Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.

No studies have identified the direct effect of changes in cerebral blood flow (CBF) on cognitive function at rest and during exercise. In this study, we manipulated CBF using hypercapnic gas to examine whether an increase in CBF improves cognitive function during prolonged exercise. The speed and the accuracy of cognitive function were assessed using the Stroop color-word test. After the Stroop test at rest, the subjects began exercising on a cycling ergometer in which the workload was increased by 0.5 kilopond every minute until a target heart rate of 140 beats/min was achieved. Then, the subjects continued to cycle at a constant rate for 50 min. At four time points during the exercise (0, 10, 20, 50 min), the subjects performed a Stroop test with and without hypercapnic respiratory gas (2.0% CO2), with a random order of the exposures in the two tests. Despite a decrease in the mean blood flow velocity in the middle cerebral artery (MCA Vmean), the reaction time for the Stroop test gradually decreased during the prolonged exercise without any loss of performance accuracy. In addition, the hypercapnia-induced increase in MCA Vmean produced neither changes in the reaction time nor error in the Stroop test during exercise. These findings suggest that the changes in CBF are unlikely to affect cognitive function during prolonged exercise. Thus, we conclude that improved cognitive function may be due to cerebral neural activation associated with exercise rather than global cerebral circulatory condition.

Granulocyte colony-stimulating factor (G-CSF) prevents left ventricular remodeling after myocardial infarction, but its effect on atherosclerosis is unknown. We examined two kinds of rabbit atherosclerosis models. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHL-MI) rabbits were treated with G-CSF or saline for 7 days from 14 months old. The vascular injury models were created by inflating angioplasty balloon in the iliac artery of rabbits and were divided into G-CSF and saline group. G-CSF significantly reduced the stenosis score of coronary artery and lipid plaque area of thoracic aorta in WHHL-MI rabbits at 4 weeks after the treatment. In the vascular injury model, G-CSF significantly prevented an increase in neointima/media ratio at 4 weeks after the treatment. G-CSF accelerated the reendothelialization of denuded arteries, and the pretreatment with nitric oxide synthase inhibitor significantly inhibited it. These results suggest that G-CSF has a therapeutic potential for the progression of atherosclerosis.

Recently, we developed a benzofuran derivative for the imaging of β-amyloid plaques, 5-(5-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)-N-methylpyridin-2-amine (18F-FPYBF-2) (Ono et al., J Med Chem 54:2971-9, 2011). The aim of this study was to assess the feasibility of 18F-FPYBF-2 as an amyloid imaging PET tracer in a first clinical study with healthy volunteers and patients with various dementia and in comparative dual tracer study using 11C-Pittsburgh Compound B (11C-PiB).

Liver-type fatty acid-binding protein (L-FABP) is a key regulator of fatty acid metabolism, but serum L-FABP levels are not well investigated in chronic liver diseases. We aimed to elucidate the prognostic ability of serum L-FABP in human chronic liver diseases and compare it with the albumin-bilirubin (ALBI) score. In 242 chronic liver disease patients, including chronic hepatitis (CH, n = 100), liver cirrhosis (LC, n = 142), and presence of hepatocellular carcinoma (HCC, n = 144), serum L-FABP levels were correlated with liver function (P < 0.0001), increased in LC compared with CH (P < 0.01), and correlated to ALBI score (P < 0.0001). Serum L-FABP levels were increased in the presence of HCC (P < 0.0001), correlating to des-gamma-carboxy prothrombin (P < 0.0001), alpha-fetoprotein (P = 0.009), and Barcelona-Clinic Liver Cancer stage. In the average follow-up period of 1,054 days, serum L-FABP levels were elevated (P < 0.0001) in patients who eventually died. The area under the curve (AUC) of serum L-FABP (0.764) was higher than that of ALB (0.709), and the patients with serum L-FABP ≤ 6.8 ng/mL had significantly longer rates of survival (P < 0.0001). Serum L-FABP (hazard ratio [HR] 4.0; P < 0.001), HCC (HR 3.7; P = 0.001), ALBI score (HR 2.7; P < 0.001), and age (HR 1.0; P = 0.049) were independent predictors of survival. In the subgroup who maintained liver function, the AUC of serum L-FABP (0.751) was higher than that of ALB (0.643). In this subgroup, serum L-FABP (HR 4.4; P = 0.002) and HCC (HR 13.9; P < 0.001) were independent predictors of survival. Conclusion: Serum L-FABP is a possible predictor of survival in chronic liver diseases from CH to LC and HCC, including any subgroup that maintains liver function.

There are two types of pyloric gland-like metaplasia in the corpus of stomach: pyloric and pseudopyloric metaplasias. They show the same morphology as the original pyloric glands in H&E staining. Pseudopyloric metaplasia is positive for pepsinogen (PG) I immunohistochemically, whereas pyloric metaplasia is negative. Recently, spasmolytic polypeptide-expressing metaplasia (SPEM) is proposed for pyloric gland-like metaplasia mainly in animal experiments. SPEM expresses trefoil factor family 2 (TFF2) and is often considered synonymous with pseudopyloric metaplasia. We reviewed consecutive 22 Japanese patients with autoimmune gastritis (AIG) to investigate TFF2 expression in pyloric and pseudopyloric metaplasias by counting all pyloric gland-like glands in biopsy specimens taken from greater curvature of the middle corpus according to the Updated Sydney System. Pyloric metaplasia was seen in all the 22 cases, and pseudopyloric metaplasia was found in 15 cases. Of 1567 pyloric gland-like glands in all the cases, 1381 (88.1%) glands were pyloric metaplasia glands, and the remaining 186 (11.9%) glands were pseudopyloric metaplasia glands. TFF2 expression was observed in pyloric or pseudopyloric metaplasia glands in 20 cases. TFF2 expression was recognized in 409 of 1381 (26.9%) pyloric metaplasia glands and 27 of 186 (14.5%) pseudopyloric metaplasia glands (P<0.01, chi-square test). In conclusion, SPEM was not always the same as pseudopyloric metaplasia in human AIG, and the majority of metaplasia in AIG was not pseudopyloric but pyloric metaplasia.

Pioglitazone (PGZ), a PPARγ agonist, has been used for diabetic patients as an insulin-sensitizing agent. Recent studies have demonstrated that PGZ increases adiponectin (APN) levels and provides vascular protection in ischemic conditions. This study was designed to assess the neuroprotective effects of PGZ against cerebral ischemia-reperfusion injury via an APN-related mechanism. Type 2 diabetic leptin-deficient mice (db/db) were administered PGZ for 1 week, and plasma insulin and APN levels were measured. These mice received a middle cerebral artery occlusion and reperfusion injury, and they were evaluated for the infarct volume and by immunohistochemistry and western blotting analysis at several time points after ischemia. PGZ-administered db/db mice showed improved insulin sensitivity, and the hemorrhagic rate and infarct volume were decreased (P < 0.05). In the PGZ-administered group, plasma APN levels increased compared with the vehicle group. In the db/db group, PGZ administration significantly suppressed inflammatory reactions and oxidative stress after reperfusion (P < 0.05). PGZ may be applicable for acute cerebral ischemia treatment in metabolic syndrome patients as well as antidiabetic agents.

The surface topography of Titanium (Ti) combined toughness and biocompatibility affects the attachment and migration of cells. Limited information of morphological characteristics, formed by precise machining in micron order, is currently available on the Ti that could promote osteoconduction. In the present study, a pure Ti membrane was pierced with precise 25 μm square holes at 75 μm intervals and appear burrs at the edge of aperture. We defined the surface without burrs as the "Head side" and that with burrs as the "Tail side". The effects of the machining microtopography on the proliferation and differentiation of the preosteoblasts (MC3T3-E1 cells) were investigated. The cells were more likely to migrate to, and accumulate in, the aperture of holes on the head side, but grew uniformly regardless of holes on the tail side. The topography on the both surfaces increased osteopontin gene expression levels. Osteocalcin expression levels were higher on the head side than one on the blank scaffold and tail side (p < 0.05). The osteocalcin protein expression levels were higher on the tail side than on the head side after 21 days of cultivation, and were comparable to the proportion of the calcified area (p < 0.05). These results demonstrate the capacity of a novel microporous Ti membrane fabricated using a precise mechanical punching process to promote cell proliferation and activity.

A strong therapeutic target of ischemic stroke is controlling brain inflammation. Recent studies have implicated the critical role of C-C chemokine receptor 5 (CCR5) in neuroinflammation during ischemic stroke. It has been reported that the expression of the matrix metalloproteinases, MMP-3, MMP-12, and MMP-13, is controlled by CCR5; however, their expressional regulation in the infarct brain has not been clearly understood. This study investigated the mRNA expression of Mmp-3, -12, and -13 in the ischemic cerebral cortex of photothrombosis mouse model. The three Mmps were highly upregulated in the early stages of ischemic stroke and were expressed in different types of cells. Mmp-3 and Mmp-13 were expressed in blood vessel endothelial cells after ischemia-induction, whereas Mmp-12 was expressed in activated microglia. The expression of Mmp-13 in resting microglia and in neurons of uninjured cerebral cortex was lost in the infarct region. Therefore, the MMPs responding to CCR5 are differentially regulated during ischemic stroke.

With the extension of life expectancy, cancer has been increasing in elderly populations. Postoperative pneumonia can negatively influence immediate mortality following gastrectomy for elderly patients, but its impact on long-term survival remains unclear. We retrospectively reviewed the cases of patients aged ≥75 years who underwent curative gastrectomy for gastric cancer from 2000 to 2014 to determine the long-term effects of postoperative pneumonia and to identify independent risk factors along with physical status and surgical procedure. Of 250 patients, 32 (12.8%) developed postoperative pneumonia. Patients with postoperative pneumonia had significantly worse overall survival (OS) than those without postoperative pneumonia (P<.001). A multivariate analysis identified postoperative pneumonia as a prognostic factor for OS (hazard ratio, 2.06; 95% CI, 1.05-3.75; P=.036). Significant risk factors for the development of postoperative pneumonia were male gender (P=.026) and D2 lymphadenectomy (P<.001). D2 lymphadenectomy was associated with poorer OS than D1 or D1+lymphadenectomy in patients with an American Society of Anesthesiologists (ASA) score 3 (P=.026), but did not influence OS negatively in patients with an ASA score ≤2. Limited lymphadenectomy did not affect the cancer-specific survival of elderly patients with ASA score 3. Postoperative pneumonia following gastrectomy has an adverse impact on the long-term survival of elderly gastric cancer patients. A limited lymphadenectomy during curative resection should be considered to prevent postoperative pneumonia in frail elderly patients with ASA score 3. Postoperative pneumonia following gastrectomy has an adverse impact on the long-term survival of elderly gastric cancer patients. Extent of lymph node dissection during curative resection should be limited to prevent postoperative pneumonia, based on the patient's frailty.

There are few prospective studies on cold forceps polypectomy (CFP) using jumbo cup forceps. Therefore, we examined patients with diminutive polyps (5 mm or smaller) treated with CFP using jumbo cup forceps to achieve an adenoma-free colon and also assessed the safety of the procedure and the recurrence rate of missed or residual polyp after CFP by performing follow-up colonoscopy 1 year later.

We have recently reported that the serotonin (5-HT) projections from the midbrain's raphe nuclei that contains 5-HT cell bodies may play a role both in heat production and in heat loss. The purpose of the present study was to clarify the involvement of 5-HT in the ventral tegmental area (VTA), where 5-HT is suggested to participate in thermoregulation, using the combined methods of telemetry, microdialysis, and high performance liquid chromatography, with a special emphasis on regulation of the body temperature (Tb) in freely moving rats. First, we measured changes in Tb, tail skin temperature (Ttail; an index of heat loss), heart rate (HR; an index of heat production), locomotor activity (Act), and levels of extracellular monoamines in the VTA during cold (5°C) or heat (35°C) exposure. Subsequently, we perfused citalopram (5-HT re-uptake inhibitor) into the VTA and measured the thermoregulatory parameters and monoamines release. Although Tb, Ttail, and HR changed during both exposures, significant changes in extracellular level of 5-HT (138.7±12.7% baseline, p<0.01), but not dopamine (DA) or noradrenaline (NA) were noted in the VTA only during heat exposure. In addition, perfusion of citalopram into the VTA increased extracellular 5-HT levels (221.0±52.2% baseline, p<0.01), but not DA or NA, while Tb decreased from 37.4±0.1°C to 36.8±0.2°C (p<0.001),Ttail increased from 26.3±0.4°C to 28.4±0.4°C (p<0.001), and HR and Act remained unchanged. Our results suggest that the VTA is a key area for thermoregulation, and 5-HT, but not DA or NA, modulates the heat loss system through action in the VTA.

We and others have previously reported that granulocyte colony-stimulating factor (G-CSF) prevents left ventricular remodeling and dysfunction after myocardial infarction in animal models and human. We have also reported that G-CSF inhibits the progression of atherosclerosis in animal models, but its precise mechanism is still elusive. So, we examined the effects of G-CSF on atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Twelve-week-old male ApoE(-/-) mice were subcutaneously administrated with 200 μg/kg of G-CSF or saline once a day for 5 consecutive days per a week for 4 weeks. Atherosclerotic lesion of aortic sinus was significantly reduced in the G-CSF-treated mice compared with the saline-treated mice (35% reduction, P<0.05). G-CSF significantly reduced the expression level of interferon-γ by 31% and increased the expression level of interleukin-10 by 20% in atherosclerotic lesions of aortic sinus. G-CSF increased the number of CD4(+)CD25(+) regulatory T cells in lymph nodes and spleen, and enhanced the suppressive function of regulatory T cells in vitro. G-CSF markedly increased the number of Foxp3-positive regulatory T cells in atherosclerotic lesions of aortic sinus. Administration of anti-CD25 antibody (PC61) that depletes regulatory T cells abrogated these atheroprotective effects of G-CSF. Moreover, in ApoE(-/-)/CD28(-/-) mice, that lack regulatory T cells, the protective effects of G-CSF on atherosclerosis were not recognized. These findings suggest that regulatory T cells play an important role in the atheroprotective effects of G-CSF.

Somatosensory neurons are classified into three main types according to their modalities: nociceptive, thermal, and mechanosensory. Within each modality group, neurons can be further divided into morphologically and functionally distinct subclasses. Here we show that heparan sulfate D-glucosaminyl 3-O-sulfotransferase 2 (HS3ST-2) is a marker for specific subsets of TrkC-expressing cutaneous low-threshold mechanosensory and proprioceptive mechanosensory neurons. Two-color in situ analysis revealed that almost all HS3ST-2 signals colocalized with TrkC but not with TrkA or TrkB mRNA. To visualize the morphological subtypes of HS3ST-2/TrkC-expressing neurons, we generated a HS3ST-2-hPLAP knock-in mouse line, in which HS3ST-2-expressing neurons and their projections are labeled by human placental alkaline phosphatase (hPLAP). AP staining in these mice demonstrated that sensory endings of muscle spindles and Golgi tendon organs as well as the cutaneous mechanosensory Merkel and longitudinal lanceolate endings in the whiskers are strongly positive for hPLAP activity. In contrast, no nociceptive endings are labeled. In the glabrous and hairy skin, rare Merkel endings and transverse lanceolate endings are weakly stained. During development, each type of nerve endings forms at different time point. Muscle innervations differentiate first, followed by formation of cutaneous sensory endings. Our results revealed the subtype identities of TrkC-positive mechanosensory neurons and demonstrated the usefulness of HS3ST-2 as a genetic marker for these subclasses of neurons.

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.

Junctional adhesion molecules (JAMs) are expressed in diverse types of stem and progenitor cells, but their physiological significance has yet to be established. Here, we report that JAMs exhibit a novel mode of interaction and biological activity in adipose-derived stromal/stem cells (ADSCs). Among the JAM family members, JAM-B and JAM-C were concentrated along the cell membranes of mouse ADSCs. JAM-C but not JAM-B was broadly distributed in the interstitial spaces of mouse adipose tissue. Interestingly, the JAM-C ectodomain was cleaved and secreted as a soluble form (sJAM-C) in vitro and in vivo, leading to deposition in the fat interstitial tissue. When ADSCs were grown in culture plates coated with sJAM-C, cell adhesion, cell proliferation and the expression of five mesenchymal stem cell markers, Cd44, Cd105, Cd140a, Cd166 and Sca-1, were significantly elevated. Moreover, immunoprecipitation assay showed that sJAM-C formed a complex with JAM-B. Using CRISPR/Cas9-based genome editing, we also demonstrated that sJAM-C was coupled with JAM-B to stimulate ADSC adhesion and maintenance. Together, these findings provide insight into the unique function of sJAM-C in ADSCs. We propose that JAMs contribute not only to cell-cell adhesion, but also to cell-matrix adhesion, by excising their ectodomain and functioning as a niche-like microenvironment for stem and progenitor cells.

Obesity, a known risk factor for various types of cancer, reduces the number and function of cytotoxic immune cells in the tumor immune microenvironment (TIME). However, the impact of obesity on CD4+ T cells remains unclear. Therefore, this study aimed to clarify the impact of obesity on CD4+ T cells in the TIME. A tumor-bearing obese mouse model was established by feeding with 45% high-fat diet (HFD), followed by inoculation with a colon cancer cell line MC38. Tumor growth was significantly accelerated compared to that in mice fed a control diet. Tumor CD4+ T cells showed a significant reduction in number and an increased expression of programmed death-1 (PD-1), and decreased CD107a expression and cytokine such as IFN-γ and TNF-α production, indicating dysfunction. We further established CD4+ T cell-depleted HFD-fed model mice, which showed reduced tumor infiltration, increased PD-1 expression in CD8+ T cells, and obesity-induced acceleration of tumor growth in a CD4+ T cell-dependent manner. These findings suggest that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells to ultimately accelerate the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity.

N-tert-Butoxycarbonylmethamphetamine (BocMA), a masked derivative of methamphetamine (MA), converts into MA under acidic condition and potentially acts as a precursor to MA following ingestion. To investigate the metabolism and excretion of BocMA, metabolism tests were conducted using human liver microsomes (HLM), rat liver microsomes (RLM) and rat.

Pirfenidone (PFD) is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis by inducing G₁ cell cycle arrest in fibroblasts. We hypothesize that PFD can induce G₁ cell cycle arrest in different types of cells, including cancer cells. To investigate the effects of PFD treatment on the growth of human prostate cancer (PCa) cells, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines (androgen-low-sensitive E9 and F10 cells and androgen-insensitive AIDL cells), as well as an androgen-insensitive human PCa cell line (PC-3). PFD treatment suppressed the growth of all PCa cells. Transforming growth factor β1 secretion was significantly increased in PFD-treated PCa cells. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G₀/G₁ phase, which was accompanied by a decrease in the S/G₂ cell population. CDK2 protein expression was clearly decreased in PFD-treated LNCaP and PC-3 cells, whereas p21 protein expression was increased in only PFD-treated LNCaP cells. In conclusion, PFD may serve as a novel therapeutic drug that induces G₁ cell cycle arrest in human PCa cells independently of androgen sensitivity. Thus, in the tumor microenvironment, PFD might target not only fibroblasts, but also heterogeneous PCa cells of varying androgen-sensitivity levels.

Transforming growth factor β1 (TGFβ1) regulates a variety of cellular functions, including cell growth, apoptosis and differentiation. The aim of the current study was to investigate the alterations of phenotypic events in the long-term exposure of prostate cancer (PCa) cells to TGFβ1 and its effect on macrophage-differentiated cells. The PCa cell line, PC-3, and the subclone, M1, were exposed to TGFβ1 for short- or long-term periods. TGFβ1 signaling was assessed by Smad3 phosphorylation, and non-canonical signaling was analyzed by quantitative polymerase chain reaction-based regulatory gene expression profiles. TGFβ1-exposed PCa cells were also co-cultured with phorbol 12-myristate 13-acetate (PMA)-treated THP-1 macrophages as a model of the tumor microenvironment. The phosphorylation of Smad3 in the PCa cells with long-term exposure was lower than that in the PCa cells with short-term exposure. Interleukin-6 mRNA expression in the PMA-treated THP-1 macrophages was significantly downregulated by co-culture with the PCa cells with long-term exposure. Cyclooxygenase-2 expression in the long-term TGFβ1-exposed PCa cells was lower than that in the control PCa cells, and the production of prostaglandin E2 (PGE2) in the long-term TGFβ1-exposed PCa cells was also significantly lower. The results of the current study demonstrated that the long-term TGFβ1 exposure of PCa cells induces phenotypic changes, including the downregulation of PGE2 production. This indicates that prolonged TGFβ-exposed PCa cells may change the cytokine production of macrophages in the tumor microenvironment.