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Competing Interests: Declaration of Competing Interest none

Purpose: The purpose of this study was to determine the utility of compressed sensing (CS) with deep learning reconstruction (DLR) for improving spatial resolution, image quality and focal liver lesion detection on high-resolution contrast-enhanced T1-weighted imaging (HR-CE-T1WI) obtained by CS with DLR as compared with conventional CE-T1WI with parallel imaging (PI)., Methods: Seventy-seven participants with focal liver lesions underwent conventional CE-T1WI with PI and HR-CE-T1WI, surgical resection, transarterial chemoembolization, and radiofrequency ablation, followed by histopathological or >2-year follow-up examinations in our hospital. Signal-to-noise ratios (SNRs) of liver, spleen and kidney were calculated for each patient, after which each SNR was compared by means of paired t-test. To compare focal lesion detection capabilities of the two methods, a 5-point visual scoring system was adopted for a per lesion basis analysis. Jackknife free-response receiver operating characteristic (JAFROC) analysis was then performed, while sensitivity and false positive rates (/data set) for consensus assessment of the two methods were also compared by using McNemar's test or the signed rank test., Results: Each SNR of HR-CE-T1WI was significantly higher than that of conventional CE-T1WI with PI (p < 0.05). Sensitivities for consensus assessment showed that HR-CE-MRI had significantly higher sensitivity than conventional CE-T1WI with PI (p = 0.004). Moreover, there were significantly fewer FP/cases for HR-CE-T1WI than for conventional CE-T1WI with PI (p = 0.04)., Conclusion: CS with DLR are useful for improving spatial resolution, image quality and focal liver lesion detection capability of Gd-EOB-DTPA enhanced 3D T1WI without any need for longer breath-holding time., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: We aimed to evaluate the outcomes of the Radiation Therapy Oncology Group 8502 "QUAD shot" regimen using volumetric modulated arc therapy (VMAT) for incurable head and neck cancer (HNC)., Materials and Methods: We included 105 patients with HNC in the study, undergoing at least one QUAD shot regimen cycle. We planned the radiotherapy using VMAT with 6 MV photons. One QUAD shot cycle included 14.8 Gy in 4 fractions with at least 6-hour intervals over 2 consecutive days, repeated every 3-6 weeks up to 3 cycles., Results: We completed 1, 2, and 3 cycles in 11 (10 %), 17 (16 %), and 77 (73 %) patients, respectively. We concurrently performed systemic therapy in 13 (12 %) patients. Tumor response was observed in 92 (88 %) patients and at least one symptom relief in 51 (71 %) of 72 patients. We observed an overall response (tumor response or symptom relief) in 98 (93 %) patients with all patients who completed 3 cycles achieving it. The median overall survival (OS) was 6.8 months. Our multivariate analysis revealed that non-squamous cell carcinoma (p < 0.001), T category of 0-2 (p = 0.021), and 3 QUAD shot cycles (p < 0.001) were independent prognostic factors of better OS. We observed Grade 3 toxicity in 2 (2 %) patients while no ≥ Grade 4 acute or ≥ Grade 3 late toxicity., Conclusions: The QUAD shot regimen using VMAT exerts appropriate palliative effect in patients with incurable HNC. Treatment with higher QUAD shot cycle number would be recommended for better treatment outcomes., 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 © 2024 Elsevier Ltd. All rights reserved.)

Total reflection X-ray fluorescence (TXRF) analysis is one of the useful techniques for determining trace elements. Owing to the high detection sensitivity of X-ray fluorescence in small residues, the hydrophobic substrates are generally used to form dot-type residue. However, when measuring low-Z elements in sample solutions with high concentrations of matrix elements, obtaining sufficient measurement sensitivity can be difficult. X-ray fluorescence is absorbed by the matrix elements, which means that the absorption effect increases in the thicker dried residue compared to the thinner dried residue. To avoid this absorption effect, we have proposed a thin film-type residue. In this study, the glass substrate was treated with an ammonia-hydrogen peroxide mixture (APM). The hydrogen peroxide decomposes organic components, and ammonia slightly etches glass materials. The APM-treated region was limited to a diameter of 6 mm by using a polytetrafluoroethylene mask placed on the glass substrate. As a result, the surface is refreshed to expose the hydroxyl group to make it superhydrophilic. The measured contact angle was 5°. By using the APM-treated superhydrophilic substrate to prepare dried residue, the net intensities in low Z elements were improved. The ratio of Al Kα intensity, calculated as net intensity in the APM-treated substrate divided by it in the hydrophobic substrate, was 2.29. The recovery of each element in the APM-treated substrate was almost 100%; however, recoveries of elements (Al, P, K, and Ca) in the hydrophobic substrate showed significant deviations from 100% (the recovery of Al was approximately 32%). In summary, we successfully improved both analytical sensitivity and accuracy in TXRF analysis by using the APM treated substrate., 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 © 2023. Published by Elsevier B.V.)

The effectiveness of the presence of a prehospital physician for patients with out-of-hospital cardiac arrest (OHCA) undergoing extracorporeal cardiopulmonary resuscitation (ECPR) remains unknown. In this multicenter, retrospective, observational study, we enrolled patients aged ≥18 years who developed OHCA and received ECPR. The primary outcome was the 1-month favorable neurological outcome. We estimated the impact of the presence of a prehospital physician on outcomes using a propensity score analysis with inverse probability weighting. We enrolled 1269 patients. Favorable neurological outcomes occurred in 25 of 316 (7.9%) patients with prehospital physicians and 94 of 953 (9.9%) patients without prehospital physicians. In the propensity score analysis, favorable neurological outcomes did not differ between 2 groups (odds ratio = 0.72; 95% confidence interval: 0.44-1.17). The 1-month favorable neurological outcome was not associated with the presence of a prehospital physician for patients with OHCA who underwent EPCR., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: We aimed to investigate the association between blood urea nitrogen to creatinine ratio (BCR) and survival with favourable neurological outcomes in patients with out-of-hospital cardiac arrest (OHCA)., Methods: This prospective, multicentre, observational study conducted in Osaka, Japan enrolled consecutive OHCA patients transported to 16 participating institutions from 2012 through 2019. We included adult patients with non-traumatic OHCA who achieved a return of spontaneous circulation and whose blood urea nitrogen and creatinine levels on hospital arrival were available. Based on BCR values, they were divided into: 'low BCR' (BCR <10), 'normal BCR' (10 ≤ BCR < 20), 'high BCR' (20 ≤ BCR < 30), and 'very high BCR' (BCR ≥ 30). We evaluated the association between BCR values and neurologically favourable outcomes, defined as cerebral performance category score of 1 or 2 at one month after OHCA., Results: Among 4415 eligible patients, the 'normal BCR' group had the highest favourable neurological outcome [19.4 % (461/2372)], followed by 'high BCR' [12.5 % (141/1127)], 'low BCR' [11.2 % (50/445)], and 'very high BCR' groups [6.6 % (31/471)]. In the multivariable analysis, adjusted odds ratios for 'low BCR', 'high BCR', and 'very high BCR' compared with 'normal BCR' for favourable neurological outcomes were 0.58 [95 % confidence interval (CI 0.37-0.91)], 0.70 (95 % CI 0.49-0.99), and 0.40 (95 % CI 0.21-0.76), respectively. Cubic spline analysis indicated that the association between BCR and favourable neurological outcomes was non-linear (p for non-linearity = 0.003). In subgroup analysis, there was an interaction between the aetiology of arrest and BCR in neurological outcome (p for interaction <0.001); favourable neurological outcome of cardiogenic OHCA patients was lower when the BCR was higher or lower, but not in non-cardiogenic OHCA patients., Conclusions: Both higher and lower BCR were associated with poor neurological outcomes compared to normal BCR, especially in cardiogenic OHCA patients., Competing Interests: Declaration of competing interest All the authors declare that they have no potential conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) differs in terms of cellular and molecular biological characteristics from HPV-negative HNSCC. However, differences in circulating tumor cells (CTCs) between HPV-positive and -negative HNSCC remain unclear., Materials and Methods: We first analyzed eight epithelial-mesenchymal transition (EMT)-related genes (VIM, CDH1, CDH2, SNAI1, SNAI2, TWIST1, ZEB1, and ZEB2) using The Cancer Genome Atlas (TCGA) database. Next, we isolated CTCs from patients with HNSCC using CD45-negative selection and expression analysis of epithelial-related genes (EPCAM, EGFR, and MET) by RT-qPCR. CTC-positive samples were further analyzed for EMT-related genes. In addition, we investigated the proportion of circulating T cell subsets and CD38+ T cells using flow cytometry and their involvement in CTCs., Results: Compared with HPV-negative HNSCC, expression of CDH1, SNAI1, SNAI2, TWIST1, and ZEB1 was downregulated in HPV-positive HNSCC, as determined by TCGA analysis. CTCs were detected in 19 (52.8 %) of 36 HPV-positive and 26 (68.4 %) of 38 HPV-negative patients with HNSCC. EPCAM-positive and MET-positive CTCs were significantly more frequent in patients with HPV-negative HNSCC. HPV-positive patients with HNSCC exhibited significantly high SNAI1 and ZEB2 expression in CTCs. Interestingly, differences in SNAI1 expression levels differed markedly between CTCs and TCGA based on HPV status. Moreover, HPV-positive patients with HNSCC exhibiting SNAI1-high CTCs showed a superior prognosis and a lower proportion of CD38+ T cells than those with SNAI1-low CTCs., Conclusion: Our results provide novel insights into the EMT-MET spectrum of CTCs and may contribute to the development of prognostic biomarkers for HPV-positive HNSCC., 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 © 2022 Elsevier Ltd. All rights reserved.)

Objectives: Exosome-mediated reciprocal crosstalk between tumor and stromal cells plays a crucial role in tumor development and progression. This study investigated whether exosomes released from head and neck squamous cell carcinoma (HNSCC) tumor cells can convert normal fibroblasts into cancer-associated fibroblasts (CAF)-like cells and further analyzed the functional characterization of fibroblasts educated by tumor-derived exosomes., Materials and Methods: Exosomes secreted from HNSCC cell lines were isolated and normal fibroblasts were established from normal oropharyngeal mucosa. The effects of the exosomes on fibroblasts were examined by proliferation and migration assays, and exosome-educated fibroblasts were analyzed for the expression of eight genes (IL1B, IL6, CXCL8, TGFB1, ACTA2, FAP, CD274, and PDCD1LG2) by RT-qPCR. Moreover, T cells or CD14-positive cells were co-cultured with culture supernatants from exosome-educated fibroblasts. T-cell proliferation and macrophage polarization were examined using flow cytometry. Then, RNA sequencing (RNA-seq) of exosome-educated fibroblasts and the corresponding control fibroblasts was performed., Results: Tumor-derived exosomes enhanced fibroblast proliferation and migration. Moreover, gene expression analysis revealed upregulation of the gene expression of proinflammatory cytokines and immunoregulatory genes, and activated fibroblast marker genes. The culture supernatants of tumor-derived exosome-educated fibroblasts suppressed T cell proliferation and the induction of protumoral macrophages compared with those of control fibroblasts. Next, comprehensive RNA-seq analysis data revealed the activation of 11 signaling pathways, including IL-6- and IL-17-related signaling., Conclusion: These results indicate that HNSCC tumor cells induce and/or differentiate into CAFs through exosome-based cell-to-cell communication to create an inflammatory tumor microenvironment., 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 © 2022 Elsevier Ltd. All rights reserved.)

Background: Inhaled medication is the cornerstone of pharmacological treatment for asthma. Poor inhaler techniques are associated with poor asthma control and an increased risk of exacerbations. Leaflets and videos are provided by several pharmaceutical companies to explain the proper techniques to patients. We investigated the effects of instructional materials (leaflet and video) on the patient's inhaler techniques., Methods: The subjects were 67 medical students at Kagoshima University. They all were provided with two types of inhalation devices: a dry powder inhaler (DPI) and a pressurized metered-dose inhaler (pMDI). The students were assigned to three groups: (1) leaflet, (2) video, and (3) combination (leaflet + video). Pulmonologists observed and assessed the students' use of the devices by using a validated checklist. The percentage of subjects who avoided critical errors was also assessed. Critical errors were errors that affected drug delivery to the lungs substantially., Results: The percentage of overall competence and the number of steps that were mastered out of the 11 steps were higher in the combination group than those in the other two groups. However, only 40% in the combination group were able to successfully execute every critical step. The percentage of subjects who avoided critical errors was also higher in the combination group than in the other groups., Conclusions: A single form of instructional materials (leaflet or video) was insufficient for acquiring proper inhaler techniques. The combination of two learning materials may help patients with asthma acquire proper inhaler techniques and subsequently, improve their asthma control., Competing Interests: Conflict of Interest H. Inoue reports grants from Boehringer Ingelheim, Novartis, GlaxoSmithKline, personal fees from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Kyorin, Sanofi, outside the submitted work. Other authors (T. Matsuyama, K. Machida, A. Hamu, K. Takagi, H. Momi, I. Higashimoto) have no conflict of interest to declare., (Copyright © 2022 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Competing Interests: Funding Support and Author Disclosures The authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Background: Rigid equinovarus foot deformities are seen in patients with cerebral palsy (CP). This retrospective study was undertaken to evaluate flexor hallucis longus tendon (FHL) transfer with gastrocsoleus recession (GSR) using motion analyses and quantitative measurement, and to investigate postoperative complications., Methods: This study included 10 hemiplegic CP patients who underwent FHL transfer with GSR, and were evaluated by motion analyses consisting of weight distribution in static standing position and three-dimensional gait analysis, both pre and post-operatively. They were assessed in terms of kinematic data, Gait Variable Scores (GVS), and Gait Profile Score (GPS)., Results: The mean age at operation was 7.3 years (range, 4-13 years), and mean follow-up duration was 35 months (range, 25-64 months) post-operatively. Weight distribution at surgical site significantly rose from 34.3% pre-operatively to 47.3% post-operatively, and abnormal asymmetry of weight distribution between surgical site and contralateral site disappeared post-operatively. Maximum ankle dorsiflexion (ADF) at initial contact rose from -20.9° to -6.28°. Similarly, Maximum ADF at both stance and swing phase rose from -13.8° to 17.7° (P = 0.0003), and from -19.5° to 1.35° (P = 0.001), respectively. Although mean GPS decreased from 15.6° pre-operatively to 11.8°, which corresponded to 2.38 times the minimal clinically important difference (MCID = 1.6°), three cases manifested talipes calcaneus at final follow-up., Conclusion: Although quantitative assessment showed that the potential value of FHL transfer with GSR was to obtain initial heel contact and maintain sufficient clearance from the ground in swing, it also revealed a risk of leading to talipes calcaneus. In the near future, we should establish accurate criteria for determination of transfer site, and consider the possibility of modification of this procedure in order to balance between recurrent equinus and significant talipes calcaneus., Study Design: Clinical comparison between preoperative and postoperative., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Background: Randomized controlled trials or observational studies showed that the use of public-access automated external defibrillator (AED) was effective for patients with out-of-hospital cardiac arrest (OHCA). However, it is unclear whether public-access AED use is effective for all patients with OHCA irrespective of first documented rhythm. We aimed to evaluate the effect of public-access AED use for OHCA patients considering first documented rhythm (shockable or non-shockable) in public locations., Methods: From the Utstein-style registry in Osaka City, Japan, we obtained information on adult patients with OHCA of medical origin in public locations before emergency-medical-service personnel arrival between 2011 and 2015. Primary outcome was 1-month survival with favorable neurological outcome. Multivariable logistic regression analysis was performed to assess the association between the public-access AED pad application and favorable neurological outcome after OHCA by using one-to-one propensity score matching analysis., Results: Among 1743 eligible patients, a total of 336 (19.3%) patients received public-access AED pad application. The proportion of patients who survived 1-month with favorable neurological outcome was significantly higher in the pad application group than in the non-pad application group (29.8% vs. 9.7%; adjusted odds ratio [AOR], 2.85; 95% confidence interval [CI], 1.73-4.68, AOR after propensity score matching, 2.46; 95% CI, 1.29-4.68). In a subgroup analysis, the AORs of patients with shockable or non-shockable rhythms were 3.36 (95% CI, 1.78-6.35) and 2.38 (95% CI, 0.89-6.34), respectively., Conclusions: Public-access AED pad application was associated with better outcome among patients with OHCA of medical origin in public locations irrespective of first documented rhythm., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The use of public-access automated external defibrillators (AEDs) has become common in Japan. To provide a strategy for appropriate public-access AED deployment, we assessed public-access defibrillation (PAD) by laypersons and the outcomes following out-of-hospital cardiac arrest (OHCA) among adult patients by location of arrest., Methods: From a nationwide, prospective, population-based registry of patients after OHCA in Japan, we enrolled adult patients with bystander-witnessed OHCA of medical origin in public locations between 2013 and 2015. The primary outcome measure was one-month favorable neurological outcome defined by cerebral performance category 1 or 2. Factors associated with favorable neurological outcome after ventricular fibrillation (VF) were assessed by multivariable logistic regression analysis., Results: A total of 20,970 adult bystander-witnessed OHCAs of medical origin occurred in public locations. Of those, the proportions of PAD by location were: 13.1% (757/5761) in public areas, 15.9% (333/2089) at workplaces, 26.0% (544/2095) in recreation/sports areas, 36.1% (112/310) in educational institutions, and 5.8% (241/4151) on streets/highways. In a multivariable analysis of VF arrests, both bystander cardiopulmonary resuscitation [adjusted odds ratio (AOR), 1.78; 95% confidence interval (CI), 1.54-2.07] and PAD (AOR, 2.33; 95% CI, 2.05-2.66), and emergency medical service (EMS) response time (AOR, 0.89; 95% CI, 0.87-0.90) were associated with improved outcomes. Earlier PAD initiated by bystanders before EMS arrival was also associated with better outcomes after OHCA., Conclusions: In Japan, where public-access AEDs are well-disseminated, the PAD program worked effectively for adult OHCA of medical origin occurring in public locations. Notably, the proportions of PAD differed substantially according to specific public locations., (Copyright © 2019 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Thermoresponsive core-corona type nanoparticles were prepared exhibiting biomolecule recognition ability on their surfaces. These thermoresponsive nanoparticles were prepared from a poly(N-isopropylacrylamide) (PNIPAAm) macro-initiator and styrene (St) in a polar solvent via atom transfer radical dispersion polymerization. The PNIPAAm macro-initiator contains an alkyl halide and/or phthalimide group on the terminated group of the polymer chain, and thus, the grafting of PNIPAAm on the PSt core resulted in terminating phthalimide end groups. These terminal groups were utilized to immobilize biomolecule recognition units, and the dispersion stabilities of the nanoparticles were found to change in aqueous solution at room temperature due to alteration of the terminating PNIPAAm groups by the presence of biomolecules at different concentrations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alkaline phosphatase (AP) is a gate-keeper of innate immune system responses by detoxifying inflammation triggering moieties released from endogenous and external sources. We examined whether AP's broad mechanism of action constitutes a safe therapeutic, either as single agent or combined with methotrexate (MTX), for chronic inflammatory disorders, for example, rheumatoid arthritis (RA). A rat model for RA was used with repeated intra-articular methylated bovine serum albumin (mBSA) injections in 1 knee ("arthritic" knee), with the contralateral knee serving as internal control. AP (200 µg, subcut) was administered before mBSA injections (prophylactic setting) or after arthritis induction (therapeutic setting) or combined with MTX (0.3 mg/kg or 1 mg/kg; intraperitoneally). As end point of treatment outcome, macrophage infiltration in knees, liver, and spleen was assessed by immunohistochemistry (ED1 and ED2 expression), immunofluoresence (macrophage marker folate receptor-β [FRβ]), and [ 18 F]fluoro-polyethylene glycol-folate positron emission tomography (PET) (macrophage imaging) and ex vivo tissue distribution. Single-agent AP treatment and combinations with MTX were well tolerated. Both prophylactic and therapeutic AP markedly reduced synovial macrophage infiltration in arthritic knees (ED1: 3.5- to 4-fold; ED2: 3.5- to 6-fold), comparable with MTX treatment. AP-MTX combinations slightly improved on single agent effects. PET monitoring and ex vivo tissue distribution studies corroborated the impact of AP, MTX, and AP-MTX on reducing synovial macrophage infiltration. Beyond localized articular effects, AP also revealed systemic anti-inflammatory effects by a 2-fold reduction of ED1, ED2, and FRβ + macrophages in liver and spleen of arthritic rats. Collectively, single-agent AP and AP combined with MTX elicited local and systemic anti-arthritic activity in arthritic rats., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2 kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: 8q24.21 is a frequently amplified genomic region in colorectal cancer (CRC). This region is often referred to as a 'gene desert' due to lack of any important protein-coding genes, highlighting the potential role of noncoding RNAs, including long noncoding RNAs (lncRNAs) located around the proto-oncogene MYC. In this study, we have firstly evaluated the clinical significance of altered expression of lncRNAs mapped to this genomic locus in CRC., Patients and Methods: A total of 300 tissues, including 280 CRC and 20 adjacent normal mucosa specimens were evaluated for the expression of 12 lncRNAs using qRT-PCR assays. We analyzed the associations between lncRNA expression and various clinicopathological features, as well as with recurrence free survival (RFS) and overall survival (OS) in two independent cohorts., Results: The expression of CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 were elevated in cancer tissues (P = 0.039, <0.001, 0.018, <0.001, 0.002, respectively). Among these, high expression of CCAT1 and CCAT2 was significantly associated with poor RFS (P = 0.049 and 0.022, respectively) and OS (P = 0.028 and 0.015, respectively). These results were validated in an independent patient cohort, in which combined expression of CCAT1 and CCAT2 expression was significantly associated with a poor RFS (HR:2.60, 95% confidence interval [CI]: 1.04-6.06, P = 0.042) and a poor OS (HR:8.38, 95%CI: 2.68-37.0, P < 0.001). We established a RFS prediction model which revealed that combined expression of CCAT1, CCAT2, and carcinoembryonic antigen was a significant determinant for efficiently predicting RFS in stage II (P = 0.034) and stage III (P = 0.001) CRC patients., Conclusions: Several lncRNAs located in 8q24.21 locus are highly over-expressed in CRC. High expression of CCAT1 and CCAT2 significantly associates with poor RFS and OS. The expression of these two lncRNAs independently, or in combination, serves as important prognostic biomarkers in CRC., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The number of people living in high-rise buildings has recently been increasing in Japan, and delayed transport time by emergency-medical-service (EMS) personnel from higher floors could lead to lower survival after out-of-hospital cardiac arrest (OHCA). However, there are no clinical studies assessing the association between the floor where patients reside and neurologically favorable outcome after OHCA., Methods: This was a prospective, population-based study conducted in Osaka City, Japan that enrolled adults aged >=18years suffering an OHCA of cardiac origin before EMS arrival between 2013 and 2014. The primary outcome measure was one-month survival with neurologically favorable outcome. We divided OHCA patients into the following groups: those residing on >=3 floors (the high floor group) and <3 floors (the low floor group). Multiple logistic regression analysis was used to assess factors associated with neurologically favorable outcome., Results: A total of 2979 patients were eligible for analysis. Of them, 1885 (62.3%) occurred below the third floor and 1094 (37.4%) occurred at or above the third floor. The proportion of neurologically favorable outcome after OHCA was significantly lower in the high floor group than in the low floor group (2.7% [30/1094] versus 4.8% [91/1885], P=0.005). In a multivariate analysis, neurologically favorable outcome after OHCA was significantly lower in the high floor group than in the low floor group (adjusted odds ratio, 0.59 [95% confidence interval, 0.37-0.96])., Conclusions: In this population, one-month survival with neurologically favorable outcome from OHCA was lower in the high floor group than in the low floor group., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Based on results of hematopoietic stem cell transplantation in animal models of stroke, clinical trials with hematopoietic stem cells administered intra-arterially or intravenously have been initiated in patients. Although intra-arterial injection is expected to deliver transplanted cells more directly to the ischemic tissue, the optimal route for enhancing clinical outcomes has not been identified in the setting of stroke. In this study, we compared the therapeutic potential of intra-arterial versus intravenous injection of bone marrow derived-mononuclear cells (BM-MNCs) and CD133-positive (CD133(+)) cells in a murine stroke model. We have found that intra-arterial injection of BM-MNCs exaggerates inflammation with accompanying loss of microvascular structures in poststroke brain and no improvement in cortical function. In contrast, intravenous injection of BM-MNCs did not similarly enhance inflammation and improved cortical function. Our results indicate that the optimal route of cell transplantation can vary with different cell populations and highlight possible issues that might arise with intra-arterial cell administration for acute ischemic cerebrovascular disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sema4D, originally identified as a negative regulator of axon guidance during development, is involved in various physiological and pathological responses. In this study, we evaluated the effect of Sema4D-deficiency on oligodendrocyte restoration after the cerebral ischemia/reperfusion using direct ligation of the middle cerebral artery followed by reperfusion. In both Sema4D(+/+) wild-type and Sema4D(-/-) null mutant mice, the peri-infarct area showed a decrease in the number of oligodendrocytes at 3 days post-reperfusion. Subsequently, the number of oligodendrocytes was observed to gradually recover in both groups. Sema4D-deficient mice, however, showed an enhanced recovery of oligodendrocytes and an upregulation of oligodendrocyte progenitor cells at days 14 and 28 of reperfusion. Cell proliferation identified by incorporation of bromodeoxyuridine was enhanced in Sema4D(-/-) mice from days 3 to 14 post-reperfusion compared to the Sema4D(+/+) mice. Furthermore, apoptotic cell death of oligodendrocytes was reduced at days 7 post-reperfusion in Sema4D(-/-) mice compared to Sema4D(+/+) mice. These findings indicate that enhanced proliferation of progenitor cells and survival of oligodendrocytes resulted in improved oligodendrocyte recovery in Sema4D(-/-) mice. This may provide a new approach for neurorestorative treatment in patients with stroke, which aims to manipulate endogenous oligodendrogenesis and thereby to promote brain repair after stroke., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Ayu Plecoglossus altivelis altivelis are one of the most economically important fish for freshwater aquaculture in Japan. We conducted expressed sequence tag analyses of three leukocyte subpopulations, thrombocytes, neutrophils, and B lymphocytes in ayu using a next generation sequencer. The sequencing and de novo assembly yielded 22,494, 22,733, and 16,505 contigs from the thrombocyte, neutrophil, and B lymphocyte cDNA libraries, respectively. Pathways involving endocytosis, phagosomes, and lysosomes, were found in all three cDNA libraries using pathway analysis. The thrombocyte cDNA library contained 2894 unique sequences, including CXC chemokine receptor 4 and MHC class II. Cytokine and cytokine receptor genes such as interleukin (IL)-1β, IL-8, IL-1 receptor (IL-1R), IL-8RA, and IL-8RB were found among the 3056 unique sequences of the neutrophil cDNA library. Typical B lymphocyte related genes such as B cell linker protein, immunoglobulin (Ig) M, IgD and transforming growth factor β were found in the 1590 unique sequences of the B lymphocyte cDNA library. In summary, a large number of immune-related genes were identified from the three leukocyte cDNA libraries. Our results represent a valuable sequence resource for understanding the immune system function in ayu., (Copyright © 2015 Elsevier B.V. All rights reserved.)

When performing minimally invasive coronary artery bypass grafting (MICS CABG), it is difficult to access the right internal thoracic artery (ITA) under direct vision. We successfully performed off-pump MICS CABG using the bilateral in situ ITAs through a 8-cm left thoracotomy under direct vision for a 76-year-old man. His postoperative course was uneventful and all the grafts were patent. This novel, minimally invasive approach safely preserves the sternum and delivers the benefits of bilateral in situ ITA grafting, providing an alternative to conventional CABG and off-pump CABG., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Responsiveness to methotrexate (MTX), the "anchor drug" for treating rheumatoid arthritis (RA), varies among individual patients. In this study we investigated the effects of folate transporter gene expression levels on disease activity among 56 Japanese patients with RA who were undergoing MTX therapy. We also assessed gene expression levels for 15 healthy control subjects. The mRNA expression levels of reduced folate carrier 1 (RFC1) and proton-coupled folate transporter (PCFT) in PBMCs from these patients and controls were determined using real-time quantitative polymerase chain reaction (PCR). Compared with PCFT, there were large individual differences in RFC1 mRNA expression levels in both RA patients and healthy controls. RFC1 mRNA expression levels and RA disease activity scores were significantly negatively correlated, as disease activity scores were lower for patients with higher RFC1 mRNA expression levels. However, RFC1 mRNA levels were not correlated with MTX doses. Thus, the clinical efficacy of MTX for Japanese RA patients was associated with the expression level of a folate transporter gene. Increased RFC1 expression may increase MTX uptake by immune cells, such as lymphocytes, and as a result, RA disease activity would be reduced., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

We prepared transformable thermoresponsive nanoparticles with variable core softness, controlled by the nanoparticle core's glass transition temperature (Tg). The nanoparticles were prepared by the dispersion copolymerization of butyl methacrylate (BMA) and/or methyl methacrylate (MMA) with a poly(N-isopropylacrylamide) (PNIPAAm) macromonomer in a polar solvent. The shape of the nanoparticle core changed with temperature. We then prepared poly(vinyl alcohol) (PVA) films with dispersed thermoresponsive nanoparticles, to elongate the nanoparticles through a uniaxial stretching of the films at 60°C. In this manner, the nanoparticle shape changed from spherical to rod-like morphologies, depending on the degree of film extension. Additionally, the rod-shaped nanoparticles only changed back to spheres with temperature modulation. The nanoparticle core's Tg value affected the rate of its physical transformation from rods to spheres at 37°C, with a slower rate observed for increased Tg. As the nanorod shape change was relatively minor at 37°C, we could control the shape of these transformable nanoparticles under various physiological conditions, a highly desirable feature for drug delivery applications., (Copyright © 2014 Elsevier B.V. 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.)

To simulate the clinical and pathologic situation in patients with stroke, as well as to evaluate future potential therapeutic approaches, it is essential to have a highly reproducible model that displays long-term survival. Though a range of rodent models has been employed in the literature, there are questions regarding reproducibility, especially in terms of ischemic zone (i.e., degree of ischemia) and long-term survival. We have developed a highly reproducible stroke model that produces a consistent ischemic zone as a result of direct transient occlusion of the middle cerebral artery (MCA) in CB-17 (CB-17/Icr-+/+Jcl) mice. The model employs a thin monofilament to twist the artery resulting in complete interruption of blood flow. Transient ischemia can be induced for up to 240min and the survival rate at 7 days post-ischemia was more than 60%, even in mice subjected to 240min of transient ischemia resulting in hemorrhagic infarction in most animals. Our method can be used to model several pathologic conditions, such as reversible reperfusion injury, delayed neuronal death, necrotic brain injury and hemorrhagic infarction. We believe this preclinical model provides a step forward for testing future therapeutic approaches applicable to patients with ischemic brain injury., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Methotrexate (MTX) exhibits large inter-individual and inter-ethnic differences in the dose required for its anti-rheumatic effect. To maintain low disease activity, patients may require increased MTX doses or co-administration of biologic disease-modifying anti-rheumatic drugs (bDMARDs). The availability of a marker predicting the effect of MTX will make it possible to increase the MTX dose and prescribe bDMARDs to patients at an early stage. To establish individualized medication for rheumatoid arthritis (RA), we investigated genetic polymorphisms of the folate pathway in Japanese RA patients. Eighty-nine patients were treated with MTX alone (MTX group). MTX and bDMARDs were co-administered to 81 patients because of insufficient MTX efficacy (MTX + bDMARDs group); an equally stable therapeutic effect was achieved in both groups. Polymorphism analyses using bDMARD co-treatment as the objective variable revealed a significant association between age and the G80A polymorphism of the reduced folate carrier 1 gene (RFC1) as an explanatory variable. Compared to patients with the A allele, patients with the G allele may have less intracellular MTX uptake and, therefore, poor efficacy; a greater number of them were found to be bDMARD concomitant cases. The results of this study suggest that the RFC1 G80A polymorphism may be a useful marker for predicting MTX efficacy in Japanese patients with RA.

Background: Unicuspid aortic valve is a rare anatomic variant, but patients may require intervention for severe valve dysfunction at a young age. We introduce a new reconstructive technique for diseased unicuspid valve by tricuspidization with glutaraldehyde-treated autologous pericardium., Methods: From April 2007 through January 2011, we performed 304 cases of aortic valve reconstruction using glutaraldehyde-treated autologous pericardium. During the same period, we encountered 9 patients with unicuspid aortic valve, including 8 male patients and 1 female patient. Mean age was 48.9±19.9 years (14-78 years). Two patients had aortic stenosis (AS), 1 had aortic regurgitation (AR), and 6 patients had both. Our original aortic valve reconstruction technique is characterized by the independent replacement of 3 leaflets with separate measurement of length between each commissure. In the case of a unicuspid aortic valve, we create a new commissure at a higher point along the raphe at the same level with the 1 normally existing commissure., Results: No early mortality or major morbidity was recorded. Postoperative echocardiography showed trivial or no AR, with the peak pressure gradients averaging 10.6±3.3 mm Hg. One-year follow-up echocardiography revealed that the peak pressure gradients averaged 8.6±3.7 mm Hg, with trivial or no AR. The mean follow-up period was 551.1±51.4 days. All 9 patients are in good condition. No reoperation or any additional intervention has been necessary., Conclusions: Diseased unicuspid aortic valves were treated by our original aortic valve reconstruction technique with excellent early results. We continue to study the long-term results., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background Aims: Human mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populations., Methods: Given these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD model., Results: Our study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD., Conclusions: Our results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.

Coronary ostial stenosis is usually treated by conventional coronary artery bypass graft surgery. Although patch angioplasty is a widely accepted alternative surgical treatment, it has been reported sporadically. We encountered bilateral ostial stenosis with Takayasu disease. This report describes successful patch angioplasty using glutaraldehyde-treated autologous pericardium of bilateral coronary ostial stenosis owing to Takayasu disease., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

A novel single-nucleotide polymorphism (SNP) in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene with enhanced expression was identified in 2001. In 2007, it was reported that this SNP, DHFR C829T, was located close to a microRNA binding site and contributed to the stability of mRNA. Many researchers have analyzed this SNP in several races including Asians and Caucasians. However, the mutation allele is not yet confirmed in most populations. In this study, we reinvestigated the frequency of this SNP using three methods. First, this SNP in genomic DNA was analyzed by a PCR-restriction fragment length polymorphism method. Second, this SNP in mRNA was analyzed by a single nucleotide extension method following a reverse transcription reaction. Third, the mRNA expression level was analyzed by a real-time PCR method. The findings in our study, regarding the discovery of this SNP, suggest that the SNP is an artifact caused by contamination by the genomic DNA of the pseudogene DHFRP1. This study is a reinvestigation of a newly discovered genetic polymorphism.

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

CADM2, a candidate gene for psoriasis, was identified by a genome-wide association study using microsatellites in the Japanese population (561 cases and 561 controls). Moreover, haplotype analysis included an additional 68 SNPs and indicated that a 110-kb haplotype block was detected for the protective risk haplotype of psoriasis. We also identified an initial exon of novel splicing variants in this haplotype block. A functional analysis by qRT-PCR using RNAs from the blood of 56 cases and 64 controls significantly demonstrated an inverse correlation between expression frequencies in a novel splicing variant and the number of alleles associated with psoriasis. To confirm these results, we must perform replication studies using other ethnic groups and more functional analysis particularly for skin tissues., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Quadricuspid aortic valves represent a very rare pathology. Most cases have been discovered incidentally during heart operations or at autopsy. Patients may become symptomatic with aortic regurgitation. We encountered a symptomatic patient with aortic regurgitation and a quadricuspid aortic valve. Successful aortic valve plasty was done with our original technique of tricuspid replacement by glutaraldehyde-treated autologous pericardium., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Recent linkage analyses of nondiabetic African-American patients with focal segmental glomerulosclerosis (FSGS) have identified MYH9, encoding nonmuscle myosin heavy chain IIA (NMMHC-IIA), as a gene having a critical role in this disease. Abnormalities of the MYH9 locus also underlie rare autosomal dominant diseases such as May-Hegglin anomaly, and Sebastian, Epstein (EPS), and Fechtner (FTNS) syndromes that are characterized by macrothrombocytopenia and cytoplasmic inclusion bodies in granulocytes. Among these diseases, patients with EPS or FTNS develop progressive nephritis and hearing disability. We analyzed clinical features and pathophysiological findings of nine EPS-FTNS patients with MYH9 mutations at the R702 codon hot spot. Most developed proteinuria and/or hematuria in early infancy and had a rapid progression of renal impairment during adolescence. Renal histopathological findings in one patient showed changes compatible with FSGS. The intensity of immunostaining for NMMHC-IIA in podocytes was decreased in this patient compared with control patients. Thus, MYH9 R702 mutations display a strict genotype-phenotype correlation, and lead to the rapid deterioration of podocyte structure. Our results highlight the critical role of NMMHC-IIA in the development of FSGS.

We analyzed the subcellular distributions and gene structures of interferon regulatory factor 3 (IRF3) transcription factor in 50 cases of human primary lung cancer. The immunohistochemical analyses revealed substantially aberrant IRF3 expression specific to the cancer lesions (2 and 6 tumors with nuclear staining, and 4 and 5 tumors with negative staining, in adenocarcinoma and squamous cell carcinoma, respectively), while the morphologically normal region around the tumors exhibited only cytoplasmic staining. In addition, we determined the sequence of the entire IRF3 coding region, and found two novel variants with the amino acid changes (S(175)(AGC)-->R(175)(CGC) and A(208)(GCC)-->D(208)(GAC)). The R(175) variant was also detected in a morphologically normal region around the nuclear staining squamous cell carcinoma, and exhibited almost the same functions as the wild type IRF3. On the other hand, the D(208) variant, found in the negative staining squamous cell carcinoma cases, reduced the nuclear translocation in response to IkappaB kinase epsilon stimulation, as compared to the wild type IRF3, but the same variant was detected in the surrounding morphologically normal region. The aberrant expression of IRF3 and the novel D(208) variant may provide clues to elucidate the etiology of primary lung cancer., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing., (2010 Elsevier Inc. All rights reserved.)

Objective: An early vascular response for angiogenesis is essential for the normal progression of bone defect healing. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis. The aim of this study was to evaluate the effects of a poly (L,D-lactic-co-glycolic acid) (PLGA) membrane with VEGF encapsulated into PLGA microspheres on bone regeneration at bone defects in rat calvaria., Study Design: Microspheres of PLGA incorporating VEGF(165) (VEGF microspheres) were prepared, and critical-size bone defects were created in rat calvaria. The VEGF microspheres, PLGA microspheres, or VEGF microspheres plus PLGA membrane were applied to the defects. Bone regeneration was evaluated using image analysis based on soft radiographic and histologic examination., Results: Mature thick bone regeneration was observed in selected sites at bone defects that had been applied with VEGF microspheres/PLGA membrane compared with those that had been applied with the other treatments., Conclusion: A combination of VEGF microspheres and a PLGA membrane effectively enhances bone regeneration., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

Dihydrofolate reductase gene (DHFR) 19-bp deletion polymorphisms result in varied DHFR enzymatic activity affecting the risk for preterm delivery, spina bifida, and the efficacy of methotrexate (MTX). Ethnic differences in DHFR 19-bp polymorphisms may be responsible for the divergent findings in previous genetic studies. We compared genotype and allele frequency of DHFR intronic 19-bp deletion polymorphisms in ethnically homogenous East Asians (from Japan) and others by polymerase chain reaction assay conducted on 277 healthy Japanese individuals. The genotype distribution was as follows: wild/wild, 11.9% (n=33); wild/deletion, 40.1% (n=111); deletion/deletion, 48.0% (n=133). The frequencies of wild type and deletion alleles were 0.32 and 0.68, respectively. The obtained genotype distribution was consistent with those calculated by Hardy-Weinberg equilibrium. The genotype distribution and allele frequencies in the Japanese population were significantly different from those previously reported for other ethnic populations. Determination of intronic 19-bp deletion polymorphisms of DHFR may be useful for monitoring the efficacy and side effects of MTX for the treatment of diseases such as rheumatoid arthritis and childhood acute leukemia in the Japanese population because the frequency of the deletion allele is higher.

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.

There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199-338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence.

A positive selection system for gene disruption using a sucrose-sensitive transgenic rhizobium was established and used for the molecular characterization of mutations induced by ion beam irradiations. Single nucleotide substitutions, insertions, and deletions were found to occur in the sucrose sensitivity gene, sacB, when the reporter line was irradiated with highly accelerated carbon and iron ion beams. In all of the insertion lines, fragments of essentially the same sequence and of approximately 1188bp in size were identified in the sacB regions. In the deletion lines, iron ions showed a tendency to induce larger deletions than carbon ions, suggesting that higher LET beams cause larger deletions. We found also that ion beams, particularly "heavier" ion beams, can produce single gene disruptions and may present an effective alternative to transgenic approaches.

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam.

Constitutive activity of NF-kappaB is associated with various human cancers including adult T-cell leukemia (ATL). In this study, we have found Tgat that activates NF-kappaB by screening a cDNA expression library derived from ATL cells. We previously identified Tgat as the oncogene, which consists of the Rho-guanine nucleotide exchange factor (Rho-GEF) domain and the unique C-terminal region, as a consequence of alternative splicing of the Trio transcript. Tgat activated the IKK activity by binding with the IkappaB kinase (IKK) complex. The Tgat mutants lacking the C-terminal region failed to associate with the IKK complex suggesting an essential role of the unique sequence. The mutation causing the loss of GEF activity also abolished the NF-kappaB activation. Moreover, co-expressed p100 was efficiently processed into p52 in the Tgat-expressing cells, suggesting the co-involvement of non-canonical pathway.

Receptor activator of nuclear factor-kappaB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue.

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.

Although the chromosomal localization (9q34) of the gene encoding the human form of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13) and its exclusive expression in the liver have been established, the cells that produce this enzyme are yet to be determined. We investigated the expression of ADAMTS13 mRNA and protein in fresh frozen specimens obtained during liver biopsies of 8 patients with liver diseases. In situ hybridizations to localize ADAMTS13 mRNA showed positive signals exclusively in perisinusoidal cells with irregularly elongated dendritic processes extending between hepatocytes. Furthermore, ADAMTS13 was detected immunohistochemically in perisinusoidal cells, whereas no staining was observed in hepatocytes. The positive cells varied in shape from unipolar to dendritic with irregularly elongated cytoplasmic processes, features common to hepatic stellate cells (HSCs). Double-labeling experiments revealed that the ADAMTS13-positive cells also expressed alpha-smooth muscle actin, confirming that these cells were activated HSCs. These results suggest that HSCs may be major cells producing ADAMTS13 in human liver.