Non-invasive transcriptomic analysis using mRNAs in skin surface lipids obtained from children with mild-to-moderate atopic dermatitis.

BACKGROUND: Specimens for analysing the molecular pathology of skin disease are generally obtained through invasive methods, such as biopsy. However, less burdensome methods are desirable for paediatric patients. We recently established a method that comprehensively analyses RNA present in sebum (skin surface lipid-RNAs: SSL-RNAs) using a next-generation sequencer. Using this method, biological information can be obtained from the skin in a completely non-invasive manner. OBJECTIVES: To verify the applicability of the SSL-RNA method for analysis of paediatric skin and analyse the molecular pathology of mild-to-moderate atopic dermatitis (AD) in children. METHODS: We collected sebum specimens from the whole faces of 23 healthy children and 16 children with mild-to-moderate AD (eczema area and severity index (EASI) score: 5.9 ± 2.6) ranging in age from 6 months to 5 years, using an oil-blotting film. We then extracted SSL-RNAs from the samples and performed an AmpliSeq transcriptomic analysis. RESULTS: The expressions of genes related to keratinization (LCE, PSORS1C2, IVL and KRT17), triglyceride synthesis and storage (PLIN2, DGAT2 and CIDEA), wax synthesis (FAR2), ceramide synthesis (GBA2, SMPD3 and SPTLC3), antimicrobial peptides (DEFB1) and intercellular adhesion (CDSN), all of which are related to the skin barrier, are lower in children with AD than in healthy children. The children with AD also have higher expression of CCL17, a Th2-cytokine and an increased Th2-immune response as demonstrated by a gene set variation analysis. Moreover, KRT17 and CCL17 expression levels are significantly correlated with the EASI score. CONCLUSIONS: Molecular changes associated with abnormal immune responses and the epidermal barrier in children with mild-to-moderate AD can be determined using the SSL-RNA method. This non-invasive method could therefore be a useful means for understanding the molecular pathology of paediatric AD.

Developing and validating the Japanese version of professional attitude scale for nurses.

AIM: We developed and psychometrically tested the Japanese version of the Professional Attitude Scale for Nurses (PASN-J). BACKGROUND: Nurses must recognize the importance of their professionalism; therefore, it is critical to quantitatively measure nurses' professional attitudes. INTRODUCTION: This instrument validation study was designed to generate an itemized scale and examine its content validity/psychometric testing using a sample of Japanese nurses. METHODS: Based on a trait approach focusing on the characteristic traits of the nursing profession, a 59-item draft scale was generated. During November 2017, 2657 nurses from 29 facilities in Japan were surveyed. The questionnaire included demographics, the 59-item draft scale, and a self-report scale of nurses' professional behaviour and nursing practice ability. Using exploratory and confirmatory factor analyses, we evaluated the construct, criterion-related, concurrent, and known-groups validity, and reliability of the PASN-J. RESULTS: Data from 1716 participants were analysed. The analyses yielded a 38-item, 3-factor scale that adequately fit the data. PASN-J scores were positively correlated with nurses' professional behaviour and nursing practice ability. CONCLUSION: The 38-item PASN-J has good reliability and validity, making it useful for measuring the current condition of nursing professionalism and evaluating nursing education. Implications for Nursing and Health Policy: This scale can evaluate nursing education and promote nurses' professionalism. The PASN-J will help identifying the elements of undergraduate nursing education that require further emphasis. Additionally, the PASN-J could facilitate the development of nursing policies to promote professional development in nurses. Ultimately, evaluating nursing education with the PASN-J enhances nurses' professional attitudes and subsequently improves their quality of nursing, nursing efficiency and patient outcomes.

Cytological features of solid variants of papillary thyroid carcinoma: a fine needle aspiration cytology study of 18 cases.

OBJECTIVE: Solid variants of papillary thyroid carcinoma (SV-PTC) are rare, and there have been few reports describing the cytological findings of such variants. METHODS: The cytological features of cellular specimens aspirated from 18 histologically confirmed SV-PTC cases were evaluated, retrospectively. RESULTS: Solid and small papillary clusters were observed in 14 (77.8%) and 13 (72.2%) cases, respectively. The incidences of large papillary clusters (11.1%) and sheet-like arrangements (11.1%) were low. Nuclear features were consistent with conventional PTC. The background was clean, and there were no colloid materials, foamy histiocytes, multinucleated giant cells, psammoma bodies, or necrotic materials. CONCLUSIONS: Solid clusters and small papillary clusters in conjunction with a clean background are diagnostic clues that indicate SV-PTC cytologically. It is thought that small papillary clusters reflect the micropapillary growth pattern seen within the lumen of middle-sized follicular structures. The presence of nuclear findings typical of conventional PTC and the absence of mitotic figures and necrotic materials are important for distinguishing SV-PTC from poorly differentiated carcinoma.

Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan.

UNLABELLED: Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4 527 samples of DNA from ticks in Japan, which were then analysed by real-time PCR and nested PCR. Francisella DNA was detected by real-time PCR in 2·15% (45/2 093) of Ixodes ovatus, 0·66% (14/2 107) of I. persulcatus, 8·22% (6/73) of I. monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I. ovatus and I. persulcatus, respectively, which were positive according to real-time PCR. Phylogenetic analysis showed that the sequence from I. ovatus and I. persulcatus were clustered with F. tularensis type B strains distributed in Eurasia. Microinjected live F. tularensis persisted in ticks, whereas heat-killed F. tularensis decreased. Microinjected F. tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F. tularensis contributes to the adaptation or survive of bacterial infection in ticks. SIGNIFICANCE AND IMPACTS OF THE STUDY: Francisella tularensis has been detected in ticks, suggesting that it is a tick-borne pathogen. However, F. tularensis has not been detected in ticks in Japan since 1991. In this study, we performed a large-scale analysis of DNA isolated from ticks in Japan and detected F. tularensis by real-time polymerase chain reaction (PCR) and nested PCR. We found that F. tularensis could survive in ticks based on an experimental tick-infection model. We also identified a bacterial factor that contributes to survival in ticks. Our results suggest that ticks are candidate vectors that mediate F. tularensis infection in Japan.

Biodegradation of 2,4-dichlorophenol by shiitake mushroom (Lentinula edodes) using vanillin as an activator.

The white-rot shiitake mushroom, Lentinula edodes, was used to degrade an environmentally hazardous compound, 2,4-dichlorophenol (DCP), using vanillin as an activator. Vanillin increased the mycelial growth from 74 to 118 mg/150 ml culture and accelerated laccase and Mn-peroxidase production from the maximum on days 24-28 without vanillin to days 10-14. It eliminated 92% of 100 mM DCP with 50 mg vanillin/l compared with only 15% without vanillin. GC-MS revealed that a diaryl ether dimer of DCP was formed in the culture without vanillin, whereas dimer formation was diminished with vanillin addition. This indicates that vanillin enhances the degradation of DCP and disrupts the formation of the toxic dimer. Therefore, lignin-derived phenol such as vanillin can be used as natural and eco-friendly activators to control white-rot mushrooms, thereby facilitating the effective degradation of environmentally hazardous compounds.

Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.

AIMS/HYPOTHESIS: In rodents and humans, the rate of beta cell proliferation declines rapidly after birth; formation of the islets of Langerhans begins perinatally and continues after birth. Here, we tested the hypothesis that increasing levels of E-cadherin during islet formation mediate the decline in beta cell proliferation rate by contributing to a reduction of nuclear ß-catenin and D-cyclins. METHODS: We examined E-cadherin, nuclear ß-catenin, and D-cyclin levels, as well as cell proliferation during in vitro and in vivo formation of islet cell aggregates, using ß-TC6 cells and transgenic mice with green fluorescent protein (GFP)-labelled beta cells, respectively. We tested the role of E-cadherin using antisense-mediated reductions of E-cadherin in ß-TC6 cells, and mice segregating for a beta cell-specific E-cadherin knockout (Ecad [also known as Cdh1] ßKO). RESULTS: In vitro, pseudo-islets of ß-TC6 cells displayed increased E-cadherin but decreased nuclear ß-catenin and cyclin D2, and reduced rates of cell proliferation, compared with monolayers. Antisense knockdown of E-cadherin increased cell proliferation and levels of cyclins D1 and D2. After birth, beta cells showed increased levels of E-cadherin, but decreased levels of D-cyclin, whereas islets of Ecad ßKO mice showed increased levels of D-cyclins and nuclear ß-catenin, as well as increased beta cell proliferation. These islets were significantly larger than those of control mice and displayed reduced levels of connexin 36. These changes correlated with reduced insulin response to ambient glucose, both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: The findings support our hypothesis by indicating an important role of E-cadherin in the control of beta cell mass and function.

The challenge of acute rejection in intestinal transplantation.

Early diagnosis and treatment of acute cellular rejection (ACR) after intestinal transplantation (ITx) is challenging. We report the outcome of three patients: two presented mild ACR improved with steroids. One presented steroid-resistant severe rejection, improved after rabbit anti-thymocyte globulin (r-ATG), but unfortunately died for encephalitis caused by opportunistic infections.

Minimally invasive plate osteosynthesis for distal radius fractures with a palmar locking plate.

PURPOSE: Since 2006, we have been performing minimally invasive plate osteosynthesis with a palmar locking plate and without division of the pronator quadratus muscle for repairing distal radial fractures. The purpose of this study was to present the surgical technique we have developed and to retrospectively evaluate the clinical outcomes. METHODS: Twenty patients were treated with this technique between January and December 2007. The range of motion of the wrist and forearm, grip strength, and the quick disability of the arm, shoulder, and hand score were assessed at the latest follow-up examination, and postoperative complications were evaluated. RESULTS: The average ranges of flexion and extension of the wrist were 55° and 60°, respectively. The average ranges of supination and pronation of the forearm were 88° and 86°, respectively. The average grip strength of the treated side was 71 % of that of the uninjured side. The average quick disability of the arm, shoulder, and hand score was 13.4 points. No patient had loss of fracture reduction, implant failure, deep infection, or tendon or nerve problems. CONCLUSIONS: The small skin incisions of this technique are advantageous from the aesthetic viewpoint. Minimally invasive plate osteosynthesis is one of the options for the treatment of distal radial fractures.

Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG-IFN-alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non-SVR patients with greater accuracy.

Endoscopic treatment for early stage colorectal tumors: the comparison between EMR with small incision, simplified ESD, and ESD using the standard flush knife and the ball tipped flush knife.

BACKGROUND: Early stage colorectal tumors can be removed by endoscopic mucosal resection but larger such tumors (20 mm) may require piecemeal resection. Endoscopic submucosal dissection (ESD) using newly developed endo-knives has enabled en-block resection of lesions regardless of size and shape. However ESD for colorectal tumor is technically difficult. Therefore, we performed EMR with small incision (EMR with SI) for more reliable EMR, ESD with snaring (simplified ESD) and ESD using the standard Flush knife and the novel ball tipped Flush knife (Flush knife BT) for easier and safer colorectal ESD. AIMS: The aims of our study were (1) to compare the treatment results of the following 3 methods (EMR with SI/si-mplified ESD/ESD) for early stage colorectal tumors, and (2) to assess the performance of Flush knife BT in colorectal ESD. METHODS: We treated 24/44/468 colorectal tumors and examined the clinicopathological features and treatment results such as tumor size, resected specimen size, procedure time, en-bloc resection rate, complication rate. We also treated 58 colorectal tumors (LST-NG:20, LST-G:36, other:2) using standard Flush knife and 80 colorectal tumors (LST-NG:32, LSTG:44, other:2) using Flush knife BT, and examined the clinicopathological features and treatment results mentioned above and also the procedure speed. RESULT: The median tumor size (mm) (EMR with SI/ simplified EMR/ESD) was 20/17/30 (EMR with SI vs. simplified ESD: p = n.s, simplified ESD vs. ESD: p < 0.0001). The median resected specimen size (mm) was 22.5/26/41 (EMR with SI vs. simplified ESD: p = 0.0018, simplified ESD vs. ESD: p < 0.0001). The procedure time (min.) was 19/27/60 (EMR with SI vs. simplified ESD: p = n.s, simplified ESD vs. ESD: p < 0.0001) The en-block resection rate (%) was 83.3/90.9 /98.9. The complication rate (post-operative bleeding rate/perforation p=n.s). In the treatment results of ESD for LSTs by knives, there was no difference between standard Flush knife and Flush knife BT for clinicopathological features and treatment results (procedure time, complication rate and en bloc R0 resection rate). However, procedure speed (cm2/min.) of LST-G was significantly faster in the Flush knife BT than in standard Flush knife. (standard Flush knife: 0.21 vs. Flush knife BT: 0.27, p = 0.034). CONCLUSION: EMR with small incision (EMR with SI) and ESD with snaring (simplified ESD) are good option to fill the gap between EMR and ESD in the colorectum, and also considered to become the nice training for the introduction of ESD. Flush knife BT appears to improve procedure speed compared with standard Flush knife, especially for LST-G in colo-rectal ESD.

Role of lymph node dissection in the treatment of urothelial carcinoma of the upper urinary tract: multi-institutional relapse analysis and immunohistochemical re-evaluation of negative lymph nodes.

AIM: To determine the role of lymph node dissection (LND) in the treatment of urothelial carcinoma (UC) of the upper urinary tract (UUT). PATIENTS AND METHODS: [Study-1] A retrospective multi-institutional study evaluated 293 patients undergoing predominantly nephroureterectomy for UC of the UUT. Of 293 patients, 267 patients had pure UC and 26 demonstrated other histological components. Regarding the pathological node status, 130 patients had pN0 disease, 141 patients had pNx disease and 22 patients had pN+ disease. The sites of initial recurrence and time to first recurrence were reviewed. The sites of recurrence were classified as locoregional or distant recurrence. The relationship between node status and future recurrence was analyzed. [Study-2] Fifty-one patients treated by nephroureterectomy at Hokkaido University Hospital were included. All had LND and all LNs were negative on hematoxylin and eosin staining. We re-evaluated the presence of micrometastasis in LND specimens by anti-cytokeratin immunohistochemistory. RESULTS: [Study-1] Of 293 patients, 76 developed disease relapse. Regional lymph node recurrence was the most common site (34 patients). On multivariate analyses that adjusted for the effect of tumor stage and tumor grade, pNx (skipping LND) was an adverse factor not only for locoregional recurrence, but also for distant relapse. [Study-2] Immunohistochemistry identified micrometastases in 7 (14%) of 51 patients. Regarding survival, 5 of these 7 patients with micrometastases were alive at last follow-up. CONCLUSIONS: On relapse analysis, skipping LND was an adverse factor not only for locoregional recurrence, but also for distant relapse. Immunohistochemistry detected micrometastases in about 14% of patients previously diagnosed as pN0. These findings further support a potential therapeutic benefit of LND by eliminating micrometastases.

Loss of Hus1 sensitizes cells to etoposide-induced apoptosis by regulating BH3-only proteins.

The Rad9-Rad1-Hus1 (9-1-1) cell cycle checkpoint complex plays a key role in the DNA damage response. Cells with a defective 9-1-1 complex have been shown to be sensitive to apoptosis induced by certain types of genotoxic stress. However, the mechanism linking the loss of a functional 9-1-1 complex to the cell death machinery has yet to be determined. Here, we report that etoposide treatment dramatically upregulates the BH3-only proteins, Bim and Puma, in Hus1-deficient cells. Inhibition of either Bim or Puma expression in Hus1-knockout cells confers significant resistance to etoposide-induced apoptosis, whereas knockdown of both proteins results in further resistance, suggesting that Bim and Puma cooperate in sensitizing Hus1-deficient cells to etoposide treatment. Moreover, we found that Rad9 collaborates with Bim and Puma to sensitize Hus1-deficient cells to etoposide-induced apoptosis. In response to DNA damage, Rad9 localizes to chromatin in Hus1-wild-type cells, whereas in Hus1-deficient cells, it is predominantly located in the cytoplasm where it binds to Bcl-2. Taken together, these results suggest that loss of Hus1 sensitizes cells to etoposide-induced apoptosis not only by inducing Bim and Puma expressions but also by releasing Rad9 into the cytosol to augment mitochondrial apoptosis.

Detection of Kobe-type Babesia microti associated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China.

Field rodent surveys for Babesia infection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China. Babesia microti was identified by microscopical examination and/or PCR in 1 Rattus coxinga and 1 Crocidura horsfieldii in central Taiwan and in 13 Niviventer confucianus and 1 Apodemus agrarius in Zhejiang and Fujian Provinces of southeastern China. Of 15 B. microti samples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.

Genetic mapping of genes for susceptibility to black spot disease in Japanese pears.

Black spot disease, which is caused by the Japanese pear pathotype of Alternaria alternata (Fr.) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We identified the exact positions and linkage groups (LGs) of the genes for susceptibility to black spot in the Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Osa Nijisseiki' (gene Ani) and 'Nansui' (gene Ana). Segregation of susceptibility and resistance fitted the expected ratio of 1:1 in progeny of 'Nansui' but showed a slight distortion in progeny of 'Osa Nijisseiki'. We mapped the genes for susceptibility to black spot in both populations using a genome scanning approach. The simple sequence repeat (SSR) markers CH04h02 and CH03d02 showed tight linkage to Ani and Ana. Although Ani and Ana are derived from different sources, both genes are located at the top region of LG 11. Information about the positions of the susceptibility genes and the molecular markers linked to them will be useful for marker-assisted selection in pear breeding programs.

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells.

We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.

The effect of a valine-rich diet on intestinal adaptation to massive small bowel resection in the rat.

BACKGROUND/PURPOSE: Recently, valine, which is one of the branched chain amino acids, has been reported to enhance liver regeneration after hepatectomy in the rat. The aim of this study was to investigate the effect of enteral valine supplementation on intestinal adaptation. MATERIALS/METHODS: Seven-week-old male Lewis rats underwent a 90% small bowel resection. The rats were randomly divided into two groups: group V (valine-rich diet) and group S (standard rat chow), according to the diet. The rats were sacrificed at the operation day and on postoperative days (POD) 7, 14, 30, and 60. The metrics were body weight (BW), blood amino acids, urine organic acids, and morphology of the residual small intestine. RESULTS: The BW and the intestinal wet weight, jejunal crypt depth, and proliferating cell nuclear antigen-positive cells in group V at POD 7 were significantly higher than those values in group S, while those in group V at POD 30 and 60 were smaller than those in group S. The urine methylmalonic acid (MMA) level in group V at POD 30 and 60 was much higher than in group S. CONCLUSION: Valine enhanced intestinal adaptation after massive small bowel resection in the acute phase. However, the long-term supplementation disturbed intestinal adaptation, which might be due to the high production of MMA.

Relationship between real-time monitoring of the graft motility and mucosal histology in swine intestinal transplantation.

We studied the correlation between the motility and the mucosal histology of the small bowel seeking to detect rejection in an early stage by real-time monitoring using a swine model. Intestinal transplantation (ITx) was performed orthotopically using FK506 immunosuppression. The distal about 20 cm segment of the allograft was exteriorized as a Thiry-Vella stoma for biopsies. Strain gauge (SG) force transducers were attached to the graft for real-time monitoring of graft motility. Pigs without ITx were used as controls (group 1). Rejection was classified into four groups by histologic findings: nonrejection (group 2), mild rejection (group 3), moderate rejection (group 4), and severe rejection (group 5). Migrating motor complex (MMC) phase III was analyzed for the following parameters: duration, amplitude, interval, motility index, velocity, and frequency of propagation. In group 2, all parameters were almost the same as those for group 1. In contrast, groups 4 and 5 showed most parameters significantly lower than those in group 1. In group 3, the contractility of the MMC was not significantly altered, but the frequency of the propagation was decreased significantly. In conclusion, graft motility detected by a real-time SG method correlated with the grade of mucosal histology. This method is useful to detect rejection at an early stage by examining the frequency of MMC propagation.