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Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is essential for human development, and DYRK1A haploinsufficiency is associated with a recognizable developmental syndrome and variable clinical features. Here, we present a patient with DYRK1A haploinsufficiency syndrome, including facial dysmorphism, delayed motor development, cardiovascular system defects, and brain atrophy. Exome sequencing identified a novel de novo heterozygous mutation of the human DYRK1A gene (c.1185dup), which generated a translational termination codon and resulted in a C-terminally truncated protein (DYRK1A-E396ter). To study the molecular effect of this truncation, we generated mammalian cell and Drosophila models that recapitulated the DYRK1A protein truncation. Analysis of the structure and deformation energy of the mutant protein predicted a reduction in protein stability. Experimentally, the mutant protein was efficiently degraded by the ubiquitin-dependent proteasome pathway and was barely detectable in mammalian cells. More importantly, the mutant kinase was intrinsically inactive and had little negative impact on the wild-type protein. Similarly, the mutant protein had a minimal effect on Drosophila phenotypes, confirming its loss-of-function in vivo. Together, our results suggest that the novel heterozygous mutation of DYRK1A resulted in loss-of-function of the kinase activity of DYRK1A and may contribute to the developmental delay observed in the patient.

Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes in vitro and in vivo. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in Drosophila, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.

Sarcopenia is a syndrome characterized by progressive loss of muscle mass and function during aging. Although mitochondrial dysfunction and related metabolic defects precede age-related changes in muscle, their contributions to muscle aging are still not well known. In this study, we used a Drosophila model to investigate the role of lipophorin receptors (LpRs), a Drosophila homologue of the mammalian very low-density lipoprotein receptor (VLDLR), in mitochondrial dynamics and muscle aging. Muscle-specific knockdown of LpR1 or LpR2 resulted in mitochondrial dysfunction and reduced proteostasis, which contributed to muscle aging. Activation of AMP-activated protein kinase (AMPK) ameliorated muscle dysfunction induced by LpR1 knockdown. These results suggest that LpR1/VLDLR is a novel key target that modulates age-dependent lipid remodeling and muscle homeostasis.

Background and Objectives Although it is well known that hypoxic culture conditions enhance proliferation of human mesenchymal stem cells, the exact mechanism is not fully understood. In this study, we investigated the effect of fibroblast growth factor (FGF)-17 from hypoxic human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on cell proliferation at late passages. Methods and Results hWJ-MSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) in normoxic (21% O2) and hypoxic (1% O2) conditions. Protein antibody array was performed to analyze secretory proteins in conditioned medium from normoxic and hypoxic hWJ-MSCs at passage 10. Cell proliferation of hypoxic hWJ-MSCs was increased compared with normoxic hWJ-MSCs from passage 7 to 10, and expression of secretory FGF-17 was highly increased in conditioned medium from hypoxic hWJ-MSCs at passage 10. Knockdown of FGF-17 in hypoxic and normoxic hWJ-MSCs decreased cell proliferation, whereas treatment of hypoxic and normoxic hWJ-MSCs with recombinant protein FGF-17 increased their proliferation. Signal transduction of FGF-17 in hypoxic and normoxic hWJ-MSCs involved the ERK1/2 pathway. Cell phenotypes were not changed under either condition. Differentiation-related genes adiponectin, Runx2, and chondroadherin were downregulated in normoxic hWJ-MSCs treated with rFGF-17, and upregulated by siFGF-17. Expression of alkaline phosphatase (ALP), Runx2, and chondroadherin was upregulated in hypoxic hWJ-MSCs, and this effect was rescued by transfection with siFGF-17. Only chondroadherin was upregulated in hypoxic hWJ-MSCs with rFGF-17. Conclusions In hypoxic culture conditions, FGF-17 from hypoxic hWJ-MSCs contributes to the maintenance of high proliferation at late passages through the ERK1/2 pathway.

Background and objectives There have been contradictory reports on the pro-cancer or anti-cancer effects of mesenchymal stem cells. In this study, we investigated whether conditioned medium (CM) from hypoxic human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (H-CM) showed enhanced anti-cancer effects compared with CM from normoxic hUC-MSCs (N-CM). Methods and results Compared with N-CM, H-CM not only strongly reduced cell viability and increased apoptosis of human cervical cancer cells (HeLa cells), but also increased caspase-3/7 activity, decreased mitochondrial membrane potential (MMP), and induced cell cycle arrest. In contrast, cell viability, apoptosis, MMP, and cell cycle of human dermal fibroblast (hDFs) were not significantly changed by either CM whereas caspase-3/7 activity was decreased by H-CM. Protein antibody array showed that activin A, Beta IG-H3, TIMP-2, RET, and IGFBP-3 were upregulated in H-CM compared with N-CM. Intracellular proteins that were upregulated by H-CM in HeLa cells were represented by apoptosis and cell cycle arrest terms of biological processes of Gene Ontology (GO), and by cell cycle of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In hDFs, negative regulation of apoptosis in biological process of GO and PI3K-Akt signaling pathway of KEGG pathways were represented. Conclusions H-CM showed enhanced anti-cancer effects on HeLa cells but did not influence cell viability or apoptosis of hDFs and these different effects were supported by profiling of secretory proteins in both kinds of CM and intracellular signaling of HeLa cells and hDFs.

This study investigates the anti-cancer effects of cervical cancer (HeLa) cells in a conditioned medium (CM) obtained from normoxic and hypoxic cultured human adult dermal fibroblasts (HDFs). The HeLa cells showed decreased cell viability, arrested cell cycles, and increased apoptosis in CM from hypoxic cultured HDFs (H-CM) compared with normoxic cultured HDFs (N-CM). In up-regulated (> 2-fold) proteins of H-CM compared with N-CM, the top enriched term of biological process of gene ontology (GO) was GO:0006955~immune response. In intracellular down-regulated (> 2-fold) proteins of HeLa cells treated with H-CM compared with N-CM, the top enriched term of biological process of GO was GO:0016579~protein deubiquitination, and the terms of the KEGG pathway were determined to be hsa05166:HTLV-I infection, hsa03410:base-excision repair, and hsa05340:primary immunodeficiency. Among down-regulated hub proteins with ≥ 5 edges (ESR1 MCL1, TBP, CD19, LCK, PCNA, CHEK1, and POLA1) of HeLa cells treated with H-CM, the top enriched term of biological process of GO and the KEGG pathway were GO:0006272~leading strand elongation and hsa05166:HTLV-I infection. H-CM displayed not only enhanced anti-cancer effects on HeLa cells compared with N-CM, but also induced intracellular signaling patterns with 9 hub proteins.

Background Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a significant pathogenic factor in Down syndrome (DS), wherein DYRK1A is overexpressed by 1.5-fold because of trisomy of human chromosome 21. Thus, DYRK1A inhibition is considered a therapeutic strategy to modify the disease. Purpose This study aims to identify a novel DYRK1A inhibitor and validate its therapeutic potential in DS-related pathological conditions. Study design In order to identify a novel DYRK1A inhibitor, we carried out two-step screening: a structure-based virtual screening of > 300,000 chemical library (first step) and cell-based nuclear factor of activated T-cells (NFAT)-response element (RE) promoter assay (second step). Primary hits were evaluated for their DYRK1A inhibitory activity using in vitro kinase assay and Tau phosphorylation in mammalian cells. Confirmed hit was further evaluated in pathological conditions including DYRK1A-overexpressing fibroblasts, flies, and mice. Results We identified aristolactam BIII, a natural product derived from herbal plants, as a novel DYRK1A inhibitor. It potently inhibited the kinase activity of DYRK1A in vitro (IC50 = 9.67 nM) and effectively suppressed DYRK1A-mediated hyperphosphorylation of Tau in mammalian cells. Aristolactam BIII rescued the proliferative defects of DYRK1A transgenic (TG) mouse-derived fibroblasts and neurological and phenotypic defects of DS-like Drosophila models. Oral administration of aristolactam BIII acutely suppressed Tau hyperphosphorylation in the brain of DYRK1A TG mice. In the open field test, aristolactam BIII significantly ameliorated the exploratory behavioral deficit of DYRK1A TG mice. Conclusion Our work revealed that aristolactam BIII as a novel DYRK1A inhibitor rescues DS phenotypes in cells and in vivo and suggested its therapeutic potential for the treatment of DYRK1A-related diseases.

Ischemic limb diseases are induced by different obstructions of peripheral arteries. These obstructions result in insufficient nutrient and oxygen supplies to the extremities, thereby leading to severe tissue damage that is in turn related to severe morbidities and mortalities. Mesenchymal stem cells (MSCs) have been isolated from various sources. These cells are multipotent with respect to differentiation and are also characterized by migration, immune suppression, and secretion of paracrine factors. Mesenchymal stem cells have been proposed to have therapeutic potential for the treatment of ischemic limb diseases. In preclinical experiments, injection of single MSCs has been shown to increase angiogenesis and blood flow in ischemic hindlimb animal models; several molecular mechanisms of angiogenesis have also been elucidated. Furthermore, modified strategies have been developed for enhancing angiogenesis and the efficacy of MSCs. These strategies have demonstrated significant effects in pre-clinical studies. In clinical trials, MSCs have shown significant effects in the treatment of ischemic limb diseases. In this review, we focus on the therapeutic properties of human MSCs and the modified methods for enhancing angiogenesis in pre-clinical experiments. We also discuss the clinical applications of MSCs for treating limb ischemia.

Calcium (Ca2+) homeostasis is essential for neuronal function and survival. Altered Ca2+ homeostasis has been consistently observed in neurological diseases. How Ca2+ homeostasis is achieved in various cellular compartments of disease-relevant cell types is not well understood. Here we show in Drosophila Parkinson's disease (PD) models that Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria through the ER-mitochondria contact site (ERMCS) critically regulates mitochondrial Ca2+ (mito-Ca2+) homeostasis in dopaminergic (DA) neurons, and that the PD-associated PINK1 protein modulates this process. In PINK1 mutant DA neurons, the ERMCS is strengthened and mito-Ca2+ level is elevated, resulting in mitochondrial enlargement and neuronal death. Miro, a well-characterized component of the mitochondrial trafficking machinery, mediates the effects of PINK1 on mito-Ca2+ and mitochondrial morphology, apparently in a transport-independent manner. Miro overexpression mimics PINK1 loss-of-function effect, whereas inhibition of Miro or components of the ERMCS, or pharmacological modulation of ERMCS function, rescued PINK1 mutant phenotypes. Mito-Ca2+ homeostasis is also altered in the LRRK2-G2019S model of PD and the PAR-1/MARK model of neurodegeneration, and genetic or pharmacological restoration of mito-Ca2+ level is beneficial in these models. Our results highlight the importance of mito-Ca2+ homeostasis maintained by Miro and the ERMCS to mitochondrial physiology and neuronal integrity. Targeting this mito-Ca2+ homeostasis pathway holds promise for a therapeutic strategy for neurodegenerative diseases.

It has been studied that mesenchymal stem cells (MSCs) have the capability to promote angiogenesis. Furthermore, there is strong evidence that hypoxic conditions can enhance angiogenesis and immune modulation mediated by MSCs, a notion that has been applied in many fields of clinical application. In the present study, we compared the efficacy of hypoxia preconditioned human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and normoxia conditioned hUC-MSCs for the treatment of ischemic injury in hindlimbs of an immunodeficient mouse model. Expression of negative markers for MSC such as CD31, CD34, and CD45 or positive markers such as CD44, CD73, CD90, and CD105 was not significantly changed in hypoxia preconditioned hUC-MSCs compared with hUC-MSCs cultured in normoxic condition. Expression of angiogenesis-related genes such as COX-2, VEGF, Tie-2, and TGF-β1 was increased compared with hUC-MSCs cultured in normoxic conditions. In the in vivo model, CD31 expression as a marker of angiogenesis was significantly increased in the ischemic limbs at 1 month after injection with hypoxic hUC-MSCs. Angiogenesis-related genes such as Ang-1, COX-1, PIGF, and MCP-1 were significantly upregulated in the muscle of ischemic hindlimbs treated with hypoxic hUC-MSCs than normoxic hUC-MSCs. Expression of proinflammatory genes such as IL-1, and IL-20 was reduced, whereas TGF-β1, which has an anti-inflammatory effect, was strongly increased. In conclusion, hypoxic culture conditions could induce expression of angiogenesis related genes in hUC-MSCs, and hypoxia preconditioned hUC-MSCs showed enhancing effects by inducing angiogenesis and low inflammatory immune response compared with normoxic hUC-MSCs in the ischemia injured hindlimb of immunodeficient mice.

Synthetic vessel grafts have been used as vascular substitutes for cardiovascular bypass procedures. In this study, we developed a novel tubular double-layered poly(l-lactide-co-ϵ-caprolactone) scaffold that did not require pretreatment with cell seeding by promoting autologous tissue regeneration by inducing the proliferation and differentiation of endothelial and smooth muscle progenitor cells after implantation. The patency and mechanical properties were maintained for one year after implantation, although 95% of the poly(l-lactide-co-ϵ-caprolactone) scaffolds had degraded. After this period, there was a lining of endothelial cells, an accumulation of collagen and elastin, and the development of neovascularization inside the poly(l-lactide-co-ϵ-caprolactone).

DYRK1A is important in neuronal development and function, and its excessive activity is considered a significant pathogenic factor in Down syndrome and Alzheimer9s disease. Thus, inhibition of DYRK1A has been suggested to be a new strategy to modify the disease. Very few compounds, however, have been reported to act as inhibitors, and their potential clinical uses require further evaluation. Here, we newly identify CX-4945, the safety of which has been already proven in the clinical setting, as a potent inhibitor of DYRK1A that acts in an ATP-competitive manner. The inhibitory potency of CX-4945 on DYRK1A (IC 50 =6.8 nM) in vitro was higher than that of harmine, INDY or proINDY, which are well-known potent inhibitors of DYRK1A. CX-4945 effectively reverses the aberrant phosphorylation of Tau, amyloid precursor protein (APP) and presenilin 1 (PS1) in mammalian cells. To our surprise, feeding with CX-4945 significantly restored the neurological and phenotypic defects induced by the overexpression of minibrain , an ortholog of human DYRK1A, in the Drosophila model. Moreover, oral administration of CX-4945 acutely suppressed Tau hyperphosphorylation in the hippocampus of DYRK1A-overexpressing mice. Our research results demonstrate that CX-4945 is a potent DYRK1A inhibitor and also suggest that it has therapeutic potential for DYRK1A-associated diseases.

Background and objectives The clinical benefits of stem cell therapy have been reported in patients with peripheral arterial occlusive disease. However, those studies had no standard reporting system to assess the outcomes, so we made a scoring system and assessed the outcomes of the limbs that underwent whole bone marrow stem cell (WBMSC) therapy. Methods and results Between July 4 and June 2009, 90 limbs of 67 patients with symptomatic thromboangiitis obliterans (TAO) were enrolled. Autologous whole bone marrow was implanted into the limb by intramuscular injections. The primary outcomes were defined by the clinical and angiographic improvement in all the limbs and the secondary outcomes were the clinical improvement and the amputation-free rates in the critical ischemic limbs (CILs). Clinical improvement and angiographic improvement was observed in 55.6% and 43.2% of all the limbs and in 50% and 50%of the CILs, respectively. The 1, 3 and 5-year amputation-free rates were 91.9%, 88.5% and 84.6% for all the limbs, respectively, and 83.9%, 77.5% and 70.4% for the CILs, respectively. A history of sympathectomy/sympathetic block was shown to be a negative prognostic factor for clinical improvement in all the limbs and in the CILs. In addition, a history of sympathetic block/sympathectomy and the smoking state were the major predictors of amputation for the CILs. Conclusions This study indicated that autologous WMBSC therapy improves the clinical status and reduces amputation factors in the limbs with symptomatic TAO and a history of sympathetic block/sympathectomy and the smoking state are useful prognostic factors.

Objective The aims of our study were to confirm the effectiveness via animal study and safety through clinical trials of using human cord blood-mononuclear cells (HCB-MNCs). Design We performed a dose–response animal study (HCB-MNCs: 4 × 106, 4 × 107 and 4 × 108) using a limb ischaemia model in dogs to assess angiogenic responses. Safety assessment in humans in terms of graft-versus-host-disease was also done by observing an uncontrolled case series. Materials and methods Twelve animal ischaemic limbs and seven patients with thromboangiitis obliterans were treated with HCB-MNCs. These cells (4 × 108) were injected into the ischaemic limb muscle of patients. The results were analysed at 8 weeks for the animal study and at 6 months for patients. Results In the animal ischaemic models, the number of capillaries, angiogenic gene expression and the angiogenic factors were increased after HCB-MNC injection. In the clinical study, the seven patients experienced no graft-versus-host-disease or cardiac/cerebral complications during the follow-up period. Conclusion This preliminary study suggests that HCB-MNC might be a safe source of stem cells for treating ischaemic limbs. However, further clinical studies are needed to establish the long-term safety and the clinical efficacy of HCB-MNC transplantation in patients with ischaemic limbs.

Purpose: Inflammatory cells are known to be associated with the progression of atherosclerosis and plaque rupture. However, the relation to inflammatory cells and apolipoproteins on the progression of atherosclerosis is unknown. This study was aimed at examining the different expressions of inflammatory cells and evaluate the effect of apolipoprotein (APO) C1 and APO E during the progression of atherosclerosis. Methods: Ten atherosclerotic tissues were compared with five non-atherosclerotic tissues. The presence of vascular smooth muscle cells (VSMCs), macrophages, T-cells, APO C1, and APO E were identified by Western blotting and immunohistochemical analysis with antibodies. The senescence was analyzed by senescence-associated β-galactosidase. Results: The protein expression and senescence of macrophages, APO C1 and APO E were significantly higher in the main atherosclerotic lesion than the non-atherosclerotic lesion. A high concentration of inflammatory cells and the paucity of VSMCs were present in the shoulder area. In addition, macrophage and T-cells are expressed in the early stage of atherosclerotic development and more expanded in advanced atherosclerotic plaques. APO C1 was expressed mainly within the necrotic core, and APO E existed mostly around the necrotic core and the fibrous cap in advanced atherosclerotic plaques. Conclusion: Our study indicated that the expression and the senescence of macrophage and T-cells may be closelyrelated to induction and deposition of APO C1 and APO E. This contributes to the development and progression of atherosclerotic plaque by expanding the necrotic core.

The Drosophila sNPF gene regulates growth through the ERK-insulin pathway. sNPF encodes a precursor protein that is processed and produces biologically active sNPF peptides. However, the functions of these peptides are not known. In Drosophila neuronal cells in culture and in flies in vivo, sNPF1 and sNPF2 activated the ERK-insulin pathway and regulated body growth. In addition, the sNPF precursor and the processed sNPF peptide were co-localized in the neurons of the central nervous system. These results indicate that sNPF1 and sNPF2 peptides processed from the sNPF precursor are sufficient for regulating body growth through the ERK-insulin pathway in Drosophila.

Insulin and insulin growth factor have central roles in growth1,2, metabolism3 and ageing4,5 of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain3,6. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.

It has been studied that mesenchymal stem cells (MSCs) have the capability to promote angiogenesis. Furthermore, there is strong evidence that hypoxic conditions can enhance angiogenesis and immune modulation mediated by MSCs, a notion that has been applied in many fields of clinical application. In the present study, we compared the efficacy of hypoxia preconditioned human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and normoxia conditioned hUC-MSCs for the treatment of ischemic injury in hindlimbs of an immunodeficient mouse model. Expression of negative markers for MSC such as CD31, CD34, and CD45 or positive markers such as CD44, CD73, CD90, and CD105 was not significantly changed in hypoxia preconditioned hUC-MSCs compared with hUC-MSCs cultured in normoxic condition. Expression of angiogenesis-related genes such as COX-2, VEGF, Tie-2, and TGF-β1 was increased compared with hUC-MSCs cultured in normoxic conditions. In the in vivo model, CD31 expression as a marker of angiogenesis was significantly increased in the ischemic limbs at 1 month after injection with hypoxic hUC-MSCs. Angiogenesis-related genes such as Ang-1, COX-1, PIGF, and MCP-1 were significantly upregulated in the muscle of ischemic hindlimbs treated with hypoxic hUC-MSCs than normoxic hUC-MSCs. Expression of proinflammatory genes such as IL-1, and IL-20 was reduced, whereas TGF-β1, which has an anti-inflammatory effect, was strongly increased. In conclusion, hypoxic culture conditions could induce expression of angiogenesis related genes in hUC-MSCs, and hypoxia preconditioned hUC-MSCs showed enhancing effects by inducing angiogenesis and low inflammatory immune response compared with normoxic hUC-MSCs in the ischemia injured hindlimb of immunodeficient mice.

The abnormal assembly of β-amyloid (Aβ) peptides into neurotoxic, β-sheet-rich amyloid aggregates is a major pathological hallmark of Alzheimer's disease (AD). Light-induced photosensitizing molecules can regulate Aβ amyloidogenesis. Multiple photochemical analyses using circular dichroism, atomic force microscopy, dot blot, and native gel electrophoresis verified that photoactivated meso-tetra(4-sulfonatophenyl)porphyrin (TPPS with M = 2H(+), Zn(2+), Cu(2+), Mn(2+)) successfully inhibits Aβ aggregation in vitro. Furthermore, Aβ toxicity was relieved in the photoexcited-TPPS-treated Drosophila AD model. TPPS suppresses neural cell death, synaptic toxicity, and behavioral defects in the Drosophila AD model under blue light illumination. Behavioral phenotypes, including larval locomotion defect and short lifespan caused by Aβ overexpression, were also rescued by blue light-excited TPPS.

We hypothesized that angiogenesis can be triggered by autologous whole bone marrow stem cell transplantation. Twenty-seven patients (34 lower limbs) with Buerger's disease, who were not candidates for surgical revascularization or radiologic intervention, were enrolled in this study. Six sites of the tibia bone were fenestrated using a 2.5-mm-diameter screw under fluoroscopic guidance. Clinical status and outcome were determined using the “Recommended Standards for Reports.” To mobilize endothelial progenitor cells (EPCs) from bone marrow, recombinant human granulocyte colony-stimulating factor (r-HuG-CSF) was injected subcutaneously as a dose of 75 μg, preoperatively. During the follow-up period (19.1 ± 3.5 months), one limb showed a markedly improved outcome (+3), and 26 limbs showed a moderately improved outcome (+2). Thirteen limbs among 17 limbs with nonhealing ulcers healed. Postoperative angiograms were obtained for 22 limbs. Two limbs showed marked (+3), five limbs moderate (+2), and nine limbs slight (+1) collateral development. However, six limbs showed no collateral development (0). Peripheral blood and bone marrow samples were analyzed for CD34 and CD133 molecules to enumerate potential EPCs, and EPC numbers were found to be increased in peripheral blood and in bone marrow after r-HuG-CSF injection. In conclusion, the transplantation of autologous whole BMCs by fenestration of the tibia bone represents a simple, safe, and effective means of inducing therapeutic angiogenesis in patients with Buerger's disease.

We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay.

Intimal hyperplasia is a major cause of restenosis after arterial bypass and balloon angioplasty. Induction of rapid re-endothelialization has been proposed to reduce intimal hyperplasia. The aim of this study was to evaluate the inhibitory effect of mesenchymal stem cells on intimal hyperplasia.Male New Zealand white rabbits were fed 1% cholesterol diets from 1 week before balloon angioplasty to the day of harvest. After dissection of rabbit carotid arteries, balloon angioplasty was performed with a 2F Fogarty embolectomy catheter. The injured carotid artery was coated with a mixture of 7 × 10(6) human umbilical cord mesenchymal stem cells (HUC-MSCs) and fibrin matrix. The carotid arteries were harvested 2, 4, and 8 weeks thereafter, and immunofluorescent staining and quantitative real-time polymerase chain reaction analysis were performed.The intima/media ratio was significantly reduced in the group treated with HUC-MSCs compared with the nontreated group (Student t-tests, *P.05). The area of re-endothelialization was significantly higher (Student t-tests, *P.05) in the group treated with HUC-MSCs than in the nontreated group. Expression of angiogenic genes such as vascular endothelial growth factor, platelet-derived growth factor, kinase insert domain receptor 1, angiopoietin 1, and angio-associated migratory cell protein was increased (analysis of variance, P.05) in the group treated with HUC-MSCs relative to the nontreated group.Our study showed that HUC-MSCs reduce the formation of intimal hyperplasia through rapid re-endothelialization. This result might be applied to development of stem cell-coated stents as well as to development of a stem cell-containing sheet coat for inhibition of intimal hyperplasia after angioplasty or surgery.

Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors. Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a target genes of sNPF and NPY signaling and regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, as well as increased food intake. Our findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila melanogaster and mammals., Author Summary Feeding behavior is one of the most essential activities in animals. Abnormal feeding behaviors cause metabolic syndromes including obesity and diabetes. Neuropeptides regulate feeding behavior in animals from nematode to human. Here, we presented molecular genetic evidences of how neuropeptides regulate food intake using fruit fly and mouse model systems. Drosophila short neuropetide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) are produced from neurons in the brain of fruit fly and mouse, respectively. These neuropeptides turned on the minibrain, in mammals also called Dyrk1a, a target gene through the PKA-CREB pathway. Then, this Mnb/Dyrk1a enzyme activated Sir2/Sirt1 enzyme, which activated FOXO transcriptional factor, turning on the expression of a sNPF/NPY target gene. The increased sNPF/NPY increased food intake in fruit flies and mice. On the contrary, increased food intake induced insulin and activated insulin signaling. When insulin signaling is activated, FOXO transcriptional factor inhibited expression of a sNPF/NPY target gene. The inhibited sNPF/NPY reduced food intake. These findings indicate that FOXO transcription factor acts as a gatekeeper for fasting–feeding transition by regulating sNPF/NPY expression in Drosophila and mammals.

Heparin-conjugated fibrin (HCF) is suitable for the release and localization of bFGF. We analyzed the effects of a bFGF delivery system using HCF with human bone marrow-derived mesenchymal stem cells (HBM- MSCs) in a dog ischemic limb model.Animals were divided into HBM-MSCs, HBM-MSCs＋HCF, bFGF-HCF, and HBM-MSCs＋ bFGF-HCF groups. A total of 1×10(7) HBM-MSCs were injected per animal, and the amount of bFGF was 1 mg per dog. Ischemic muscles were harvested at eight weeks and six months after injection of cells. The HBM-MSCs＋ bFGF-HCF group exhibited decreased proportions of capillaries and arterioles six months after transplantation. However, there were more cells positive for the angiogenic factors, VEGF and PDGF, in the eight-week specimens compared with those harvested six months after transplantation.Our results demonstrated that a single injection of HBM-MSCs did not have significant long-term angiogenic effects; however, a bFGF delivery system using HCF exerted prolonged angiogenic effects when combined with HBM-MSCs.

Methionine sulfoxide reductase A (msrA) was previously found to increase resistance to oxidative stress and longevity in animals. We identified Drosophila msrA (dmsrA), a Drosophila homolog of human msrA, as a downstream effector of forkhead box O (FOXO) signaling in Drosophila, which enhances resistance to oxidative stress and increases survival under stressed conditions. Additionally, overexpression of dmsrA in neurons extended the lifespan of flies. Moreover, overexpression of dmsrA in fat body cells caused FOXO to translocate to the nucleus, implying that this possible positive feedback loop between dmsrA and FOXO could potentiate the antioxidant activity of dmsrA and increase the lifespan in Drosophila.