Development and evaluation of a point-of-care test with a combination of EZ-Fast DNA extraction and real-time PCR and LAMP detection: evaluation using blood samples containing the bovine leukaemia DNA.

Along with progress in globalization of society, the spread of infectious diseases has accelerated worldwide. The deployment of highly sensitive genetic tests is essential for early diagnosis and early containment of potential outbreaks and epidemics, as well as routine surveillance, although tedious and expensive nucleic acid extraction steps represent a major drawback. Here we developed a simple and rapid DNA extraction method, named as an EZ-Fast kit, applicable to the field setting. The kit does not require advanced laboratory equipment or expensive DNA extraction kits and achieves crude DNA extraction within 10 min at extremely low cost and can easily be performed in field settings. When combined with real-time PCR and LAMP analyses, the performance of the POCT, using 183 bovine blood samples, was similar to that of the existing DNA extraction method: 92·5% (135/146) (real-time PCR) and 93·7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic specificities. The developed POCT provides a powerful tool to facilitate on-site diagnosis in a field setting.

Combination of low-dose total skin electron beam therapy and subsequent localized skin electron beam therapy as a therapeutic option for advanced-stage mycosis fungoides.

Electron beam therapy (EBT) is an established treatment for mycosis fungoides (MF), but evidence for the use of EBT in advanced cutaneous conditions is limited, and optimal scheduling of the regimen for such conditions remains unclear. We report the case of a 44-year-old woman diagnosed with MF with widespread cutaneous lesions, including multiple huge tumours in the craniofacial area. Low-dose total skin (TS)EBT and subsequent localized skin (LS)EBT achieved striking improvements in eruptions. Oral etretinate was also administered during therapy. Our experience implies that combined TSEBT and LSEBT may be worth attempting when a patient presents with both widespread lesions and prominent tumours, even when the tumours are extremely large.

Outcomes and Radiographic Findings of Isolated Spontaneous Superior Mesenteric Artery Dissection.

OBJECTIVES: This study aimed to investigate the features, treatments, and prognosis of patients with symptomatic and asymptomatic isolated SMA dissection. METHODS: Data from 35 consecutive patients in whom isolated SMA dissection was diagnosed on computed tomography angiography (CTA) between 2004 and 2015 at two general hospitals in Japan, were collected retrospectively. Nineteen symptomatic patients were compared, and 16 asymptomatic patients with incidentally revealed SMA dissection were also compared. In addition, the vascular remodelling and outcomes during follow-up were evaluated. RESULTS: The patient characteristics in the symptomatic and incidental groups were comparable except for age; mean ages were 55.9 ± 13.9 and 65.3 ± 10.9 years, respectively. Most of the symptomatic patients were managed conservatively (including antiplatelet therapy, anticoagulants, blood pressure control, or bowel rest). In addition, one patient was initially treated by endovascular intervention because of intestinal ischaemia, and another was switched from conservative to surgical treatment. The in-hospital outcome was good with no mortality. In the incidental group, all 16 patients were observed as outpatients without additional treatment. Complete remodelling of the false lumen was observed in 31% of patients with follow-up CTA, and was associated with the presence of symptoms and the absence of false lumen with blood flow at diagnosis. Neither recurrent or new onset abdominal pain, intervention for SMA dissection, nor SMA related death was observed in either group during the follow-up period (750 ± 779 and 1200 ± 951 days). CONCLUSIONS: The characteristics of asymptomatic patients with incidentally revealed SMA dissection were comparable with those of symptomatic patients, except for age. During follow-up, factors favouring complete remodelling of false lumens were the presence of symptoms, and the absence of false lumen blood flow at diagnosis.

Age is a significant predictor of early and late improvement in semen parameters after microsurgical varicocele repair.

Accumulating evidence indicates that varicocele repair improves sperm quality. However, longitudinal changes in sperm parameters and predictors of improved semen characteristics after surgery have not been fully investigated. We retrospectively reviewed data from 100 men who underwent microsurgical subinguinal varicocele repair at a single centre. Follow-up semen examinations were carried out at 3, 6 and 12 months post-operatively. Logistic regression was used to identify predictors of early (3 months) and late (≥6 months) improvement in semen parameters after varicocele repair. At 3 months post-operatively, 76.1% of the patients had improved total motile sperm counts, which continued to improve significantly up to 12 months post-operatively (p = .016). When comparing changes in semen parameters between younger (<37 years) and older (≥37 years) men, post-operative improvements in sperm concentration and motility were greater among younger men. Multivariate analysis showed that younger age was associated with early (p = .043) and late (p = .010) post-operative improvement in total motile sperm count. Our findings indicate that early varicocele repair improved semen parameters after surgery.

h-BN nanosheets as simple and effective additives to largely enhance the activity of Au/TiO2 plasmonic photocatalysts.

The activity of Au nanoparticle-loaded P25 TiO2 (Au/P25) plasmonic photocatalysts, evaluated by the oxidative decomposition of formic acid in water under visible light irradiation, was enhanced up to 3 times by simply mixing Au/P25 with photocatalytically inactive h-BN nanosheets as a result of electron transfer from photoexcited Au/TiO2 to the h-BN nanosheets and retardation of the charge recombination.

An innovative masticatory efficiency test using odour intensity in the mouth as a target marker: a feasibility study.

A large number of studies have reported the findings for masticatory efficiency tests; however, some objective masticatory efficiency tests have a drawback, in that subjects are required to spit out the test material. This study examined the possibility of a masticatory efficiency test that evaluates the intensity of odours released when chewing an odour compound-containing material. A total of 20 volunteers were used in this study. The odour intensity in the breath after chewing a gum was measured by portable odour sensor device. The odour intensity after chewing the gum was measured over four chewing durations and at four intervals between spitting out and measurement of the odour intensity. The volume of stimulated saliva was measured by calculating the difference in the weight of the gauze before and after chewing to examine the effect of saliva flow. With an increase in chewing duration, odour intensity reduced. The odour intensity was the highest immediately after chewing. There was a positive correlation between odour intensity and gummy jelly-related masticatory efficiency test value (G-METV), which was significant for 10-s chewing. The regression equation was calculated from three objective variables of odour intensity and G-METV as dependent variable. Pearson's correlation coefficient between G-METV and the odour intensity-related masticatory efficiency value (O-METV) was 0·68. The coefficient of variation of O-METV was significantly lower than that of G-METV. These results suggest that the masticatory performance can be estimated by measuring the odour intensity immediately after chewing food containing odour compounds for 10 s.

Derivation of integration-free iPSCs from a Klinefelter syndrome patient.

PURPOSE: Klinefelter syndrome (KS) (47, XXY) is the most common sex chromosome abnormality in humans. KS is characterized by gynecomastia, tall stature, small testes, low testosterone levels, learning disabilities, and behavioral problems. KS is also associated with infertility due to non-obstructive azoospermia (NOA). The mechanism underlying NOA is still poorly understood, and although there is no current treatment, the use of microdissection testicular sperm extraction (micro-TESE) followed by in vitro fertilization can result in successful conception. The generation of induced pluripotent stem (iPS) cells derived from KS patients may be useful for studying the disease mechanism and identifying novel therapies. METHODS: Cells from a KS patient were transduced with Sendai viral vectors encoding four transcription factors, OCT4, SOX2, KLF4, and C-MYC, and the transduced cells were analyzed for in vitro and in vivo pluripotency. RESULTS: KS patient-derived iPS cells were successfully generated and shown to produce teratomas in the testes of SCID mice. In vitro differentiation of the iPS cells into cardiomyocyte-like cells was confirmed by the presence of clusters of beating cells. CONCLUSIONS: KS patient-derived iPS cells that could differentiate into cardiomyocyte-like cells were established.

Clinical significance of ERG rearrangement subtype and its association with increased p53 expression in Japanese and German prostate cancer.

UNLABELLED: This study investigated differences in prevalence of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) and ETS transcription factor family member, v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion gene (TMPRSS2-ERG fusions) in clinically localized prostate cancer Japanese and German patients. A total of 105 specimens, including 69 Japanese and 36 German patients, were collected. The status of TMPRSS2-ERG fusion was determined by fluorescence in situ hybridization, and correlations of the TMPRSS2-ERG fusion with clinicopathological characteristics and immunohistochemistry were studied. Gene fusions were identified in 20% (14/69) of Japanese and 53% (19/36) of German patients (P < 0.001). The difference in the type of gene fusion between the two ethnic groups was statistically significant (P=0.024). Overexpression of ERG protein was significantly associated with gene fusion. Biochemical recurrence was significantly higher in patients with ERG overexpression than in those without, and not related to TMPRSS2-ERG fusion status. Interestingly, two types of gene fusions (deletion and increase of copy number) were significantly associated with increased p53 expression (P = 0.005). Association of specific gene fusions harboring higher genomic alterations with p53 expression levels suggests that p53 mutation might drive more aggressive arrangements of TMPRSS2-ERG fusion in prostate cancer. KEYWORDS: ERG, p53, prostate cancer, TMPRSS2-ERG fusion.

Rural Victorian Telestroke project.

BACKGROUND: Intravenous thrombolysis improves functional outcomes in acute ischaemic stroke. However, many rural stroke patients are denied thrombolysis because of a rural neurologist shortage. 'Telestroke' facilitates thrombolysis by providing remote access to neurologists via videoconferencing systems. AIMS: To develop a safe and feasible Telestroke system in a rural Victorian hospital that facilitates delivery of intravenous thrombolysis to acute ischaemic stroke patients. METHODS: A pilot videoconferencing Telestroke system was set up between Royal Melbourne Hospital and Northeast Health Wangaratta. Acute stroke patients presenting within 4.5 h of symptom onset without intracranial haemorrhage were eligible for Telestroke. However, eligible patients were excluded from Telestroke if they had haemorrhagic risk factors. Data were collected from intervention (October 2009-September 2010) and control group (October 2008-September 2009) by medical file audit. Primary outcome measure was percentage of patients thrombolysed. Secondary outcome measures included incidence of symptomatic intracerebral haemorrhage and door-to-computed tomography time. RESULTS: One hundred and forty-five acute stroke patients presented in control year and 130 patients in intervention year. Fifty-four patients in intervention and 36 patients in control group were eligible for thrombolysis. In intervention group, 24 patients had Telestroke activated and 8 patients underwent thrombolysis. There was no thrombolysis in the control group. There were neither symptomatic intracerebral haemorrhages nor deaths attributable to thrombolysis. Median door-to-computed tomography time did not significantly differ between eligible patients in control and intervention groups. CONCLUSION: Telestroke has the potential to bridge the gap of rural-metropolitan inequality in acute stroke care. Our Telestroke system successfully introduced safe thrombolysis and early specialist review of acute stroke patients in rural Victoria.

Main occluding area in partially edentulous patients: changes before and after implant treatment.

The 'main occluding area', the location where food crushing occurs during the first stroke of mastication, is reported to be an important concept; however, it is currently limited to findings in individuals with normal dentition. The purpose of this study was to assess the changes in the location, area and bite force of the main occluding area before and after implant treatments. We enrolled 50 partially edentulous and 22 normally dentate subjects. To identify the location of the main occluding area, each subject was instructed to freely bite once on a dental stopping using the partially edentulous side or the normally dentate area. The location, occluding contact area and bite force of the main occluding area before and after the implant treatments were analysed. The main occluding area was located at a reproducible location in the partially edentulous and normally dentate subjects. This location was principally the first molar region, and for the partially edentulous patients with missing teeth in the molar regions, it moved from the premolar region to the first molar region after treatment. The occluding contact area and bite force for the main occluding area increased (P < 0·05) after the implant treatment in the partially edentulous patients with missing teeth in the molar regions. These results suggest that the main occluding area can be restored to the first molar region after implant treatment and may be an important factor in the assessment of prosthodontic treatment.

Transgenic mice overproducing human thioredoxin-1, an antioxidative and anti-apoptotic protein, prevents diabetic embryopathy.

AIMS/HYPOTHESIS: Experimental studies have suggested that apoptosis is involved in diabetic embryopathy through oxidative stress. However, the precise mechanism of diabetic embryopathy is not yet clear. Thioredoxin (TRX) is a small, ubiquitous, multifunctional protein, which has recently been shown to protect cells from oxidative stress and apoptosis. Using transgenic mice that overproduce human TRX-1 (TRX-Tg mice), we examined whether oxidative stress is involved in fetal dysmorphogenesis in diabetic pregnancies. METHODS: Non-diabetic and streptozotocin-induced diabetic (DM) female mice were mated with male TRX-Tg mice. Pregnant mice were killed either at day 10 or day 17 of gestation, and viable fetuses and their placentas were recovered, weighed and assessed for gross and histological morphology, biochemical markers and gene expression. RESULTS: In both wild-type (WT) and transgenic (Tg) groups, fetal and placental weights in the diabetic group were significantly decreased compared with the non-diabetic group. The incidence of malformation was higher in the diabetic group, and was significantly decreased in the TRX-Tg group (DM-WT vs DM-Tg; 28.6% vs 10.4%). Oxidative stress markers such as thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine were increased in DM-WT group fetuses but were decreased in fetuses from the DM-Tg group. Furthermore, immunohistochemically assayed apoptosis and cleaved caspase-3 production in embryonic neuroepithelial cells was significantly increased in the DM-WT group, and was significantly decreased in the DM-Tg group. CONCLUSIONS/INTERPRETATION: These results indicate that oxidative stress is involved in diabetic embryopathy, and that the antioxidative protein TRX at least partially prevents diabetic embryopathy via suppression of apoptosis.

Subjective Evaluation of Denture Adhesives: A Multicenter Randomized Controlled Trial.

INTRODUCTION: Many reports show that denture adhesives improve the retention and stability of dentures. However, few randomized controlled trials have examined the effects of denture adhesives. OBJECTIVE: This 10-center randomized controlled trial with parallel groups involving 200 edentulous patients wearing complete dentures aimed to evaluate the effects of short-term use of cream and powder denture adhesives. METHODS: Patients were allocated into 2 cream- and powder-type adhesive groups and 1 control group. Intervention groups were treated with the 2 adhesives (1 each), and the control group received saline solution. Adhesive or control was applied to the denture-mucosal surface for 4 d, and data at baseline and after day 4 of intervention (i.e., 8 meals) were obtained. Patient satisfaction was evaluated with a 100-mm visual analog scale. Oral health-related quality of life was measured with the Japanese version of the Oral Health Impact Profile for Edentulous Patients. Perceived chewing ability was evaluated by a questionnaire regarding ease of chewing and swallowing food. Between-group comparisons were performed with Kruskal-Wallis tests with the Mann-Whitney U test adjusted by Bonferroni correction. Within-group comparisons of pre- and postintervention measurements were performed with the Wilcoxon signed-rank test. Intention-to-treat analysis was also performed. RESULTS: Between-group comparisons showed no significant differences for general satisfaction or Oral Health Impact Profile for Edentulous Patients. However, significant differences in satisfaction with various denture functions with cream- and powder-type adhesives were seen in pre- and postintervention comparisons (P < 0.05). Significant differences were also observed for perceived chewing ability of hard foods (P < 0.05). CONCLUSION: These results suggest that although denture adhesives do not invariably improve denture function, they do affect subjective evaluations and possibly chewing of hard foods. Therefore, the effects of denture adhesive use are insufficient to resolve any fundamental dissatisfaction with dentures ( ClinicalTrials.gov NCT01712802 ). KNOWLEDGE TRANSFER STATEMENT: The results of this study suggest that denture adhesives should be applied under certain conditions; however, an appropriate diagnosis is important before application. These practice-based data provide information to establish evidence-based guidelines for applying denture adhesives.

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

Continuous lines of basophil/mast cells derived from normal mouse bone marrow.

Nonadherent tissue culture cell lines were established from normal bone marrow of a variety of mouse strains. The lines possessed morphological and histochemical markers of the basophil/mast cell and contained committed stem cells for metachromatic cells. Their derivation from normal marrow and their lack of tumorigenicity despite long-term culture makes these cell lines potentially important for studies of the mechanisms of allergic reactions and inflammation as well as the differentiation pathways involving this subset of hematopoietic cells.

"Helium spots": caused by a diapiric magma from the upper mantle.

"Helium spots," where a significant amount of helium is present in the soil [up to 350 parts per million with a high (3)He to (4)He ratio of (8.90 +/- 0.31) x 10(-6)], have been found along the fault zone formed by the 1966 Matsushiro swarm earthquakes. The formation of the "helium spots" and the occurrence of the earthquakes are interpreted as the results of a diapiric uprise of a magma approximately 1 kilometer in diameter.

Chemical methylation of inorganic mercury with methylcobalamin, a vitamin B12 analog.

Chemical methylation of mercuric chloride with methylcobalamin has been studied. Methylated mercury was detected by gas chromatography; and analysis of the products of the reaction by thin-layer chromatography revealed that the methylation proceeded at a remarkably high rate when methylcobalamin and inorganic mercury were mixed. Dimethylmercury was an initial product of the reaction.

Interobserver agreement in endoscopic evaluation of reflux esophagitis using a modified Los Angeles classification incorporating grades N and M: a validation study in a cohort of Japanese endoscopists.

The Los Angeles classification system is the most widely employed criteria associated with the greatest interobserver agreement among endoscopists. In Japan, the Los Angeles classification system has been modified (modified LA system) to include minimal changes as a distinct grade of reflux esophagitis, rather than as auxiliary findings. This adds a further grading M defined as minimal changes to the mucosa, such as erythema and/or whitish turbidity. The modified LA system has come to be used widely in Japan. However, there have been few reports to date that have evaluated the interobserver agreement in diagnosis when using the modified LA classification system incorporating these minimal changes as an additional grade. A total of 100 endoscopists from university hospitals and community hospitals, as well as private practices in the Osaka-Kobe area participated in the study. A total of 30 video clips of 30-40 seconds duration, mostly showing the esophagocardiac junction, were created and shown to 100 endoscopists using a video projector. The participating endoscopists completed a questionnaire regarding their clinical experience and rated the reflux esophagitis as shown in the video clips using the modified LA classification system. Agreement was assessed employing kappa (kappa) statistics for multiple raters. The kappa-value for all 91 endoscopists was 0.094, with a standard error of 0.002, indicating poor interobserver agreement. The endoscopists showed the best agreement on diagnosing grade A esophagitis (0.167), and the poorest agreement when diagnosing grade M esophagitis (0.033). The kappa-values for the diagnoses of grades N, M, and A esophagitis on identical video pairs were 0.275-0.315, with a standard error of 0.083-0.091, indicating fair intraobserver reproducibility among the endoscopists. The study results consistently indicate poor agreement regarding diagnoses as well as fair reproducibility of these diagnoses by endoscopists using the modified LA classification system, regardless of age, type of practice, past endoscopic experience, or current workload. However, grade M reflux esophagitis may not necessarily be irrelevant, as it may suggest an early form of reflux disease or an entirely new form of reflux esophagitis. Further research is required to elucidate the pathophysiological basis of minimal change esophagitis.

The enhancement effect of three sugar alcohols on the fungicidal effect of benzethonium chloride toward Candida albicans.

OBJECTIVE: The purpose of this study was to evaluate if the sugar alcohols erythritol, xylitol and sorbitol enhance the fungicidal effect of benzethonium chloride (BTC) toward in vitro candidal biofilms. METHODS: An in vitroCandida albicans biofilm was formed on a plastic coverslip coated with type I collagen. The enhancement of the fungicidal effect was evaluated using microbial assay after treating the biofilm with the test solutions (sugar alcohols: erythritol, xylitol, and sorbitol, each containing BTC solution). RESULTS: No fungicidal effect was observed with sugar alcohols without BTC. The fungicidal effect of erythritol-containing BTC increased with the erythritol concentration. Further, the level of enhancement of erythritol was the highest in three sugar alcohols. CONCLUSION: Sugar alcohols, especially erythritol, enhanced the fungicidal effect of BTC toward in vitro candidal biofilms.

Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis.

BACKGROUND: The Cut1 and Cut2 proteins of the fission yeast Schizosaccharomyces pombe form a complex and are required for the separation of sister chromatids during anaphase. Polyubiquitinated Cut2 degrades at the onset of anaphase and this degradation, like that of mitotic cyclin, is dependent on the anaphase-promoting complex/cyclosome. Expression of Cut2 that cannot be degraded blocks sister chromatid separation and anaphase spindle elongation. Here, we have investigated the role of the Cut1-Cut2 interaction in sister chromatid separation. RESULTS: The carboxyl terminus of Cut2 interacts with the amino terminus of Cut1, and temperature-sensitive Cut2 mutants expressed Cut2 proteins that contain substitutions in the carboxyl terminus and fail to interact with Cut1, resulting in aberrant anaphase. Localization of Cut1 alters dramatically during the cell cycle. Cut1 is retained in the cytoplasm during interphase and moves to the mitotic spindle pole bodies and the spindle upon entry into prophase, when spindles are formed. The association between Cut2 and Cut1 is needed for the localization of Cut1 to the spindles, as Cut1 remains unbound to the spindle if complex formation is impaired. Cut2 degrades during anaphase, but Cut1 remains bound to the anaphase spindle. This association with the anaphase spindle requires the conserved carboxyl terminus of Cut1. CONCLUSIONS: Complex formation between Cut1 and Cut2 is needed for the onset of normal anaphase. Cut2 is required for loading Cut1 onto the spindle at prophase and Cut2 proteolysis is needed for the active participation of Cut1 in sister chromatid separation.

The association of DNA repair gene polymorphisms with the development and progression of renal cell carcinoma.

BACKGROUND: DNA repair enzymes repair some of the DNA damage associated with risk factors for renal cell carcinoma (RCC), including smoking. DNA repair gene polymorphisms modulate the repair capacity and might influence individual risk and progression of RCC. We examined associations between functional polymorphisms and risk, clinicopathologic characteristics and survival of RCC. PATIENTS AND METHODS: The study groups comprised 215 RCC patients and 215 age- and gender-matched healthy controls. Polymorphisms in xeroderma pigmentosum complementation groups C, D and G and X-ray repair cross-complementing groups 1 and 3 genes were genotyped. RESULTS: No significant differences in DNA repair genotype were observed between RCC cases and controls. In all patients, however, greater numbers (> or =3) of total variant alleles in all DNA repair genes studied were associated with less frequent venous extension (P = 0.0079). In smokers, some genotypes were associated with characteristics of RCC (Ps < or = 0.0067) and smokers with greater numbers of total variant alleles had improved overall survival (P = 0.040). CONCLUSION: These results suggest that DNA repair gene polymorphisms may not influence RCC susceptibility, but that some of them may influence RCC progression, especially in smokers, possibly due to altered DNA repair capacity by these polymorphisms.