs), such as SETDB1, SUV39H1, EZH2, and PR-Set7, and their respective transcriptional repressive markers, H3K9me3, H3K27me3, and H4K20me1, to cccDNA also increased in Sirt2.5-overexpressing cells. Among these, the Sirt2.5-PR-Set7 and -SETDB1 interactions increased upon HBV replication. These results demonstrate that Sirt2.5 reduces cccDNA levels and viral transcription through epigenetic modification of cccDNA via direct and/or indirect association with HKMTs, thereby exhibiting anti-HBV activity.IMPORTANCE Sirt2, a predominant cytoplasmic α-tubulin deacetylase, promotes the growth of hepatocellular carcinoma; indeed, HBV replication increases Sirt2 expression, and overexpression of Sirt2 is associated with hepatic fibrosis and epithelial-to-mesenchymal transition. Increased amounts of Sirt2 isoforms 1, 2, and 5 upon HBV replication might further upregulate HBV replication, leading to a vicious cycle of virus replication/disease progression. However, we show here that catalytically inactive nuclear Sirt2.5 antagonizes the effects of Sirt2.1 and Sirt2.2 on HBV replication, thereby inhibiting cccDNA level, transcription of cccDNA, and subsequent synthesis of replicative intermediate DNA. More Sirt2.5 was recruited to cccDNA than Sirt2.1, thereby increasing epigenetic modification by depositing transcriptional repressive markers, possibly through direct and/or indirect association with histone lysine methyltransferases, such as SETDB1, SUV39H1, EZH2, and/or PR-Set7, which represses HBV transcription. Thus, Sirt2.5 might provide a functional cure for HBV by silencing the transcription of HBV.

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner.

The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by the PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased the formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Coimmunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). In contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.IMPORTANCE The HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved arginine-proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBx-Par14/Par17 interaction stabilized HBx; promoted its translocation to the nucleus and mitochondria; and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, the Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and Par17 may represent a novel therapeutic option against HBV infection.

Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3ß/ß-Catenin Signaling Pathway.

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3ß (GSK-3ß) and increased ß-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3ß/ß-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased ß-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased ß-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates ß-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3ß/ß-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3ß, and increases ß-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases ß-catenin levels. Although HBx activates AKT to upregulate ß-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.

Longitudinal Changes in Peripapillary Retinal Nerve Fiber Layer Thickness in High Myopia: A Prospective, Observational Study.

PURPOSE: To determine longitudinal change of peripapillary retinal nerve fiber layer (pRNFL) thickness in patients with high myopia without ophthalmic disease. DESIGN: Prospective observational study. PARTICIPANTS: Participants were divided into 2 groups: a high myopia group (80 eyes) that included eyes with an axial length ≥26.0 mm and a control group (80 eyes) that included eyes with a spherical equivalent (SE) between +3.0 and -6.0 diopters (D). Both groups were further divided into age subgroups by decade: 20s, 30s, 40s, and 50s. Each subgroup included 20 eyes. METHODS: After the initial visit, pRNFL thickness measurements were performed 2 times more with at least 1-year intervals between examinations using spectral-domain OCT. The mean pRNFL thickness was fitted with linear mixed models. MAIN OUTCOME MEASURES: The pRNFL thickness and rate of pRNFL thickness reduction. RESULTS: The mean patient age and thickness of the pRNFL at the first visit were 39.5±12.5 years and 90.16±9.06 µm, and 41.5±12.2 years and 96.80±9.50 µm in the high myopia and control groups, respectively. The high myopia group showed a significant reduction in mean pRNFL thickness between the first and second visits, and between the second and third visits (P < 0.001 and P = 0.002, respectively). For individuals aged 50 to 59 years, the reduction rate was -1.69 and -0.63 µm/year in the high myopia and control groups, respectively; the interaction between group and duration was significant (P = 0.014). The reduction rate in individuals aged 40 to 49 years was -1.70 and -0.48 µm/year in the 2 groups, respectively; the interaction was also significant (P = 0.031). Among those aged 30 to 39 years and 20 to 29 years, no such significant interactions were observed (-0.95 vs. -0.57 µm/year, P = 0.086 and -0.31 vs. -0.19 µm/year, P = 0.858, respectively). CONCLUSIONS: Highly myopic eyes had a significantly greater decrease in pRNFL over 2 years than normal eyes. In addition, the reduction rate of pRNFL thickness was greater in older patients with high myopia, whereas similar values were shown in normal controls except individuals aged 20 to 29 years.

Effect of combined goniotomy and phacoemulsification on intraocular pressure in open-angle glaucoma patients.

IMPORTANCE: Although goniotomy is known to be successful in treating congenital glaucoma, its effect in adult glaucoma patients remains unclear. BACKGROUND: To evaluate the efficacy and safety of goniotomy performed simultaneously with cataract surgery in treatment of open-angle glaucoma (OAG). DESIGN: Retrospective comparative study. PARTICIPANTS: A total of 76 patients with moderately controlled OAG (intraocular pressure [IOP] ≤ 21 mmHg using medications) undergoing cataract surgery. METHODS: Comparison of patients who underwent the conventional goniotomy during cataract surgery (combined goniotomy group) with those who underwent cataract surgery alone (phaco group). MAIN OUTCOME MEASURES: Changes in IOP and medications, and complications through 12 months. RESULTS: Baseline IOP was 18.2 ± 2.4 mmHg in the combined goniotomy group and 17.4 ± 1.9 mmHg in the phaco group; number of medications was 2.6 ± 1.1 and 2.4 ± 0.9, respectively (P > 0.05). The reduction in IOP and medication use from baseline in the combined goniotomy group was significantly greater at 12 months compared to the phaco group (-3.1 ± 2.9 mmHg vs -1.3 ± 2.4 mmHg and -1.2 ± 0.9 vs -0.7 ± 0.9, respectively, both P < 0.05). The success rate was 76.7% in the combined goniotomy group and 50.0% in the phaco group at 12 months (P = 0.021). No significant complication was observed in either group. CONCLUSIONS AND RELEVANCE: Combined goniotomy and cataract surgery showed a significantly greater reduction in IOP and number of medications compared to cataract surgery alone at 1 year after surgery, with similarly favourable safety profiles.

Comparative Analysis of Human Mesenchymal Stem Cells Derived From Bone Marrow, Placenta, and Adipose Tissue as Sources of Cell Therapy.

Various source-derived mesenchymal stem cells (MSCs) with multipotent capabilities were considered for cell therapeutics of incurable diseases. The applicability of MSCs depends on the cellular source and on their different in vivo functions, despite having similar phenotypic and cytological characteristics. We characterized MSCs from different sources, including human bone marrow (BM), placenta (PL), and adipose tissue (AT), in terms of the phenotype, surface antigen expression, differentiation ability, proteome reference map, and blood flow recovery in a hindlimb ischemic disease model. The MSCs exhibit different differentiation potentials depending on the cellular source despite having similar phenotypic and surface antigen expression. We identified approximately 90 differentially regulated proteins. Most up- or down-regulated proteins show cytoskeletal or oxidative stress, peroxiredoxin, and apoptosis roles according to their functional involvement. In addition, the PL-MSCs retained a higher therapeutic efficacy than the BM- and AT-MSCs in the hindlimb ischemic disease model. In summary, we examined differentially expressed key regulatory factors for MSCs that were obtained from several cellular sources and demonstrated their differentially expressed proteome profiles. Our results indicate that primitive PL-MSCs have biological advantages relative to those from other sources, making PL-MSCs a useful model for clinical applications of cell therapy.

Well-defined differentiation of hESC-derived hemangioblasts by embryoid body formation without enzymatic treatment.

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.

Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington's disease patient.

HD (Huntington's disease) is a devastating neurodegenerative genetic disorder caused by abnormal expansion of CAG repeats in the HTT (huntingtin) gene. We have recently established two iPSC (induced pluripotent stem cell) lines derived from a HD patient carrying 72 CAG repeats (HD-iPSC). In order to understand the proteomic profiles of HD-iPSCs, we have performed comparative proteomic analysis among normal hESCs (human embryonic stem cells; H9), iPSCs (551-8) and HD-iPSCs at undifferentiated stages, and identified 26 up- and down-regulated proteins. Interestingly, these differentially expressed proteins are known to be involved in different biological processes, such as oxidative stress, programmed cell death and cellular oxygen-associated proteins. Among them, we found that oxidative stress-related proteins, such as SOD1 (superoxide dismutase 1) and Prx (peroxiredoxin) families are particularly affected in HD-iPSCs, implying that HD-iPSCs are highly susceptible to oxidative stress. We also found that BTF3 (basic transcription factor 3) is up-regulated in HD-iPSCs, which leads to the induction of ATM (ataxia telangiectasia mutated), followed by activation of the p53-mediated apoptotic pathway. In addition, we observed that the expression of cytoskeleton-associated proteins was significantly reduced in HD-iPSCs, implying that neuronal differentiation was also affected. Taken together, these results demonstrate that HD-iPSCs can provide a unique cellular disease model system to understand the pathogenesis and neurodegeneration mechanisms in HD, and the identified proteins from the present study may serve as potential targets for developing future HD therapeutics.

Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication.

Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.

Licochalcone H Induces Cell Cycle Arrest and Apoptosis in Human Skin Cancer Cells by Modulating JAK2/STAT3 Signaling.

Licochalcone H (LCH) is a phenolic compound synthetically derived from licochalcone C (LCC) that exerts anticancer activity. In this study, we investigated the anticancer activity of LCH in human skin cancer A375 and A431 cells. The 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay was used to evaluate the antiproliferative activity of LCH. Cell cycle distribution and the induction of apoptosis were analyzed by flow cytometry. Western blotting assays were performed to detect the levels of proteins involved in cell cycle progression, apoptosis, and the JAK2/STAT3 signaling pathway. LCH inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay revealed that LCH induced apoptosis, and the LCH-induced apoptosis was accompanied by cell cycle arrest in the G1 phase. Western blot analysis showed that the phosphorylation of JAK2 and STAT3 was decreased by treatment with LCH. The inhibition of the JAK2/STAT3 signaling pathway by pharmacological inhibitors against JAK2/STAT3 (cryptotanshinone (CTS) and S3I-201) simulated the antiproliferative effect of LCH suggesting that LCH induced apoptosis by modulating JAK2/STAT3 signaling.

The hsa-miR-5739 modulates the endoglin network in endothelial cells derived from human embryonic stem cells.

MicroRNAs are small, noncoding RNAs that bind to seed sequences on the 3' untranslated regions of their target genes and then negatively regulate gene expressions via the RISC complex. The novel miRNA, hsa-miR-5739, was cloned and characterized its function and cellular expression in current study. The hsa-miR-5739 downregulated endothelial cells that were derived from human ES cells significantly suppressed the translational level of endoglin. This study showed that characterized hsa-miR-5739 expression by performing expression during endothelial differentiation and demonstrate potential roles of hsa-miR-5739 in human endothelial cell differentiation.

Enhancement of wound healing by secretory factors of endothelial precursor cells derived from human embryonic stem cells.

BACKGROUND AIMS: Stem cells have been shown to have a therapeutic effect in several ischemic animal models, including hindlimb ischemia and chronic wound. We examined the wound-healing effect of secretory factors released by human embryonic stem cell (hESC)-derived endothelial precursor cells (EPC) in cutaneous excisional wound models. METHODS: hESC-EPC were sorted by CD133/KDR, and endothelial characteristics were confirmed by reverse transcription (RT)-polymerase chain reaction (PCR), Matrigel assay and ac-LDL uptake. Conditioned medium (CM) of hESC-EPC was prepared, and concentrated hESC-EPC CM was applied in a mouse excisional wound model. RESULTS: hESC-EPC CM accelerated wound healing and increased the tensile strength of wounds after topical treatment and subcutaneous injection. In addition, hESC-EPC CM treatment caused more rapid re-formation of granulation tissue and re-epithelialization of wounds compared with control vehicle medium and CB-EPC CM-treated wounds. In vitro, hESC-EPC CM significantly improved the proliferation and migration of dermal fibroblasts and epidermal keratinocytes. hESC-EPC CM also increased the extracellular matrix synthesis of fibroblasts. Analysis of hESC-EPC CM with a multiplex cytokine array system indicated that hESC-EPC secreted distinctively different cytokines and chemokines, such as epidermal growth factor (EGF), fibroblast growth factor (bFGF), fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, platelet-derived growth factor-AA (PDGF-AA) and vascular endothelial growth factor (VEGF), which are well known to be important in normal angiogenesis and wound healing. CONCLUSIONS: This study has demonstrated the wound-healing effect of hESC-EPC CM and characterized the spectrum of cytokines released by hESC-EPC that are functionally involved in the wound-healing process. These results suggest that secretory factors released from stem cells could be an important mediator of stem cell therapy in ischemic tissue diseases.

Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation.

BACKGROUND: In vitro maturation (IVM) of mammalian oocytes is divided into the GV (germinal vesicle stage), MI (metaphase I stage) and MII (metaphase II stage) stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood.The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. RESULT: A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. CONCLUSION: These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy.

Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90ß and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy.