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Background: Post-stroke dysphagia (PSD) is a common complication after stroke. Early PSD prediction is essential for patient stratification for intensive oral intake rehabilitation. We aimed to develop a PSD prediction score using clinical data obtained at admission., Methods: We examined consecutive patients with acute ischemic stroke between 2018 and 2019. The dysphagia status 14 days after admission was assessed using the Functional Oral Intake Scale (FOIS). PSD was defined as FOIS 1-3, which represents tube-dependent nutrition. Using multivariable logistic regression analysis, we constructed the Enteral tube Nutrition for Geriatric post-stroke dysphagia Evaluation (ENGE) score. The discriminative performance of the ENGE score was analyzed by receiver operating curve analysis. The reproducibility of the ENGE score was validated using patient data in 2020., Results: PSD developed in 84 of 488 patients (median age 78 years; 57% males). The ENGE score ranged from 0 to 6, with 1 point assigned for older age (≥78 years), 1 for high premorbid modified Rankin Scale (mRS) (≥1), 3 for high NIHSS score (≥12), and 1 for low serum albumin (<3.0 mg/dl). The area under the curve (AUC) of the ENGE score for discriminating PSD was 0.88 (95% confidence interval [CI] 0.83-0.92), and a score of 3 or more had a higher positive likelihood ratio. In the validation cohort, the AUC of the ENGE score for PSD was 0.85 (95% CI 0.78-0.91), which was similar to the derivation cohort (p = 0.491)., Conclusions: The ENGE score predicts severe PSD after acute ischemic stroke with good reproducibility., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We developed a novel light optics system installed in a scanning transmission electron microscope (STEM) to introduce a focused light accurately adjusted at the electron beam irradiation position using a parabolic mirror. With a parabolic mirror covering both the upper and lower sides of the sample, the position and focus of the light beam can be evaluated by imaging the angular distribution of the transmitted light. By comparing the light image and the electron micrograph, the irradiation positions of the electron beam and the laser beam can be accurately adjusted to each other. The size of the focused light was confirmed to be within a few microns from the light Ronchigram, which is consistent with the simulated light spot size. The spot size and position alignment were further confirmed by laser-ablating only a targeted polystyrene particle without damaging the surrounding particles. When using a halogen lamp as the light source, this system allows investigating optical spectra in comparison with cathodoluminescence (CL) spectra at exactly the same location., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: We sometimes experience disinhibition during bronchoscopy with sedation. However, the impact of adding pethidine on disinhibition has not yet been investigated. This study aimed to examine the additive impact of pethidine on disinhibition during bronchoscopy with midazolam., Methods: This retrospective study involved consecutive patients who underwent bronchoscopy between November 2019 and December 2020 (sedated with midazolam: Midazolam group) and between December 2020 and December 2021 (sedated with midazolam plus pethidine: Combination group). The severity of disinhibition was defined as follows: moderate, disinhibition that always needed restraints by assistants; and severe, disinhibition that needed antagonization of sedation by flumazenil to continue bronchoscopy. One-to-one propensity score matching was used to match baseline characteristics between both groups., Results: After propensity score matching with depression, the type of bronchoscopic procedure, and the dose of midazolam, 142 patients matched in each group. The prevalence of moderate-to-severe disinhibition significantly decreased from 16.2% to 7.8% (P = 0.028) in the Combination group. The Combination group had significantly better scores for sensation after bronchoscopy and feelings toward bronchoscopy duration than did the Midazolam group. Although the minimum SpO 2 during bronchoscopy was significantly lower (88.0 ± 6.2 mmHg vs. 86.7 ± 5.0 mmHg, P = 0.047) and the percentage of oxygen supplementation significantly increased (71.1% vs. 86.6%, P = 0.001) in the Combination group, no fatal complications were observed., Conclusions: Adding pethidine could reduce disinhibition occurrence in patients undergoing bronchoscopy with midazolam, with better subjective patient outcomes during and after bronchoscopy. However, whether more patients may need oxygen supplementation and whether hypoxia occurs during bronchoscopy should be considered., Clinical Trial Registration: UMIN000042635., Competing Interests: Conflict of Interest The authors have no conflict of interest., (Copyright © 2023 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: Disinhibition is sometimes experienced during bronchoscopy with sedation. However, data on disinhibition during bronchoscopy are scarce. We examined the prevalence and characteristics of disinhibition during bronchoscopy with midazolam., Methods: This retrospective study analyzed consecutive patients who underwent bronchoscopy between November 2019 and December 2020. The severity of disinhibition was defined as follows: mild, disinhibition sometimes requiring restraints by assistants; moderate, disinhibition always requiring restraints by assistants; and severe, disinhibition requiring antagonization of sedation by flumazenil to continue bronchoscopy., Results: Among 251 eligible patients who were sedated using midazolam, 36 (14.3%; 95% confidence interval [CI], 10.5%-19.2%), 42 (16.7%; 95% CI, 12.6%-21.8%), and 7 (2.8%; 95% CI, 1.4%-5.6%) experienced mild, moderate, and severe disinhibition, respectively. Depression (odds ratio [OR] 2.77; 95% CI, 1.20-6.41), endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (OR 10.23; 95% CI, 1.02-103.01, referred to brushing/bronchial washing/observation), and increased administration of midazolam (OR 1.20; 95% CI, 1.02-1.42, per 1-mg increase) were independently associated with moderate-to-severe disinhibition. Patients experiencing moderate disinhibition reported significantly better scores for discomfort during bronchoscopy. Besides the maximum systolic and diastolic blood pressures during bronchoscopy, the changes in hemodynamic and respiratory statuses during bronchoscopy or complications did not significantly differ between patients experiencing moderate-to-severe disinhibition and those experiencing none-to-mild disinhibition., Conclusions: Moderate-to-severe disinhibition occurred in 19.5% of patients during bronchoscopy with midazolam. We should focus on disinhibition when patients have depression or are planning to undergo EBUS-TBNA, and sparing the administration of midazolam might reduce the occurrence of disinhibition., Clinical Trial Registration: UMIN000038571., Competing Interests: Conflict of Interest The authors have no conflict of interest., (Copyright © 2021 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The pathology of sporadic Alzheimer's disease is hallmarked by altered signal transduction via the neurotransmitter receptor-G-protein-mediated protein kinase A (PKA) and protein kinase C (PKC) pathways. Because the accumulation of amyloid-β (Aβ) depends on its rates of synthesis and clearance, the metabolic pathway of Aβ in the brain and the entire body warrants exploration. The two major enzymes involved in Aβ degradation in the brain are believed to be the neprilysin and insulin-degrading enzyme (IDE). This study investigated whether PKA and PKC regulate the degradation of Aβ by modulating the protein levels of neprilysin and IDE in astrocytes. Activation of PKA induced a significant decrease in neprilysin protein levels in cultured astrocytes, whereas activation of PKC induced a significant decrease in the protein level of neprilysin and an increase in the protein level of IDE. Following activation of PKC, the reduction of neprilysin was achieved by its secretion into the culture media. Moreover, PKA-activated astrocytes significantly delayed the degradation of exogenous Aβ, whereas PKC-activated astrocytes significantly facilitated its degradation. These results suggest that PKA and PKC regulate Aβ degradation in astrocytes through a decrease in the protein level of neprilysin and an increase in neprilysin secretion and protein levels of IDE, respectively., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent epidemiological studies have hypothesized that the prevalence of cortical cataracts is closely related to ultraviolet radiation. However, the prevalence of nuclear cataracts is higher in elderly people in tropical areas than in temperate areas. The dominant factors inducing nuclear cataracts have been widely debated. In this study, the temperature increase in the lens due to exposure to ambient conditions was computationally quantified in subjects of 50-60 years of age in tropical and temperate areas, accounting for differences in thermoregulation. A thermoregulatory response model was extended to consider elderly people in tropical areas. The time course of lens temperature for different weather conditions in five cities in Asia was computed. The temperature was higher around the mid and posterior part of the lens, which coincides with the position of the nuclear cataract. The duration of higher temperatures in the lens varied, although the daily maximum temperatures were comparable. A strong correlation (adjusted R 2  > 0.85) was observed between the prevalence of nuclear cataract and the computed cumulative thermal dose in the lens. We propose the use of a cumulative thermal dose to assess the prevalence of nuclear cataracts. Cumulative wet-bulb globe temperature, a new metric computed from weather data, would be useful for practical assessment in different cities., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Infectious complications after endobronchial ultrasound-guided transbronchial biopsy with a guide sheath (EBUS-GS-TBB) are serious in that they may delay or change scheduled subsequent therapy. The aim of this study was to identify risk factors for infection after EBUS-GS-TBB., Research Question: What are the risk factors for infection after EBUS-GS-TBB?, Study Design and Methods: We retrospectively reviewed the medical records of 1,045 consecutive patients who had undergone EBUS-GS-TBB for peripheral lung lesions between January 2013 and December 2017 at Fujita Health University Hospital. We evaluated the following risk factors for infectious complications after EBUS-GS-TBB: relevant patient characteristics (age and comorbidities), lesion size, CT scan features of target lesion (intratumoral low-density areas [LDAs] and cavitation), stenosis of responsible bronchus observed by bronchoscopy, and laboratory data before EBUS-GS-TBB (WBC count and C-reactive protein concentration)., Results: Forty-seven of the study patients developed infectious complications (24 with pneumonia, 14 with intratumoral infection, three with lung abscess, three with pleuritis, and three with empyema), among whom the complication caused a delay in cancer treatment in 13 patients, cancellation of cancer treatment in seven patients, and death in three patients. Multivariate analysis showed that cavitation (P = .007), intratumoral LDAs (P < .001), and stenosis of responsible bronchus observed by bronchoscopy (P < .001) were significantly associated with infectious complications after EBUS-GS-TBB. Prophylactic antibiotics had been administered to 13 patients in the infection group. Propensity matched analysis could not show significant benefit of prophylactic antibiotics in preventing post-EBUS-GS-TBB infections., Interpretation: Cavitation, LDAs for CT scan features of target lesions, and stenosis of responsible bronchus observed by bronchoscopy are risk factors of post-EBUS-GS-TBB infection. In the cohort, prophylactic antibiotics failed to prevent infectious complications., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection. There is currently no effective vaccine or antiviral treatment available against DENV. In previous studies, we showed that Lactococcus lactis strain Plasma (LC-Plasma) could activate plasmacytoid dendritic cells, which play an important role against virus infection. LC-Plasma administration ameliorated symptoms of viral diseases and its effect appeared to be associated with IFN-α induction. However the precise mechanism of LC-Plasma protection remained unclear. In this study, we investigated the effects of LC-Plasma-induced humoral factors on DENV replication using HepG2 cells as an in vitro infection model. When HepG2 cells were preincubated with supernatants of LC-Plasma-stimulated bone marrow-derived dendritic cells, the replication of DENV was significantly inhibited in a dose dependent manner and its activity was evident regardless of the DENV serotype. In addition, the expression of interferon-stimulated genes, including ISG15, IFITM-1, MxA, RSAD2, and RyDEN, was significantly upregulated by humoral factors. We also compared the effects of representative strains of lactic acid bacteria and found that the ability to prevent DENV replication was unique to LC-Plasma. In addition, it was revealed that both anti-DENV replication activity and ISG induction depended on type I IFN rather than type III IFN signaling. Taken together, since LC-Plasma induces, in a more natural form, potent anti-DENV replication activities irrespective of viral serotypes via induction of type I IFN, LC-Plasma could be safely used as a prophylactic anti-DENV option., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Polymethymethacrylate (PMMA) is often used to reconstruct defects after curettage of Giant Cell Tumors (GCT). While GCTs usually originate in the epiphysis, the use of PMMA in distal femoral lesions may induce the risk of degenerative osteoarthritis (OA). We investigated the limb function of patients after curettage with PMMA beyond 20 years of follow-up., Methods: Patients with more than 20 years of follow-up who underwent curettage with PMMA for distal femoral GCTs were observed. We retrospectively investigated the radiographic assessment of OA and functional assessment of the limb. The Kellgren and Lawrence (KL) grading system was used for radiographic evaluation., Results: Five patients were included in this study. The mean age was 33 years, and the mean period from application of PMMA to final follow-up observation was 28.1 years. Four lesions were primary, and one lesion was recurrent. There were no patients with postoperative recurrence. There were no OA changes in preoperative radiographs. The shortest mean distance from PMMA to the articular cartilage was 4.6 mm on radiographs immediately after surgery. On radiographs at final follow-up observation, the KL grading were as follows: grade 1, 2 patients; grade 2, 1 patient; grade 3, 2 patients. All patients were able to independently ambulate without a crutch, and there was not enough pain to require nonsteroidal anti-inflammatory drugs. The mean flexion of the knee joint was 116°., Conclusions: Although PMMA used for distal femoral GCTs exhibited OA changes beyond a 20 year follow-up period, there were no cases requiring artificial joints, and the affected limbs demonstrated good function., (Copyright © 2018 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Fatty degeneration of skeletal muscle leads to muscle weakness and loss of function. Preventing fatty degeneration in skeletal muscle is important, but no drug has been used clinically. In this study, we performed drug repositioning using human platelet-derived growth factor receptor α (PDGFRα)-positive mesenchymal progenitors that have been proved to be an origin of ectopic adipocytes in skeletal muscle. We found that promethazine hydrochloride (PH) inhibits adipogenesis in a dose-dependent manner without cell toxicity. PH inhibited expression of adipogenic markers and also suppressed phosphorylation of cAMP response-element binding protein, which was reported to be a primary regulator of adipogenesis. We established a mouse model of tendon rupture with intramuscular fat deposition and confirmed that emerged ectopic adipocytes are derived from PDGFRα + cells using lineage tracing mice. When these injured mice were treated with PH, formation of ectopic adipocytes was suppressed significantly. Our results show that PH inhibits PDGFRα + mesenchymal progenitor-dependent ectopic adipogenesis in skeletal muscle and suggest that treatment with PH can be a promising approach to prevent fatty degeneration of skeletal muscle., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

There is a case report of a patient with overlapping Guillain-Barré syndrome and Bickerstaff brainstem encephalitis after infection with herpes simplex virus type 1 (HSV-1), who carried high titers of serum anti-GQ1b IgG antibodies. Several studies have linked viral infection to the modulation of ganglioside expression such as human T-lymphotropic virus to GD2 and simian virus 40 to GM3. Also, enhancement of the expression of GM2 on the cell membrane after cytomegalovirus infection has been reported. The objective of this study was to unveil the relationship between HSV-1 infection and the alteration of cellular ganglioside expression in neuronal and glial cell lines. In addition to these cell lines, several human tumor cell lines including astrocytoma cells, neuroblastoma cells, T-cell leukemia cells and kidney cells derived from normal human and monkey were infected with HSV-1 as well as HSV-2. To measure changes in ganglioside-related gene expressions and gangliosides levels in cells, quantitative PCR and glycosphingolipid-glycomic analysis were performed. Changes in gene expression of glycosyltransferases and sialyltransferases were observed in HSV-1- and HSV-2-infected cells, although with different trends. 39 glycosphingolipid-glycans were quantitatively analyzed. HSV-1 and HSV-2 infections resulted in changes in the total amount of gangliosides depending on the cell lines used and type of virus. Qualitative changes caused by each infection of HSV-1 and HSV-2 were almost negligible., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Naturally occurring galactomannans were sulfated to give sulfated galactomannans with degrees of substitution of 0.7-1.4 per sugar unit and molecular weights of M¯n=0.6×10(4)-2.4×10(4). Sulfated galactomannans were found to have specific biological activities in vitro such as anticoagulant, anti-HIV and anti-Dengue virus activities. The biological activities were compared with those of standard dextran and curdlan sulfates, which are polysaccharides with potent antiviral activity and low cytotoxicity. It was found that sulfated galactomannans had moderate to high anticoagulant activity, 13.4-36.6unit/mg, compared to that of dextran and curdlan sulfates, 22.7 and 10.0unit/mg, and high anti-HIV and anti-Dengue virus activities, 0.04-0.8μg/mL and 0.2-1.1μg/mL, compared to those curdlan sulfates, 0.1μg/mL, respectively. The cytotoxicity on MT-4 and LCC-MK2 cells was low. Surface plasmon resonance (SPR) of sulfated galactomannans revealed strong interaction with poly-l-lysine as a model compound of virus proteins, and suggested that the specific biological activities might originate in the electrostatic interaction of negatively charged sulfate groups of sulfated galactomannans and positively charged amino groups of surface proteins of viruses. These results suggest that sulfated galactomannans effectively prevented the infection of cells by viruses and the degree of substitution and molecular weights played important roles in the biological activities., (Copyright © 2016. Published by Elsevier B.V.)

Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8-1.6 μM; and half maximal cytotoxicity concentration [CC50], 53.6 μM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background & Aims: Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels., Methods: We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV., Results: Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo., Conclusion: We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Accurate prognoses are needed for patients with advanced cancer., Objectives: To evaluate the accuracy of physicians' clinical predictions of survival (CPS) and assess the relationship between CPS and actual survival (AS) in patients with advanced cancer in palliative care units, hospital palliative care teams, and home palliative care services, as well as those receiving chemotherapy., Methods: This was a multicenter prospective cohort study conducted in 58 palliative care service centers in Japan. The palliative care physicians evaluated patients on the first day of admission and followed up all patients to their death or six months after enrollment. We evaluated the accuracy of CPS and assessed the relationship between CPS and AS in the four groups., Results: We obtained a total of 2036 patients: 470, 764, 404, and 398 in hospital palliative care teams, palliative care units, home palliative care services, and chemotherapy, respectively. The proportion of accurate CPS (0.67-1.33 times AS) was 35% (95% CI 33-37%) in the total sample and ranged from 32% to 39% in each setting. While the proportion of patients living longer than CPS (pessimistic CPS) was 20% (95% CI 18-22%) in the total sample, ranging from 15% to 23% in each setting, the proportion of patients living shorter than CPS (optimistic CPS) was 45% (95% CI 43-47%) in the total sample, ranging from 43% to 49% in each setting., Conclusion: Physicians tend to overestimate when predicting survival in all palliative care patients, including those receiving chemotherapy., (Copyright © 2015 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Recent studies have suggested that a positive correlation exists between surgical interventions performed under general anesthesia and the risk of developing Alzheimer's disease (AD) in the late postoperative period. It has been reported that amyloid β-protein (Αβ) fibrillogenesis, which is closely related to AD, is accelerated by exposure to anesthetics. However, the mechanisms underlying these effects remain uncertain. This study was designed to investigate whether the anesthetic midazolam affects Αβ fibrillogenesis, and if so, whether it acts through GM1 ganglioside (GM1) on the neuronal surface. Midazolam treatment decreased GM1 expression in the detergent-resistant membrane microdomains of neurons, and these effects were regulated by the gamma-aminobutyric acid-A receptor. Midazolam inhibited Αβ fibril formation from soluble Αβ on the neuronal surface. In addition, midazolam suppressed GM1-induced fibril formation in a cell-free system. Moreover, midazolam inhibited the formation of Αβ assemblies in synaptosomes isolated from aged mouse brains. These finding suggested that midazolam has direct and indirect inhibitory effects on Αβ fibrillogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The psychotomimetic effects of stimulant drugs including amphetamines and cocaine are known to change during the postnatal development in humans and experimental animals. To obtain an insight into the molecular basis of the onset of stimulant-induced psychosis, we have explored the gene transcripts that differentially respond to methamphetamine (MAP) in the developing rat brains using a differential cloning technique, the RNA arbitrarily-primed PCR. We identified from the rat neocortex a novel and developmentally regulated MAP-inducible gene mrt3 (MAP responsive transcript 3) that is transcribed to a presumable non-coding RNA of 3.8kb and is located on the reverse strand of the F-box/LRR-repeat protein 17-like gene mapped on the rat chromosome Xq12. The mrt3 mRNAs are predominantly expressed in the brain and lung. Acute MAP injection upregulated the mrt3 expression in the neocortex at postnatal day 50, but not days 8, 15 and 23, in a D1 receptor antagonist-sensitive manner. This upregulation was mimicked by another stimulant, cocaine, whereas pentobarbital and D1 antagonist failed to alter the mrt3 expression. Moreover, repeated treatment with MAP for 5 days inhibited the ability of the challenge dose of MAP or cocaine to increase the neocortical mrt3 expression without affecting the basal mrt3 mRNA levels on day 14 of withdrawal. These late-developing, cocaine-cross reactive, D1 antagonist-sensitive and long-term regulations of mrt3 by MAP are similar to those of stimulant-induced behavioral sensitization, a model of the onset and relapse of stimulant-induced psychosis and schizophrenia, and therefore may be associated with the pathophysiology of the model., (Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.)

Objective: Adequate goal-setting is important in health counselling and treatment for obesity and overweight. We tried to determine the minimum weight reduction required for improvement of obesity-related risk factors and conditions in obese and overweight Japanese people, using a nationwide intervention programme database., Methods: Japanese men and women (n=3480; mean age±standard deviation [SD], 48.3±5.9 years; mean body mass index±SD, 27.7±2.5kgm(-2)) with "Obesity Disease" or "Metabolic Syndrome" participated in a 6-month lifestyle modification programme (specific health guidance) and underwent follow-up for 6 months thereafter. The relationship between percent weight reduction and changes in 11 parameters of obesity-related diseases were examined., Results: Significant weight reduction was observed 6 months after the beginning of the programme, and it was maintained for 1 year. Concomitant improvements in parameters for obesity-related diseases were also observed. One-third of the subjects reduced their body weight by ≥3%. In the group exhibiting 1% to <3% weight reduction, plasma triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), haemoglobin A1c (HbA1c), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (γ-GTP) decreased significantly, and high-density lipoprotein cholesterol (HDL-C) increased significantly compared to the control group (±1% weight change group). In addition to the improvements of these 7 parameters (out of 11), significant reductions in systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose (FPG) and uric acid (UA) (total 11 of 11 parameters) were observed in the group with 3% to <5% weight reduction. In the group with ≥5% weight reduction, the same 11 parameters also improved as those in the group with 3% to <5% weight reduction., Conclusion: The 6-month lifestyle modification programme induced significant weight reduction and significant improvement of parameters of obesity-related diseases. All the measured obesity-related parameters were significantly improved in groups with 3% to <5% and ≥5% weight reduction. Based on these findings, the minimum weight reduction required for improvement of obesity-related risk factors or conditions is 3% in obese and overweight (by WHO classification) Japanese people., (Copyright © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.)

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Solar lentigines (SLs) are characterized by hyperpigmented macules, commonly seen on sun-exposed areas of the skin. Although it has been reported that an increase in the number of melanocytes and epidermal melanin content was observed in the lesions, the following questions remain to be answered: (1) Is acceleration of melanogenesis in the epidermis caused by an increased number of melanocytes or the high melanogenic potential of each melanocyte? (2) Why does the number of melanocytes increase?, Objective: To elucidate the pathogenic mechanism of SLs by investigating the number, melanogenic potential and proliferation status of the melanocyte lineage in healthy skin and SL lesions., Methods: Immunostaining for melanocyte lineage markers (tyrosinase, MART-1, MITF, and Frizzled-4) and a proliferation marker, Ki67, was performed on skin sections, and the obtained images were analyzed by image analysis software., Results: The expression level of tyrosinase to MART-1 of each melanocyte was significantly higher in SL lesions than healthy skin. The numbers of melanocytes in the epidermis, melanoblasts in the hair follicular infundibulum and melanocyte stem cells in the bulge region were increased in SL; however, no significant difference was observed in the Ki67-positive rate of these cells., Conclusion: The melanogenic potential of each melanocyte was elevated in SL lesions. It was suggested that the increased number of melanocytes in the SL epidermis might be attributed to the abnormal increase of melanocyte stem cells in the bulge., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Pathogenesis of Alzheimer's disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK., (Copyright © 2014 Elsevier Inc. All rights reserved.)

UV radiation is a well-known inducer of epidermal pigmentation that is utilized in therapy for vitiligo, one of the skin depigmentation disorders. Although it has been reported that melanocyte stem cells (McSCs) play essential roles in hair pigmentation, the relationship between McSCs and epidermal pigmentation remains unclear. Repetitive UVB irradiation on the dorsal skin of F1 mice of HR-1 × HR/De caused apparent epidermal pigmentation, and it was characterized by increase in the number of melanocytes. Interestingly, differentiation of McSCs into melanoblasts in hair follicles was followed by induction of epidermal melanocyte differentiation. Administration of a neutralizing antibody for Kit receptor that depletes resident melanoblasts could not suppress increased number of melanocytes. UVB irradiation also induced robust expression of Wnt7a as well as Kitl in epidermis, and β-catenin translocation into nucleus in McSCs. Intradermal injection of IWR-1 (inhibitor of Wnt response 1), a chemical inhibitor of β-catenin activation, and small interfering RNA (siRNA) against Wnt7a suppressed increase in the number of epidermal melanocytes. Taken altogether, it was demonstrated that Wnt7a triggered McSCs differentiation through β-catenin activation, and Kitl might induce following migration of melanoblasts to epidermis. These findings will help in developing therapeutic technologies for vitiligo and other pigmentary disorders.

Background: Although a challenge test using non-steroidal anti-inflammatory drugs (NSAIDs) is crucial for diagnosis of aspirin-induced asthma (AIA), it also has drawbacks in terms of possible side effects. Therefore, alternative in-vitro diagnostic methods for AIA are awaited., Methods: Nineteen stable non-AIA patients (9 males and 10 females; mean age, 49.4 ± 4.8 years), and 20 AIA patients (9 males and 11 females; mean age, 51.1 ± 4.8 years) were enrolled in this study. CD11b and CD16 expressions on the peripheral-blood granulocytes after administration of aspirin and different concentrations of PGE2 in vitro were examined using flowcytometry., Results: Aspirin induced a significant increase in CD11b expression on eosinophils (CD16 negative granulocytes) in 19 AIA patients and one non-AIA patient. Increase in CD11b expression on eosinophils by aspirin administration was suppressed by PGE2 in a dose-dependent manner., Conclusions: The measurement of CD11b expression on peripheral-blood eosinophils showed very high sensitivity and specificity of (-95%) in diagnosing AIA. Although this method requires laboratory facilities for flowcytometry, it may be very useful in diagnosis of AIA without side effects. In addition, PGE2 may be involved in regulation of CD11b expression on eosinophils by aspirin administration.

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening., (Copyright © 2012 Elsevier B.V. All rights reserved.)

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-β-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-β-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-β-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-β-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish by administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ability of beta-tricalcium phosphate (beta-TCP) particles to store electric charge was confirmed by thermally stimulated depolarization current measurement as well as surface potential measurement. The efficacy of stored electrical charge on beta-TCP particles in enhancing nerve regeneration was evaluated. Bridge grafting was performed into sciatic nerve defects in Wistar rats with the following tubes: chitosan mesh tubes; chitosan mesh tubes on which beta-TCP particles with or without electrical polarization treatment had been immobilized (polarized and non-polarized tubes, respectively). As a control, isografts were used. Both motor and sensory nerve function as well as electrophysiological recovery progressed with time in each group. Immunofluorescence revealed rapider nerve regeneration in the polarized tube group compared with the non-polarized tube group. The axon density and axon area in the polarized tube group were significantly greater than those in the chitosan mesh tube and non-polarized group, and showed no significant differences from the control group. These results suggest that the stored charge on electrically polarized beta-TCP particles immobilized on chitosan mesh tubes may enhance nerve regeneration to the same extent as isografting., (2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics. To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4(+) cells and Fzd7(+) cells were different from those of Kit(+) cells (precursor of melanocytes: melanoblasts). Fzd4(+) and Fzd7(+) cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit(+) cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4(+) and Fzd7(+) cells following Kit(+) cells during differentiation. These results suggested that Fzd4(+) and Fzd7(+) cells were more immature than melanoblasts, therefore raising the possibility that Fzd4(+) and Fzd7(+) cells are MSCs., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: The existence of multipotent stem cells in subcutaneous adipose tissue has been reported. We previously confirmed that p75 neurotrophin receptor (p75NTR; CD271)-positive cells in subcutaneous adipose tissue possessed multipotency, although changes of the characteristics in p75NTR-positive adipose-derived stem cells (ASCs) with aging remain unclear., Objective: To investigate the effect of aging on p75NTR-positive ASCs., Methods: The number of p75NTR-positive ASCs in subcutaneous adipose tissue of ICR mice aged 3-24 weeks was analyzed by immunostaining and flow cytometry. Subsequently, the cells were isolated and their ability to attach to the cell culture dish, proliferation rate (doubling time) and the expression of senescence-associated beta-galactosidase (SA-beta gal), a cellular senescence marker, were assessed. Age-related changes in the differentiation potential of p75NTR-positive cells in adipogenic, osteogenic, chondrogenic and myogenic lineage were also investigated., Results: The number of ASCs per unit of tissue weight in adipose tissue and the attachment rate of isolated cells decreased with aging. No difference in the cell proliferation rate and the percentage of SA-beta gal-positive cells was detected. Although the efficacy of differentiation into adipogenic and osteogenic lineages slightly decreased with aging, the differentiation potential into chondrogenic and myogenic lineages was not changed., Conclusion: The number of ASCs per unit of tissue weight decreased in aged mice. However, the cells possessed proliferation and differentiation potentials almost equal to those of young mice even though the differentiation potentials showed a tendency of decrease. These results raise the possibility that stem cell functions, self-renewal and multipotency, are maintained regardless of aging., ((c) 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)