Extracellular α-synuclein impairs sphingosine 1-phosphate receptor type 3 (S1PR3)-regulated lysosomal delivery of cathepsin D in HeLa cells.

α-Synuclein (α-Syn)-positive intracellular fibrillar protein deposits, known as Lewy bodies, are thought to be involved in the pathogenesis of Parkinson's disease (PD). Although recent lines of evidence suggested that extracellular α-Syn secreted from pathogenic neurons contributes to the propagation of PD pathology, the precise mechanism of action remains unclear. We have reported that extracellular α-Syn caused sphingosine 1-phosphate (S1P) receptor type 1 (S1PR1) uncoupled from Gi and inhibited downstream G-protein signaling in SH-SY5Y cells, although its patho/physiological role remains to be clarified. Here we show that extracellular α-Syn caused S1P receptor type 3 (S1PR3) uncoupled from G protein in HeLa cells. Further studies indicated that α-Syn treatment reduced cathepsin D activity while enhancing the secretion of immature pro-cathepsin D into cell culture medium, suggesting that lysosomal delivery of cathepsin D was disturbed. Actually, extracellular α-Syn attenuated the retrograde trafficking of insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor, which is under the regulation of S1PR3. These findings shed light on the understanding of dissemination of the PD pathology, that is, the mechanism underlying how extracellular α-Syn secreted from pathogenic cells causes lysosomal dysfunction of the neighboring healthy cells, leading to propagation of the disease.

Identification of novel OCRL isoforms associated with phenotypic differences between Dent disease-2 and Lowe syndrome.

BACKGROUND: Although Lowe syndrome and Dent disease-2 are caused by Oculocerebrorenal syndrome of Lowe (OCRL) mutations, their clinical severities differ substantially and their molecular mechanisms remain unclear. Truncating mutations in OCRL exons 1-7 lead to Dent disease-2, whereas those in exons 8-24 lead to Lowe syndrome. Herein we identified the mechanism underlying the action of novel OCRL protein isoforms. METHODS: Messenger RNA samples extracted from cultured urine-derived cells from a healthy control and a Dent disease-2 patient were examined to detect the 5' end of the OCRL isoform. For protein expression and functional analysis, vectors containing the full-length OCRL transcripts, the isoform transcripts and transcripts with truncating mutations detected in Lowe syndrome and Dent disease-2 patients were transfected into HeLa cells. RESULTS: We successfully cloned the novel isoform transcripts from OCRL exons 6-24, including the translation-initiation codons present in exon 8. In vitro protein-expression analysis detected proteins of two different sizes (105 and 80 kDa) translated from full-length OCRL, whereas only one protein (80 kDa) was found from the isoform and Dent disease-2 variants. No protein expression was observed for the Lowe syndrome variants. The isoform enzyme activity was equivalent to that of full-length OCRL; the Dent disease-2 variants retained >50% enzyme activity, whereas the Lowe syndrome variants retained <20% activity. CONCLUSIONS: We elucidated the molecular mechanism underlying the two different phenotypes in OCRL-related diseases; the functional OCRL isoform translated starting at exon 8 was associated with this mechanism.

Rapid Lipid Modification of Endothelial Cell Membranes in Cardiac Ischemia/Reperfusion Injury: a Novel Therapeutic Strategy to Reduce Infarct Size.

PURPOSE: Plasma membranes constitute a gathering point for lipids and signaling proteins. Lipids are known to regulate the location and activity of signaling proteins under physiological and pathophysiological conditions. Membrane lipid therapies (MLTs) that gradually modify lipid content of plasma membranes have been developed to treat chronic disease; however, no MLTs have been developed to treat acute conditions such as reperfusion injury following myocardial infarction (MI) and percutaneous coronary intervention (PCI). A fusogenic nanoliposome (FNL) that rapidly incorporates exogenous unsaturated lipids into endothelial cell (EC) membranes was developed to attenuate reperfusion-induced protein signaling. We hypothesized that administration of intracoronary (IC) FNL-MLT interferes with EC membrane protein signaling, leading to reduced microvascular dysfunction and infarct size (IS). METHODS: Using a myocardial ischemia/reperfusion swine model, the efficacy of FNL-MLT in reducing IS following a 60-min coronary artery occlusion was tested. Animals were randomized to receive IC Ringer's lactate solution with or without 10 mg/mL/min of FNLs for 10 min prior to reperfusion (n = 6 per group). RESULTS: The IC FNL-MLT reduced IS (25.45 ± 16.4% vs. 49.7 ± 14.1%, P < 0.02) and enhanced regional myocardial blood flow (RMBF) in the ischemic zone at 15 min of reperfusion (2.13 ± 1.48 mL/min/g vs. 0.70 ± 0.43 mL/min/g, P < 0.001). The total cumulative plasma levels of the cardiac injury biomarker cardiac troponin I (cTnI) were trending downward but were not significant (999.3 ± 38.7 ng/mL vs. 1456.5 ± 64.8 ng/mL, P = 0.1867). However, plasma levels of heart-specific fatty acid binding protein (hFABP), another injury biomarker, were reduced at 2 h of reperfusion (70.3 ± 38.0 ng/mL vs. 137.3 ± 58.2 ng/mL, P = 0.0115).  CONCLUSION: The IC FNL-MLT reduced IS compared to vehicle in this swine model. The FNL-MLT maybe a promising adjuvant to PCI in the treatment of acute MI.

Berberine and palmatine inhibit the growth of human rhabdomyosarcoma cells.

A natural isoquinoline alkaloid, berberine, has been known to exhibit anti-tumor activity in various cancer cells via inducing cell cycle arrest. However, it has not been investigated whether berberine and its analogs inhibit the growth of rhabdomyosarcoma (RMS), which is the most frequent soft tissue tumor in children. The present study examined the anti-tumor effects of berberine and palmatine on expansions of three human embryonal RMS cell lines; ERMS1, KYM1, and RD. Intracellular incorporation of berberine was relatively higher than that of palmatine in every RMS cell line. Berberine significantly inhibited the cell cycle of all RMS cells at G1 phase. On the other hand, palmatine only suppressed the growth of RD cells. Both of berberine and palmatine strongly inhibited the growth of tumorsphere of RD cells in three-dimensional culture. These results indicate that berberine derivatives have the potential of anti-tumor drugs for RMS therapy.Abbreviations: ARMS: alveolar rhabdomyosarcoma; ERMS: embryonal rhabdomyosarcoma; RMS: rhabdomyosarcoma.

Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling.

α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein-protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P1R) from the lipid raft fractions. S1P1R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P1R became refractory to S1P stimulation required for activating inhibitory G-protein (Gi) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P1R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn-treated cells. Furthermore, cholesterol-depleting agent-induced S1P1R expulsion from the rafts also resulted in S1P1R uncoupling. Taken together, these results suggest that extracellular α-Syn-induced expulsion of S1P1R from lipid rafts promotes the uncoupling of S1P1R from Gi, thereby blocking subsequent Gi signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.

Involvement of Gßγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gßγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gßγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.

Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy.

RATIONALE: The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent. OBJECTIVE: To determine whether 3 CPC infusions have greater efficacy than 1 infusion. METHODS AND RESULTS: Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region. CONCLUSIONS: This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Chronic phase improvements in electrocardiographic and echocardiographic manifestations of left ventricular hypertrophy after alcohol septal ablation for drug-refractory hypertrophic obstructive cardiomyopathy.

After alcohol septal ablation (ASA), regression of left ventricular hypertrophy (LVH) has been observed in several studies using echocardiography or cardiac magnetic resonance, and favorable changes of myocardial excitation have been expected. However, no studies have focused on the alteration of electrocardiography (ECG) findings after ASA. Therefore, we evaluated serial changes in ECG parameters during the chronic phase after ASA for drug-refractory hypertrophic obstructive cardiomyopathy (HOCM). From 1998 to 2014, we performed 187 ASA procedures in 157 drug-refractory HOCM patients. After excluding patients who underwent dual-chamber pacing therapy and who underwent staged or repeat ASA within 2 years after the index ASA, 25 patients without bundle branch block and additional pacemaker implantation were enrolled in the main study group. ECGs, echocardiograms, and clinical follow-up data were evaluated at baseline and, 1, 6, 12, and 24 months after ASA. Patients with bundle branch block or additional pacemaker implantation were assigned in a referential group (n = 79), in which the echocardiographic changes between baseline and at 1 year were evaluated. Sokolow-Lyon index (SLi), Cornell index, and total 12-lead QRS amplitude significantly decreased during 2-year follow-up after ASA. SLi and Cornell index significantly decreased from 6 to 12 months (p < 0.05 vs. p < 0.01). Changes in SLi were significantly associated with changes in the interventricular septal thickness (r = 0.54, p < 0.005), left ventricular mass index (r = 0.40, p = 0.050), and peak creatine phosphokinase level (r = -0.41, p = 0.042), but not in the Cornell index and 12-lead QRS amplitude. In the comparison between baseline and at 1 year, significant improvements in the interventricular septal thickness, posterior wall thickness, left atrial size, E/A ratio, and E/e' were observed in the echocardiographic study. Changes of SLi reflected regression of LVH after ASA with the best correlation. During the chronic phase after ASA, LVH regression was confirmed by echocardiographic and ECG parameters.

Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.

BACKGROUND: Glomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains. METHODS: In this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains. RESULTS: We report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic. CONCLUSIONS: This is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.

Geographical predisposition influences on the distribution and tissue characterisation of eccentric coronary plaques in non-branching coronary arteries: cross-sectional study of coronary plaques analysed by intravascular ultrasound.

BACKGROUND: We investigated the influence of geographical predisposition on the spatial distribution and composition of coronary plaques. METHODS: Thirty coronary arteries were evaluated. A total of 1441 cross-sections were collected from intravascular ultrasound (IVUS) and radio-frequency signal-based virtual histology (VH-IVUS) imaging. To exclude complex geographical effects of side branches and to localise the plaque distribution, we analysed only eccentric plaques in non-branching regions. The spatial distribution of eccentric plaques in the coronary artery was classified into myocardial, lateral, and epicardial regions. The composition of eccentric plaques was analysed using VH-IVUS. RESULTS: The plaque was concentric in 723 sections (50.2%) and eccentric in 718 (49.9%). Eccentric plaques were more frequently distributed towards the myocardial side than towards the epicardial side (46.7 ± 7.5% vs. 12.5 ± 4.2%, p = 0.003). No significant difference was observed between the myocardial and lateral sides (46.7 ± 7.5% vs. 20.8 ± 5.0%) or between the lateral and epicardial sides. Eccentric thin-capped fibroatheromas were more frequently distributed towards the myocardial side than towards the lateral side (p = 0.024) or epicardial side (p = 0.005). CONCLUSION: Geographical predisposition is associated with distribution, tissue characterisation, and vulnerability of plaques in non-branching coronary arteries.

Novel α-Galactosidase A Mutation (K391E) in a Young Woman With Severe Cardiac and Renal Manifestations of Fabry Disease.

Fabry disease, an X-linked lysosomal storage disorder due to α-galactosidase A deficiency, is associated with dysfunction of various cell types and results in a systemic vasculopathy. We describe a 29-year-old woman with Fabry disease presenting with severe cardiac and renal manifestations. Gene analysis demonstrated a novel mutation (K391E) in the GLA gene. Enzyme replacement therapy (ERT) was started with agalsidase-ß after confirming the diagnosis of Fabry disease, resulting in normalization of LV systolic function and improvement of renal function. As early therapy is crucial for preventing life-threatening sequelae, clinicians should consider Fabry disease in young patients presenting with cardiac and renal disease without any likely causes.

[Computed tomography colonography image assessment using colon phantom-effects of reconstructed slice thickness].

Computed tomography colonography (CTC) is a robust and reliable imaging test of the colon. Recent studies show good sensitivity for the identification of nonpolypoid (flat) lesions as well. The purpose of this study was to determine the accuracy and reproducibility of a volume-rendering (VR) and virtual gross pathology (VGP) technique for detecting a polypoid lesion phantom by varying slice thickness. The scan of a simulated house-made phantom was performed using a 16-slice CT scanner with varying combinations of tube voltage (120 kVp), effective exposure (100 mAs), detector configuration (16×0.75 mm), rotation time (0.75 s), helical pitch (0.688, 0.938, 1.066 and 1.188), reconstruction kernel (A, B and C), and section thickness/reconstruction interval (0.8/0.4, 1.0/0.5 and 1.5/0.75 mm). All image data were transferred to a three-dimensional workstation to assess multi-planar reformation (MPR), VR and VGP. Accuracy of volume measurement using the VR technique for quantitative analysis was compared using a paired t-test. Four radiological technologists also independently evaluated the visual score using the VGP technique for qualitative analysis, and their evaluations were compared using one-way analysis of variance with Fisher's protected least significant difference post-hoc test. There was a statistically significant difference in reproducibility between the three different slice thicknesses as to volume measurement and observer performance test (p<0.01 and p<0.05, respectively). Furthermore, the reproducibility improved when using thinner slices. In conclusion, VR and VGP techniques using a slice thickness of 0.8 mm made it possible to maintain accuracy and reproducibility when using CTC to detect polypoid lesions.

Involvement of sphingosine 1-phosphate signaling in insulin-like growth factor-II/mannose 6-phosphate receptor trafficking from endosome to the trans-Golgi network.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional glycoprotein not only play roles in IGF-II degradation and pro-TGFß activation but binding to and transport M6P-bearing lysosomal enzymes from the trans-Golgi network (TGN) or the cell surface to lysosomes. At present, information regarding a retrograde transport of IGF-II/M6P receptor from endosomes to the TGN is still limited. We show here that a continuous ligand-dependent activation of sphingosine 1-phosphate receptor type 3 (S1P3R) on the endosomal membranes is required for subsequent recycling back of cargo-unloaded IGF-II/M6P receptors to the TGN. We have further clarified that Gq coupled with S1P3R plays a critical role in the activation of casein kinase 2, which phosphorylates and keeps PACS1 connector protein active for the association with IGF-II/M6P receptors, which enables transport carrier formation with the aid of other adaptor proteins toward the TGN. These findings shed light on the molecular mechanism underlying how continuous activation of the S1P receptor and subsequent downstream Gq signaling regulates the retrograde transport of the empty IGF-II/M6P receptors back to the TGN.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation. Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process. Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different. Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol.

Influence of ω3 fatty acids on maternal behavior and brain oxytocin in the murine perinatal period.

INTRODUCTION: Perinatal women often experience mood disorders and postpartum depression due to the physical load and the rapid changes in hormone levels caused by pregnancy, childbirth, and nursing. When the mother's emotions become unstable, their parental behavior (maternal behavior) may decline, the child's attachment may weaken, and the formation of mother-child bonding can become hindered. As a result, the growth of the child may be adversely affected. The objective of this study was to investigate the effect of ω3 fatty acid deficiency in the perinatal period on maternal behavior and the oxytocin concentration and fatty acid composition in brain tissue. MATERIALS AND METHODS: Virgin female C57BL/6 J mice fed a ω3 fatty acid-deficient (ω3-Def) or adequate (ω3-Adq) diet were mated for use in this study. To assess maternal behavior, nest shape was evaluated at a fixed time from gestational day (GD) 15 to postpartum day (PD) 13, and a retrieval test was conducted on PD 3. For neurochemical measurement, brains were removed from PD 1-6 dams and hippocampal fatty acids and hypothalamic oxytocin concentrations were assessed. RESULTS: Peripartum nest shape scores were similar to those reported previously (Harauma et al., 2016); nests in the ω3-Def group were small and of poor quality whereas those in the ω3-Adq group were large and elaborate. The inferiority of nest shape in the ω3-Def group continued from PD 0-7. In the retrieval test performed on PD 3, dams in the ω3-Def group took longer on several parameters compared with those in the ω3-Adq group, including time to make contact with pups (sniffing time), time to start retrieving the next pup (interval time), and time to retrieve the last pup to the nest (grouping time). Hypothalamic oxytocin concentrations on PD 1-6 were lower in the ω3-Def group than in the ω3-Adq group. DISCUSSION: Our data show that ω3 fatty acid deficiency reduces maternal behavior, a state that continued during pup rearing. This was supported by the observed decrease in hypothalamic oxytocin concentration in the ω3-Def group. These results suggest that ω3 fatty acid supplementation during the perinatal period is not only effective in delivering ω3 fatty acids to infants but is also necessary to activate high-quality parental behavior in mothers.

Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells.

Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10-30 µM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 µM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10-30 µM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

Ethnic comparison in takotsubo syndrome: novel insights from the International Takotsubo Registry.

BACKGROUND: Ethnic disparities have been reported in cardiovascular disease. However, ethnic disparities in takotsubo syndrome (TTS) remain elusive. This study assessed differences in clinical characteristics between Japanese and European TTS patients and determined the impact of ethnicity on in-hospital outcomes. METHODS: TTS patients in Japan were enrolled from 10 hospitals and TTS patients in Europe were enrolled from 32 hospitals participating in the International Takotsubo Registry. Clinical characteristics and in-hospital outcomes were compared between Japanese and European patients. RESULTS: A total of 503 Japanese and 1670 European patients were included. Japanese patients were older (72.6 ± 11.4 years vs. 68.0 ± 12.0 years; p < 0.001) and more likely to be male (18.5 vs. 8.4%; p < 0.001) than European TTS patients. Physical triggering factors were more common (45.5 vs. 32.0%; p < 0.001), and emotional triggers less common (17.5 vs. 31.5%; p < 0.001), in Japanese patients than in European patients. Japanese patients were more likely to experience cardiogenic shock during the acute phase (15.5 vs. 9.0%; p < 0.001) and had a higher in-hospital mortality (8.2 vs. 3.2%; p < 0.001). However, ethnicity itself did not appear to have an impact on in-hospital mortality. Machine learning approach revealed that the presence of physical stressors was the most important prognostic factor in both Japanese and European TTS patients. CONCLUSION: Differences in clinical characteristics and in-hospital outcomes between Japanese and European TTS patients exist. Ethnicity does not impact the outcome in TTS patients. The worse in-hospital outcome in Japanese patients, is mainly driven by the higher prevalence of physical triggers. TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique Identifier: NCT01947621.

Myogenetic Oligodeoxynucleotide (myoDN) Recovers the Differentiation of Skeletal Muscle Myoblasts Deteriorated by Diabetes Mellitus.

Skeletal muscle wasting in patients with diabetes mellitus (DM) is a complication of decreased muscle mass and strength, and is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle wasting. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8 (IL-8). iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat muscle wasting in patients with DM.

Blood flow distribution after end-to-side anastomosis with wide arteriotomy in extremity free flap surgery.

PURPOSE: Although many studies have investigated the optimal anastomotic procedure for the end-to-side (ETS) procedure with a free flap, no study has focused on the size of the arteriotomy. Some surgeons have recently described the effectiveness of ETS with wide arteriotomy, but the postoperative haemodynamics remains unclear for free flaps created using this technique. The aim of this study was to use ultrasonography to evaluate the postoperative blood flow distribution after ETS with a wide arteriotomy in extremity free flap surgery. METHODS: We evaluated 20 free flaps in 18 consecutive patients who received an ultrasonographic examination after free flap surgery using the ETS technique with wide arteriotomy for arterial anastomosis. All flaps were examined after surgery and blood flow was calculated for the flap and recipient vessels. RESULTS: All 20 flaps survived, but one flap developed asymptomatic arterial thrombosis and 19 flaps were analysed. For the ETS technique with wide arteriotomy, peripheral circulation was well preserved in all flaps. Comparison of flap types showed that blood flow was significantly higher in myocutaneous flaps than in fasciocutaneous flaps, but there was no significant difference according to the size of the arteriotomy. CONCLUSIONS: Given the range of arteriotomy performed using the ETS with a wide arteriotomy technique, the blood flow volume in the flap depended on the type of flap but not on the size of the arteriotomy. A steal phenomenon related to the creation of a wide window in the receipt artery was not found in the analysed retrospective cohort.