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Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.

Administration of cetuximab (C-mab) in combination with paclitaxel (PTX) has been used for patients with head and neck squamous cell carcinoma (SCC) clinically. In this study, we attempted to clarify the molecular mechanisms of the enhancing anticancer effect of C-mab combined with PTX on oral SCC cells in vitro. We used two oral SCC cells (HSC4, OSC19) and A431 cells. PTX alone inhibited cell growth in all cells in a concentration-dependent manner. C-mab alone inhibited the growth of A431 and OSC19 cells at low concentrations, but inhibited the growth of HSC4 cells very weakly, even at high concentrations. A combined effect of the two drugs was moderate on A431 cells, but slight on HSC4 and OSC19 cells. A low concentration of PTX enhanced the antibody-dependent cellular cytotoxicity (ADCC) induced by C-mab in all of the cells tested. PTX slightly enhanced the anticancer effect of C-mab in this ADCC model on A431 and HSC4 cells, and markedly enhanced the anticancer effect of C-mab on OSC19 cells. These results indicated that PTX potentiated the anticancer effect of C-mab through enhancing the ADCC in oral SCC cells.

Aim: We attempted to clarify the role of Peroxisome proliferator-activated receptor γ (PPARγ) and its ligand, troglitazone (TRO) on oral squamous cell carcinoma (SCC)., Materials and Methods: The expression of PPARγ gene was examined in 47 human oral SCC tissues and two human oral SCC cell lines, CA9-22 and HSC-4. The effects of TRO on the growth and cell-cycle progression of human oral SCC cells were examined., Results: PPARγ mRNA was detected in 20 of 47 oral SCC tissues and two human oral SCC cells. TRO significantly suppressed the growth of the cells, but did not induce apoptosis. CA9-22 cells treated with TRO showed an increased fraction in the G 1 phase and decreased fractions in the S and G 2 -M phases., Conclusion: TRO did not induce apoptosis in oral SCC cells, but did inhibit the growth of the cells by arresting the cell cycle at G 1 phase., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Escherichia coli is well known for its ability to undergo artificial transformation. This bacterium is not generally recognized as naturally transformable. However, several past studies have demonstrated that laboratory and natural strains of E. coli can express modest natural competence under feasible environmental conditions. Here we describe a protocol outlining natural transformation in E. coli in environmental media such as natural freshwater.

The development of carbon dioxide fixation under mild conditions is a central theme in organic synthesis. Despite the tremendous progress in the field of organocatalysis in the past two decades, the coupling reactions of epoxides with carbon dioxide that proceed at atmospheric pressure at temperatures of less than 100 °C have remained challenging. In our aspirational studies of tetraarylphosphonium salts (TAPS) catalysis, we report here the bifunctional TAPS-catalyzed synthesis of five-membered cyclic carbonates by chemical fixation using 1 atm of carbon dioxide at 60 °C. Intriguing substituent effects of TAPS were observed, in which electron-donating groups enhanced their reactivity. In addition, the mechanism was thoroughly investigated by undertaking both experimental and theoretical studies, suggesting that the electronic properties of TAPS affect carbon dioxide insertion into halohydrin intermediates. The results provided fruitful information to understand the origin of the TAPS behavior, which would contribute to the design of novel catalysts for carbon dioxide capture.

Background: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated., Methods: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA., Results: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001)., Conclusions: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.

Background: To examine the incidence of cirrhosis patients with high-risk esophageal varices (EV) who show hepatic venous pressure gradient (HVPG) < 10 mmHg and to identify their hemodynamic features. Methods: This prospective study consisted of 110 cirrhosis patients with EV, all with the candidate for primary or secondary prophylaxis. Sixty-one patients had red sign, and 49 patients were bleeders. All patients underwent both Doppler ultrasound and HVPG measurement. Results: There were 18 patients (16.4%) with HVPG < 10 mmHg. The presence of venous-venous communication (VVC) was more frequent in patients with HVPG < 10 mmHg (10/18) than in those with HVPG ≥ 10 mmHg (19/92; p = 0.0021). The flow volume in the left gastric vein (LGV) and the incidence of red sign were higher in the former (251.9 ± 150.6 mL/min; 16/18) than in the latter (181 ± 100.5 mL/min, p = 0.02; 45/92; p = 0.0018). The patients with red sign had lower HVPG (13.3 ± 4.5) but advanced LGV hemodynamics (velocity 13.2 ± 3.8 cm/s; flow volume 217.5 ± 126.6 mL/min), whereas those without red sign had higher HVPG (16.2 ± 4.6, p = 0.001) but poorer LGV hemodynamics (10.9 ± 2.3, p = 0.002; 160.1 ± 83.1, p = 0.02). Conclusion: Patients with high-risk EV with HVPG < 10 mmHg showed 16.4% incidence. Although low HVPG may be underestimated by the presence of VVC, the increased LGV hemodynamics compensates for the severity of portal hypertension, which may contribute to the development of red sign., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

: Background : Recent advances in the development of tyrosine kinase inhibitors (TKIs) have enabled patients with unresectable hepatocellular carcinoma (HCC) to receive multiple TKIs in sequence. The aim of this study was to identify predictors of good candidates for second-line treatment after disease progression during sorafenib treatment. Methods : This is a retrospective cohort study of 190 consecutive HCC patients who were treated with sorafenib in our hospital. Three criteria of good candidates for second-line TKI at the time of disease progression during sorafenib treatment were defined as follows: criterion 1 was the same as the inclusion criteria of the regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE) study, criterion 2 was the inclusion criteria of the RESORCE study plus Child-Pugh score 5, and criterion 3 was the inclusion criteria of the RESORCE study plus albumin-bilirubin (ALBI) grade 1. Factors at baseline and at week 4 during sorafenib treatment were used to predict patients fulfilling each of these three criteria. Results : The distribution of patients was 29%, 13%, and 6% in criteria 1, 2, and 3, respectively. Significant factors for meeting criterion 1 was the combination of baseline albumin >3.7 g/dL (odds ratio (OR) 2.7) plus degree of decrease in albumin (Δalbumin) at week 4 <0.2 g/dL (OR 2.6), or the combination of baseline ALBI score <-2.33 (OR 2.5) and ΔALBI at week 4 <0.255 (OR 4.9). For criterion 2, the value of baseline albumin and ALBI score was identical to criterion 1; however, Δalbumin (<0.1 g/dL) and ΔALBI score (<0.19) became stricter. For criterion 3, the value of baseline albumin (>3.8 g/dL) and ALBI (<-2.55) became stricter, as did Δalbumin (<0.1 g/dL) and ΔALBI (<0.085). Furthermore, tumor burden (>11) was selected as an additional predictor (OR 5.4). Conclusion : Predictors to satisfy the RESORCE study inclusion criteria were as follows: preserved liver function at baseline, as reflected by albumin or ALBI score, and small deterioration of liver function early during sorafenib therapy, as reflected by Δalbumin or ΔALBI at week 4. Liver function at baseline and degree of change in liver function during sorafenib treatment need to be stricter for better outcomes of liver function with disease progression., Competing Interests: The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background and Aims: The present study aimed to report our real-life experience of the TPO receptor agonist lusutrombopag for cirrhotic patients with low platelet counts., Methods: We studied platelet counts in 1,760 cirrhotic patients undergoing invasive procedures at our hospital between January 2014 and December 2017. In addition, we studied 25 patients who were administered lusutrombopag before invasive procedures between June 2017 and January 2018. Effectiveness of lusutrombopag to raise platelet counts and to avoid transfusion and treatment-related adverse events were analyzed., Results: In 1,760 cirrhotic patients without lusutrombopag prior to invasive procedures, proportion of patients whose platelet counts <50,000/μL and needed platelet transfusions were 66% (n = 27/41) for radiofrequency ablation, 43% (n = 21/49) for transarterial chemoembolization, and 55% (n = 21/38) for endoscopic injection sclerotherapy / endoscopic variceal ligation, respectively. In 25 cirrhotic patients treated by lusutrombopag prior to the invasive procedures, platelet counts significantly increased compared with baseline (82,000 ± 26,000 vs. 41,000 ± 11,000/μL) (p < 0.01). Out of 25 patients, only 4 patients (16%) needed platelet transfusion before the invasive procedures. The proportion of patients with low platelet count and who needed platelet transfusions was significantly low in patients treated with lusutrombopag compared to those not treated with lusutrombopag (16% (4/25) vs. 54% (69/128), p = 0.001). Platelet counts after lusutrombopag treatment and prior to invasive procedures were lower in patients with a baseline platelet count ≤30,000/μL (n = 8) compared with those with a baseline platelet count >30,000/μL (n = 17) (50,000 ± 20,000 vs 86,000 ± 26,000/μL, p = 0.002). Patients with a baseline platelet count ≤30,000/μL with spleen index (calculated by multiplying the transverse diameter by the vertical diameter measured by ultrasonography) ≥40 cm2 (n = 3) had a lower response rate to lusutrombopag compared to those with spleen index <40 cm2 (n = 5) (0% vs. 100%, p = 0.02). Hemorrhagic complication was not observed. Recurrence of portal thrombosis was observed and thrombolysis therapy was required in one patient who had prior history of thrombosis., Conclusions: Lusutrombopag is an effective and safe drug for thrombocytopenia in cirrhotic patients, and can reduce the frequency of platelet transfusions., Competing Interests: This study was supported by a grant from the Japan Agency of Medical Research and Development (grant number: 15fk0210007h0002, URL: http://www.amed.go.jp/en/). Additionally, NI received lecture fees from SHIONOGI & CO., LTD. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development, or marketed products to declare.

4-Hydroxyderricin (4-HD) is a major polyphenol of Angelica keiskei (Japanese name Ashitaba), exhibiting anti-allergic, anti-diabetic, anti-oxidant, and antitumor effects. The present study was designed to evaluate the effects of 4-HD on bone formation and maintenance by using cultured osteoclasts and osteoblasts. 4-HD did not affect cell proliferation of stromal ST2 cells and preosteoblast MC3T3-E1 cells at concentrations of 1-10 μM. This compound inhibited the formation of multinucleated osteoclasts from mouse splenic cells, and we identified a molecular pathway of osteoclast differentiation mediated by 4-HD, which led to inhibition of the expression of receptor activator of nuclear factor-κB ligand and macrophage-colony stimulating factor in ST2 cells. By contrast, 4-HD enhanced indices of osteoblast differentiation, such as alkaline phosphatase activity and calcium deposition by osteoblastic MC3T3-E1 cells, at concentrations of 1-10 μM. Furthermore, we found that 4-HD at 1 μM attenuated H 2 O 2 levels in MC3T3-E1 cells. Our findings indicate that 4-HD may have critical effects on bone formation and maintenance.

Aim: The aim of this study is to clarify the value of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP) for predicting hepatocellular carcinoma (HCC) in chronic hepatitis C patients who achieved sustained virologic response (SVR) by therapy with interferon-free, direct-acting antivirals (DAAs)., Methods: This is a retrospective cohort study that included 567 patients who underwent antiviral therapy with an interferon-free DAA regimen and achieved SVR. Serum WFA + -M2BP was measured after SVR. Factors predictive of HCC occurrence and recurrence were analyzed in the patients after stratification by previous treatment history of HCC., Results: Among 518 patients who had no history of HCC, 13 developed HCC. Post-SVR WFA + -M2BP ≥1.75 cut-off index (C.O.I., P < 0.001) and α-fetoprotein (AFP) level ≥6 ng/mL (P = 0.01) were significant predictors of HCC development. Multivariate analysis showed that post-SVR WFA + -M2BP ≥1.75 C.O.I. was an independent factor significantly associated with the development of HCC (hazard ratio [HR] 6.0; 95% confidence interval (CI), 1.8-19.4; P = 0.003). Among 49 patients who had a previous history of HCC, 22 had recurrence after SVR. Post-SVR AFP ≥6 ng/mL was the only factor associated with recurrence-free survival (HR 3.1; 95% CI, 1.3-7.5; P = 0.01)., Conclusions: Post-SVR WFA + -M2BP is a predictive factor for the development of HCC in patients with no previous HCC history and treated with DAAs. Post-SVR AFP was predictive for HCC recurrence after DAA therapy., (© 2018 The Japan Society of Hepatology.)

BRCA1 functions as a tumor suppressor in DNA repair and centrosome regulation. Previously, Obg-like ATPase 1 (OLA1) was shown to interact with BARD1, a heterodimer partner of BRCA1. OLA1 binds to BRCA1, BARD1, and γ-tubulin and functions in centrosome regulation. This study determined that overexpression of wild-type OLA1 (OLA1-WT) caused centrosome amplification due to centriole overduplication in mammary tissue-derived cells. Centrosome amplification induced by overexpression of the cancer-derived OLA1 mutant, which is deficient at regulating centrosome number, occurred in significantly fewer cells than in that induced by overexpression of OLA1-WT. Thus, it was hypothesized that overexpression of OLA1 with normal function efficiently induces centrosome amplification, but not that of OLA1 mutants, which are deficient at regulating centrosome number. We analyzed whether overexpression of OLA1 missense mutants of nine candidate phosphorylation residues, three residues modified with acetylation, and two ATP-binding residues caused centrosome amplification and identified five missense mutants that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1. Two phosphomimetic mutations restored the binding to BARD1 and the efficient centrosome amplification by their overexpression. Knockdown and overexpression of BARD1 also caused centrosome amplification. BARD1 mutant reported in cancer failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification and reduced its centrosomal localization. Combined, these data reveal that the OLA1-BARD1 interaction is important for the regulation of centrosome number. Implications: Regulation of centrosome number by BRCA1/BARD1 together with OLA1 is important for the genome integrity to prevent tumor development. Mol Cancer Res; 16(10); 1499-511. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Persister cells, or persisters, are a specific subpopulation of bacterial cells that have acquired temporary antibiotic-resistant phenotypes. In this study, we showed that Escherichia coli produces many more persister cells in colony-biofilm culture than in the usual liquid culture and that these persisters can be maintained in higher numbers than those from liquid culture for up to 4 weeks at 37°C in a fresh, nutrient-rich, antibiotic-containing medium, even after complete withdrawal from the colony-biofilm culture. This suggests the presence of a long-retention effect, or "memory effect", in the persister cell state of E. coli cells. We also discovered that such increases in persisters during colony-biofilm culture and their memory effects are common, to a greater or lesser degree, in other bacterial species. This is true not only for gram-negative bacteria ( Acinetobacter and Salmonella ) but also for gram-positive bacteria ( Staphylococcus and Bacillus ). This is the first report to suggest the presence of a common memory mechanism for the persister cell state, which is inscribed during colony-biofilm culture, in a wide variety of bacteria.

Background: The present study aimed to investigate the impact of pre-sarcopenia on the prognosis of patients with advanced hepatocellular carcinoma (HCC) treated with sorafenib., Methods: We enrolled 214 patients (71 ± 10 years old; 166 men and 48 women; 90% Child-Pugh grade A and 10% Child-Pugh grade B) treated with sorafenib in our hospital from July 2009 to August 2016. The muscle volume was measured from CT images just before sorafenib administration using software (SliceOmatic). Skeletal muscle mass index was calculated, and the presence of pre-sarcopenia was judged according to the standard (42 cm2/m2 for men and 38 cm2/m2 for women) proposed by the Japan Society of Hepatology., Results: Pre-sarcopenia was found in 123 patients (57%). The overall survival (OS) in patients with pre-sarcopenia tended to be worse than in patients without pre-sarcopenia (median 252 vs. 284 days, respectively; p = 0.16). Multivariate Cox hazard analysis revealed a baseline serum albumin level of ≤3.5 g/dl [hazard ratio (HR) 1.9; p = 0.0006], a baseline alpha-fetoprotein(AFP) level of ≥100 ng/ml (HR 2.1; p = 0.002), presence of lesions in bilateral hepatic lobes (HR 1.7; p = 0.03), and presence of major portal vein invasion (HR 1.8; p = 0.01) to be independent prognostic factors. In the 68 patients who had three or more negative prognostic factors, the presence of pre-sarcopenia did not correlate with prognosis. Of the 146 patients who had two or less prognostic factors, OS was significantly worse in 84 patients (58%) with pre-sarcopenia than in 62 patients without pre-sarcopenia (median 417 vs. 562 days, respectively; p = 0.047), and Cox hazard analysis revealed pre-sarcopenia to be an important prognostic factor (HR 1.6; p = 0.047)., Conclusion: In sorafenib treatment for advanced HCC, pre-sarcopenia is a significant prognostic factor in patients with two or less negative prognostic factors, and could be the target of intervention to improve prognosis., Competing Interests: The authors have declared that no competing interests exist.

Aim: Intermediate-stage hepatocellular carcinoma varies widely in tumor burden and liver function. This study aimed to clarify the importance of subclassification by the up-to-seven criteria in both clinical course and liver function deterioration in such patients., Methods: We retrospectively analyzed 224 patients with Child-Pugh grade A who underwent initial transarterial chemoembolization (TACE) for hepatocellular carcinoma. Tumor downstaging to within the Milan criteria within 1 year and liver function worsening as Child-Pugh grade deterioration from A to B were analyzed., Results: The median survival time was 35.8 months. Forty-five patients had no recurrence within 1 year after initial TACE. Of the 179 patients with at least one recurrence within a year, 44 (25%) achieved tumor downstaging to within the Milan criteria and showed significantly longer survival than non-downstaged ones (P = 0.02). Logistic regression univariate analysis revealed that up-to-seven criteria fulfillment was associated with tumor downstaging to within the Milan criteria (odds ratio 2.6; P = 0.007). The median deterioration time was 26.7 months. Multivariate analysis revealed that beyond the up-to-seven criteria (hazard ratio [HR] 1.9; P = 0.005) was an independent factor associated with Child-Pugh grade deterioration, along with serum albumin (HR 1.54; P = 0.01), serum bilirubin (HR 1.49; P = 0.02), and prothrombin time (HR 1.54; P = 0.04)., Conclusions: The up-to-seven criteria had prognostic value and could predict non-critical recurrence and maintenance of Child-Pugh grade in patients who underwent initial conventional TACE., (© 2017 The Japan Society of Hepatology.)

Background: Laboratory determination of fibrin/fibrinogen degradation products (FDP) levels, along with that of the D-dimer, is important for assessing the fibrinolytic situation. Recently, we developed a new FDP reagent "Lias Auto P-FDP", which can detect various FDP fragments. The purpose of this study was to evaluate the basic performance of the newly developed Lias Auto P-FDP and compare it with Lias Auto D-Dimer Neo assay., Methods: The within-run precision of Lias Auto P-FDP and Lias Auto D-Dimer was determined 20 times in low and high value controls. The between-day precision was evaluated five times a day for five days. The linearity study was performed by diluting high value samples for 2 - 10-fold and 2 - 8-fold. The comparative study was performed using 172 patient samples with elevated FDP values. For the discrepancy analysis, the samples were divided into three groups by the discrepancy percentage between the FDP and D-dimer values. The groups were defined as follows: lower discrepancy group, less than -20%; no discrepancy group, -20% to 20%; upper discrepancy group, more than 20%., Results: The coefficient of variation % (CV%) in within-run and between-day precision were within 3.8% for both FDP and the D-dimer. The correlation coefficients were more than 0.999 and the linearity was high. In the comparative study, the values of FDP were higher than that of the D-dimer in all samples. The median FDP and D-dimer values of lower discrepancy, no discrepancy, and upper discrepancy groups were 11.8, 20.3, and 51.4, and 8.0, 11.3, and 13.1, respectively. FDP showed an increasing tendency but D-Dimer showed constant values. Thus, the possible cause of discrepancy between FDP and D-dimer values were the elevated FDP values. In addition, the values of plasmin-α2 plasmin inhibitor complex (PIC) in the upper discrepancy group were higher than that of the lower and no discrepancy groups, indicating progression of fibrinolysis., Conclusions: In this study, we evaluated the newly developed Lias Auto P-FDP reagent and confirmed that the basic performance was acceptable. FDP was elevated in samples with high PIC values, which indicated progression of fibrinolysis. Determination of fibrinolysis conditions by FDP measurement is important.

System x c - was recently described as the most upstream node in a novel form of regulated necrotic cell death, called ferroptosis. In this context, the small molecule erastin was reported to target and inhibit system x c - , leading to cysteine starvation, glutathione depletion and consequently ferroptotic cell death. Although the inhibitory effect of erastin towards system x c - is well-documented, nothing is known about its mechanism of action. Therefore, we sought to interrogate in more detail the underlying mechanism of erastin's pro-ferroptotic effects. When comparing with some well-known inhibitors of system x c - , erastin was the most efficient inhibitor acting at low micromolar concentrations. Notably, only a very short exposure of cells with low erastin concentrations was sufficient to cause a strong and persistent inhibition of system x c - , causing glutathione depletion. These inhibitory effects towards system x c - did not involve cysteine modifications of the transporter. More importantly, short exposure of tumor cells with erastin strongly potentiated the cytotoxic effects of cisplatin to efficiently eradicate tumor cells. Hence, our data suggests that only a very short pre-treatment of erastin suffices to synergize with cisplatin to efficiently induce cancer cell death, findings that might guide us in the design of novel cancer treatment paradigms.

Preparation of a range of oxazolidinones, including enantioenriched N-aryl-substituted oxazolidinones, in which tetraarylphosphonium salts (TAPS) catalyze the [3 + 2] coupling reaction of isocyanates and epoxides effectively, is described. The key finding is a Brønsted acid/halide ion bifunctional catalyst that can accelerate epoxide ring opening with high regioselectivity. Mechanistic studies disclosed that the ylide generated from TAPS, along with the formation of halohydrins, plays a crucial role in the reaction with isocyanates.

Background: Tolvaptan is vasopressin type 2 receptor antagonist that inhibits water reabsorption. It is used in combination with standard diuretics to treat ascites unresponsive to standard diuretic therapy or hyponatremia because of liver cirrhosis. This study evaluated the effectiveness and safety of tolvaptan in clinical practice and aimed to determine the factors related to its effectiveness., Methods: Tolvaptan was administered to 88 consecutive cirrhotic patients with ascites unresponsive to standard diuretic therapy. An effective treatment response was a ≥2% reduction in body weight on day 7. The association of patient pretreatment characteristics with therapeutic effects was analyzed., Results: Mean weight reduction on day 7 of tolvaptan therapy was -2.9% ± 3.2%, and treatment was effective in 52% of patients. Multivariate analysis revealed that spot urine Na/K ratio ≥2.5 at baseline was the only factor independently related to therapeutic effect, with an odds ratio of 7.85 (95% confidence interval 2.64-23.40, p = 0.0002). Weight reduction percentage on day 7 was -4.0% ± 2.8% in patients with spot urine Na/K ≥2.5, which was significantly greater than the 0.7% ± 2.7% loss in those with urine Na/K < 2.5 (p < 0.05). A spot urine Na/K ratio ≥2.5 had a sensitivity of 85% and specificity of 60% for predicting effective treatment. No adverse events of treatment led to treatment discontinuation., Conclusions: Baseline spot urine Na/K was predictive of an effective response to tolvaptan therapy. It is simple to perform and readily available and might serve as an indicator of optimal timing of tolvaptan administration in patients with inadequate response to conventional Na diuretic therapy.

Several methods have been proposed for protein-sugar binding site prediction using machine learning algorithms. However, they are not effective to learn various properties of binding site residues caused by various interactions between proteins and sugars. In this study, we classified sugars into acidic and nonacidic sugars and showed that their binding sites have different amino acid occurrence frequencies. By using this result, we developed sugar-binding residue predictors dedicated to the two classes of sugars: an acid sugar binding predictor and a nonacidic sugar binding predictor. We also developed a combination predictor which combines the results of the two predictors. We showed that when a sugar is known to be an acidic sugar, the acidic sugar binding predictor achieves the best performance, and showed that when a sugar is known to be a nonacidic sugar or is not known to be either of the two classes, the combination predictor achieves the best performance. Our method uses only amino acid sequences for prediction. Support vector machine was used as a machine learning algorithm and the position-specific scoring matrix created by the position-specific iterative basic local alignment search tool was used as the feature vector. We evaluated the performance of the predictors using five-fold cross-validation. We have launched our system, as an open source freeware tool on the GitHub repository (https://doi.org/10.5281/zenodo.61513)., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor whose expression level is positively correlated with tumor aggressiveness and metastatic potential. However, the mechanism underlying SLPI-induced enhancement of malignant phenotype is not completely understood. The malignancy of cancer cells is highly dependent on cell migration activity. Our previous study revealed that gingival carcinoma Ca9-22 cells, but not colorectal adenocarcinoma HT-29 cells, expressed SLPI. Therefore, we investigated the migration activity of these two cell types to understand the nature of SLPI-mediated tumor aggressiveness and metastatic potential. In vitro wound healing assay indicated that HT-29 cells and SLPI-deleted Ca9-22 cells showed lower migration activity than wild-type Ca9-22 cells, suggesting that SLPI-induced cell migration plays an important role in tumor aggressiveness and metastatic potential. In addition, our gene expression profiling study based on microarray data for the three cell types identified a number of candidates, including LCP1 and GLI, that could be key molecules in the mechanism of SLPI-induced cell migration.

Objectives: This study aimed to build a prediction score of prognosis for patients with advanced hepatocellular carcinoma (HCC) after sorafenib treatment., Methods: A total of 165 patients with advanced HCC who were treated with sorafenib were analyzed. Readily available baseline factors were used to establish a scoring system for the prediction of survival., Results: The median survival time (MST) was 14.2 months. The independent prognostic factors were C-reactive protein (CRP) <1.0 mg/dL [hazard ratio (HR) =0.51], albumin >3.5 g/dL (HR =0.55), alpha-fetoprotein <200 ng/mL (HR =0.45), and a lack of major vascular invasion (HR =0.39). Each of these factors had a score of 1, and after classifying the patients into five groups, the total scores ranged from 0 to 4. Higher scores were linked to significantly longer survival (p<0.0001). Twenty-nine patients (17.6%) with a score of 4 had a MST as long as 36.5 months, whereas MST was as short as 2.4 and 3.7 months for seven (4.2%) and 22 (13.3%) patients with scores of 0 and 1, respectively., Conclusions: A novel prognostic scoring system, which includes the CRP level, has the ability to stratify the prognosis of patients with advanced stage HCC after treatment with sorafenib.

Purpose: The aim of this in vitro study was to evaluate tensile bond strengths and corrosion resistance of CoCr alloys joined with gold cylinder by a soldering system in comparison with the conventional cast-joining system., Methods: CoCr alloys joined with gold cylinder by a soldering system using a high-fusing gold solder (CoCr/Solder/Gold cylinder), gold alloy joined with gold cylinder by a cast joining system (Gold alloy/Gold cylinder) and CoCr castings were fabricated. The tensile bond strength and corrosion resistance in 0.9% NaCl solution (pH 7.4 and pH 2.3) were evaluated. Scanning electron microscopy (SEM) of the fractured surface and electron probe microanalysis (EPMA) of the joined interfaces were also performed., Results: The tensile bond strengths of the CoCr/Solder/Gold cylinder specimens showed similar values as the Gold alloy/Gold cylinder specimens. SEM observation and EPMA analyses suggested firm bonding between the CoCr alloy and gold cylinder. The released elements from the CoCr/Solder/Gold cylinder specimens were similar to ones from CoCr castings., Conclusions: Results showed that superstructures made of CoCr alloys joined with the gold cylinder using a high-fusing gold solder had sufficient bond strength and high corrosion resistance. These hybrid frameworks with cobalt-chromium alloy and gold cylinder are promising prosthesis for implant superstructures with the low cost and favorable mechanical properties instead of conventional high-gold alloys., (Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.)

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that diminishes tissue destruction during inflammation. A recent report revealed high levels of SLPI expression in the oral carcinoma cell. In addition, overexpression of SLPI up-regulates metastasis in lung carcinoma cells. On the other hand, matrix metalloproteinases (MMPs) are proteinases that participate in extracellular matrix degradation. SLPI and MMPs are involved as accelerators of the tumor invasion process; however, their exact roles are not fully understood. Understanding the mechanism of tumor invasion requires models that take the effect of microenvironmental factors into account. In one such in vitro model, different carcinoma cells have been shown to invade myoma tissue in highly distinct patterns. We have used this myoma model, as it provides a more natural stroma-like environment, to investigate the role of SLPI in tumor invasion. Our results indicate that the model provides a relevant matrix for tumor invasion studies, and that SLPI is important for the invasion of oral carcinoma Ca9-22 cells in conjunction with MMPs. Furthermore, using bioinformatics analysis, we have identified candidates as key molecules involved in SLPI-mediated tumor invasion., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Remarkable advances in high-throughput sequencing technologies have fundamentally changed our understanding of the genetic and epigenetic molecular bases underlying human health and diseases. As these technologies continue to revolutionize molecular biology leading to fresh perspectives, it is imperative to thoroughly consider the enormous excitement surrounding the technologies by highlighting the characteristics of platforms and their global trends as well as potential benefits and limitations. To date, with a variety of platforms, the technologies provide an impressive range of applications, including sequencing of whole genomes and transcriptomes, identifying of genome modifications, and profiling of protein interactions. Because these applications produce a flood of data, simultaneous development of bioinformatics tools is required to efficiently deal with the big data and to comprehensively analyze them. This review covers the major achievements and performances of the high-throughput sequencing and further summarizes the characteristics of their applications along with introducing applicable bioinformatics tools. Moreover, a step-by-step procedure for a practical transcriptome analysis is described employing an analytical pipeline. Clinical perspectives with special consideration to human oral health and diseases are also covered., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Lupus anticoagulant (LA) is an antibody that interferes with in vitro coagulation reactions. The mixing test is considered useful for LA diagnosis and is also recommended to differentiate between acquired hemophilia A (AHA) and factor deficiency. However, there has been little study to differentiate between LA and AHA. Our aims are to investigate whether we can differentiate LA and AHA by the mixing test and to establish new formulas for the mixing test to differentiate these samples clearly., Materials and Methods: We examined 27 LA-positive, 29 coagulation factor deficient, 24 unfractionated heparin and 48 AHA samples. Index of circulating anticoagulant (ICA) values, calculated from the clotting times without incubation and after 2h incubation, were defined as ICA immediate (ICAi) and ICA delayed (ICAd) respectively. ICAd/ICAi and ICAd-ICAi were also calculated to compare the sensitivity and specificity., Results: ICAd/ICAi and ICAd-ICAi for AHA samples were significantly higher than those of the other sample groups. The sensitivities to AHA in ICAi, ICAd, ICAd/ICAi and ICAd-ICAi were 66.7%, 81.3%, 93.8% and 91.7% respectively, while the specificities for AHA were 45.0%, 66.3%, 85.0% and 98.8% respectively. ICAd/ICAi and ICAd-ICAi showed high sensitivity and specificity., Conclusions: ICAd/ICAi and ICAd-ICAi were useful for LA and AHA diagnosis, because these could differentiate between LA and AHA samples. These new formulas can contribute to the rapid diagnosis and treatment of LA and AHA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lupus anticoagulant-hypothrombinemia syndrome (LAHS) is a rare disease involving hemorrhagic diathe- sis due to hypothrombinemia with lupus anticoagulant. We report a 28-week-pregnant woman at twenty years of age, who had been hospitalized with jaundice. In laboratory data, AST, ALT, and bilirubin were elevated and the prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged. Although the liver failure was improved after she delivered a baby by Caesarean section, postoperative intraperitoneal bleeding persisted. The diagnosis by liver biopsy was autoimmune hepatitis. Although the bleeding was stopped on the seventh postoperative day, the prolongation of PT and APTT remained. LA was positive in the diluted Russell's viper venom time. Anti-cardiolipin and anti-beta-2-glycoprotein anti- bodies were also positive. The prothrombin activity was reduced. A high titer of phosphatidylserine- dependent antiprothrombin antibody (aPS/PT), which causes bleeding, was observed. Based on these data, she was diagnosed with LAHS. The liver dysfunction and prolongation of PT and APTT were normalized following the administration of corticosteroids. In this case, aPS/PT may have contributed to the pathological physiology of LAHS. [Case Report].

Background: The water channel aquaporin 2 (AQP2) at the apical membrane of renal collecting duct cells mediates water reabsorption. The expression of AQP2 at the apical membrane is tightly regulated by vasopressin and was quantitated by measurement of the urinary form by a recently developed ELISA. Tolvaptan, an antagonist of vasopressin type 2 receptor, inhibits water reabsorption in cirrhosis. The aim of this study was to determine the correlation between the pharmacological effect of tolvaptan and the dynamics of urinary AQP2 levels., Methods: Tolvaptan was administered to 41 cirrhotic patients with ascites unresponsive to standard diuretic therapy. Urinary excretion of AQP2 and urinary osmolarity were measured at the baseline and at 4, 8, and 24 h after administration of tolvaptan., Results: At the baseline, urinary AQP2/creatinine ratios were significantly higher in cirrhotic patients with ascites than in healthy controls (P < 0.0001). After administration of tolvaptan, urinary AQP2/creatinine ratios decreased by 45.0 % at 4 h and 77.0 % at 8 h. Similarly, urinary osmolarity decreased by 42.0 % at 4 h and 41.5 % at 8 h. Urinary AQP2 levels and urinary osmolarity significantly correlated at the baseline and at all time points after tolvaptan administration. The degree of the decrease in urinary AQP2 levels and degree of the decrease in urinary osmolarity correlated significantly at 4 h (r = 0.452, P = 0.009) and 8 h (r = 0.384, P = 0.030) after tolvaptan administration., Conclusions: These results indicate that the vasopressin-AQP2 system plays a major role in fluid retention in cirrhosis and that the pharmacological effect of tolvaptan to inhibit water reabsorption can be monitored by measurement of the dynamics of urinary AQP2 levels.

Background & Aims: The fibrosis stage, which is evaluated by the distribution pattern of collagen fibers, is a major predictor for the development of hepatocellular carcinoma (HCC) for patients with hepatitis C. Meanwhile, the role of elastin fibers has not yet been elucidated. The present study was conducted to determine the significance of quantifying both collagen and elastin fibers., Methods: We enrolled 189 consecutive patients with hepatitis C and advanced fibrosis. Using Elastica van Gieson-stained whole-slide images of pretreatment liver biopsies, collagen and elastin fibers were evaluated pixel by pixel (0.46 μm/pixel) using an automated computational method. Consequently, fiber amount and cumulative incidences of HCC within 3 years were analyzed., Results: There was a significant correlation between collagen and elastin fibers, whereas variation in elastin fiber was greater than in collagen fiber. Both collagen fiber (p = 0.008) and elastin fiber (p < 0.001) were significantly correlated with F stage. In total, 30 patients developed HCC during follow-up. Patients who have higher elastin fiber (p = 0.002) in addition to higher collagen fiber (p = 0.05) showed significantly higher incidences of HCC. With regard to elastin fiber, this difference remained significant in F3 patients. Furthermore, for patients with a higher collagen fiber amount, higher elastin was a significant predictor for HCC development (p = 0.02)., Conclusions: Computational analysis is a novel technique for quantification of fibers with the added value of conventional staging. Elastin fiber is a predictor for the development of HCC independently of collagen fiber and F stage.

Motivation: Predictive tools that model protein-ligand binding on demand are needed to promote ligand research in an innovative drug-design environment. However, it takes considerable time and effort to develop predictive tools that can be applied to individual ligands. An automated production pipeline that can rapidly and efficiently develop user-friendly protein-ligand binding predictive tools would be useful., Results: We developed a system for automatically generating protein-ligand binding predictions. Implementation of this system in a pipeline of Semantic Web technique-based web tools will allow users to specify a ligand and receive the tool within 0.5-1 day. We demonstrated high prediction accuracy for three machine learning algorithms and eight ligands., Availability and Implementation: The source code and web application are freely available for download at http://utprot.net They are implemented in Python and supported on Linux., Contact: shimizu@bi.a.u-tokyo.ac.jp, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press.)

Aim: For patients receiving endoscopic submucosal dissection (ESD), there is urgent need pertaining to the prevention of postoperative bleeding. We conducted a retrospective propensity score-matched study that evaluated whether pre-ESD gastric lavage prevents postoperative bleeding after ESD for gastric neoplasms., Methods: From September 2002 to October 2015, the 760 consecutive patients receiving ESD for gastric neoplasm were enrolled and data regarding them were retrospectively analyzed. All patients received conventional preventive treatment against delayed bleeding after ESD, including the administration of proton pump inhibitor and preventive coagulation of visible vessels, at the end of the ESD procedure., Results: Pre-ESD risk factors for postoperative bleeding included tumor size and no gastric lavage. Using multivariate analysis tumor size >2.0 cm (HR 2.90, 95% CI 1.65-5.10, p = 0.0002) and no gastric lavage (HR 3.20, 95% CI 1.13-9.11, p = 0.029) were found to be independent risk factors. Next, we evaluated the effect of gastric lavage on the prevention of post-ESD bleeding using a propensity score-matching method. A total of 284 subjects (142 per group) were selected. Adjusted odds ratio of gastric lavage for post-ESD bleeding was 0.25 (95% CI 0.071-0.886, p = 0.032)., Conclusion: Pretreatment gastric lavage reduced postoperative bleeding in patients receiving ESD for gastric neoplasm.

Human stem cells are promising sources for regenerative therapy. To ensure safety of future therapeutic applications, the differentiation potency of stem cells has to be tested and be widely opened to the public. The potency is generally assessed by teratoma formation comprising differentiated cells from all three germ layers, and the teratomas can be inspected through high-quality digital images. The teratoma assay, however, lacks consistency in transplantation protocols and even in interpretation, which needs community-based efforts for improving the assay quality. Here, we have developed a novel database OpenTein (Open Teratoma Investigation, http://opentein.hgc.jp/) to archive and freely distribute high-resolution whole-slide images and relevant records. OpenTein has been designed as a searchable, zoomable and annotatable web-based repository system. We have deposited 468 images of teratomas derived by our transplantation of human stem cells, and users can freely access and process such digital teratoma images. Approximately, the current version of OpenTein responds within 11.2 min for processing 2.03 gigapixel teratoma images. Our system offers valuable tools and resources in the new era of stem cell biology., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

An understanding and ability to develop a strategy to prevent pre-analytical errors of laboratory tests in the hemostasis area are two of the most important skills of medical technologists and related doctors. Recently, the working group for standardization of sampling in coagulation tests is working towards a consensus. This article reviews a summary of the consensus: (1) The anticoagulant for coagulation tests is 3.13-3.2% sodium citrate at a ratio of 1:9 to whole blood and the accuracy of the ratio is within 10%. (2) Blood sampling is achieved with the use of a 21-23G needle and coagulation. Blood sampling can be achieved by both a syringe and vacuum tube system. After taking blood, laboratory tests such as of the prothrombin time (PT) and activated partial thromboplastin time (APTT) should be completed within one hour and the storage temperature should be at room temperature, not ice-cold conditions. 3) To prepare a plasma sample, citrated blood is centrifuged at 1,500 x g for 15 min at room temperature to minimize the remaining platelets in plasma (below 10,000/microL at least).

Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-κB. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of IκBβ, which indicates that it is an inhibitor of NF-κB at the protein level. Taken together, SLPI may regulate pIgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression.SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-κB reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-κB protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of IκBβ. These results demonstrate that SLPI down-regulates pIgR expression through the NF-κB signaling pathway by inhibiting degradation of IκBβ protein., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.

Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP) and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs), and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

Background: Periodontitis is a chronic inflammatory disease initiated by a synergistic and dysbiotic microbial community that elicits a gingival inflammatory response leading to tissue breakdown. Periodontitis shares many characteristics with other chronic inflammatory diseases, including abnormal glycosylation of immunoglobulin (Ig)G. The current authors have previously demonstrated that IgG from gingival crevicular fluid (GCF) of patients with chronic periodontitis contains galactose (Gal)-deficient IgG., Methods: The origin of the aberrantly glycosylated IgG was determined by measuring levels of Gal-deficient IgG in GCF and serum from patients with periodontitis and non-periodontitis controls using lectin enzyme-linked immunosorbent assay. The Ig-producing cells and the proportion of cells producing Gal-deficient IgG were immunohistochemically determined in gingival tissues from patients with periodontitis by fluorescence microscopy. The results were statistically evaluated and correlated with clinical data., Results: The results indicate that GCF of patients with periodontitis had higher levels of Gal-deficient IgG compared with controls (P = 0.002). In gingival tissues, IgG was the dominant isotype among Ig-producing cells, and 60% of IgG-positive cells produced Gal-deficient IgG. Moreover, the proportion of Gal-deficient IgG-producing cells directly correlated with clinical parameters of probing depth and clinical attachment loss (AL)., Conclusion: These results suggest that the presence of Gal-deficient IgG is associated with gingival inflammation and may play a role in the worsening of clinical parameters of periodontitis, such as AL.

A female patient in her seventies with diabetes mellitus, hyper-lipidemia and mitral regurgitation was admitted because of the acute heart failure. She was treated with diuretics and vasodilators, however these were not effective. Therefore the CHDF using heparin was required for the patients. After the introduction of CHDF, the platelet count subsequently decreased to less than 7.0 x 10(4)/μl. After stopping CHDF, the platelet count recovered. In the second CHDF treatment, the platelet count decreased again. HIT was suspected because of both the usage of heparin and five points of 4T's score in the patient. Heparin was discontinued immediately and then her platelet count improved. The HIT antibody by latex-particle-enhanced immunoturbidimetric assay was performed simultaneously, however it was not detected. After re-using heparin by heparin lock, platelet count had been decreasing. Furthermore the thrombus was observed in the infusion tube. We considered that a clinical course did not accord with the result of HIT antibody. We measured HIT antibody by another method, an enzyme immunoassay (EIA), and the positive antibody was observed. We encountered a rare case with discrepancy in the results of HIT antibody between two methods. When HIT is suspected by the results from the clinical course and 4T's score, even though the negative HIT antibody, heparin should be discontinued and the different assay for HIT antibody such as an EIA in this case should be performed.

Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1+/- adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1+/- precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1+/- precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1+/- adult mice, we found that the Gli1+/- mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed.

Following a failure of a bone graft or an implant placement, the hypothesis of a biological abnormality is rarely considered as a possible cause. A systematic search of peer-reviewed literature for dyslipidemia or vitamin D deficiency may explain this lack of consideration. Excess low-density lipoprotein cholesterol (dyslipidemia) is responsible for a slower bone metabolism or lower dental implant osseointegration. In addition, vitamin D is a key factor for linking innate and adaptive immunity. Both of these factors are compromised under the conditions of vitamin D deficiency. Therefore, vitamin D deficiency slows implant osseointegration and increases the risk of graft infection. Vitamin D is also involved in immune function and therefore allergic reactions.

High-molecular-weight kininogen (HMWK) deficiency is a very rare hereditary disorder. We herein report a case of HMWK deficiency with splenic infarction. The HMWK activity of the proband was markedly decreased (0.9%). Direct sequencing of his HMWK gene showed a homozygous "TC" insertion at c523-524 in exon 4. This insertion led to an amino acid substitution, Ser175Ser, resulting in a frameshift mutation and a premature stop codon in amino acid 183. Furthermore, the HMWK activity was also reduced in the patient's three children, who exhibited the heterozygous "TC" insertion at c523-524 in exon 4. This is the first report of this gene alteration in a patient with HMWK deficiency.

A large number of clinical laboratory technologists are qualified as diabetes educators; however, few of them actually participate in teaching patients. This is partially because it is difficult to balance their routine laboratory tasks and the work of a diabetes educator. We have introduced ultrasonic examinations of the ophthalmic artery, inspection of the R-R interval and current perceptual-threshold inspection for the early diagnosis of diabetic complications, and we have been contributing to the good medical care for diabetes. Furthermore, to care for diabetic foot lesions, clinical laboratory technologists have participated in checking diabetic patients' feet since 2007. In concrete terms, we examine the feet of diabetic patients, take digital pictures of the feet, and write a report, while preparing for thermographic examination of the patient. At the same time, we give simple guidance about foot care. Technologists cannot perform medical treatment; however, this has been accepted by medical staff because we only check foot lesions. We make use of existing medical imaging and reporting systems in the physiological laboratory, so doctors and nurses on the diabetic care team can always obtain information about the patients. Such actions have a good reputation not only among medical staff but also among diabetic patients.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1(-/-) fetuses, and the phenotype was more severe in Gli1(-/-);Gli2(-/-) newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.

Drug testing with the use of point of care testing (POCT) has been widely used in Japan, especially in the field of drug abuse, poisoning, and anticoagulant therapy with warfarin. For evidence-based medicine of POCT, an interesting report was presented by the National Academy of Clinical Biochemistry in the United States as the guideline in 2006. Users of POCT devices should understand all limitations of the devices. This strength/consensus recommendation is strong and the level of evidence is high. In this field, cyan, arsenic, paraquat, organic phosphate, methanol, acetaminophen, barbiturates, benzodiazepines, antidepressants, and some other drugs were detected by POCT devices such as Triage DOA and a detector tube system and others in Japan. The usefulness of the organophosphorus pesticide detection kit in the accident of GYOZA POISONING from china was noteworthy. In the case of toluene intoxication, the detector tube system was useful as a screening test for the gas phase test of a 2-year-old patient's vomit and excreta without any information from his parents. In warfarin treatment, a POCT device was useful for small hospitals and clinics. Although the cost is not covered by the health insurance system in Japan, the emergency centers of hospitals use these POCT devices for clinical decision-making. This is the most important problem.

Aims: Left ventricular (LV) fibrosis and stiffening play crucial roles in the development of heart failure with preserved ejection fraction (HFPEF). Plasma level of digitalis-like factors (DLFs) is increased in patients with hypertension, a principal underlying cardiovascular disease of HFPEF. Digitalis-like factors inhibit ion-pumping function of Na(+)/K(+)-ATPase and activate the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger (NCX). Digitalis-like factors are known to promote collagen production in fibroblasts. The aim of this study was to explore whether the pharmacological inhibition of the NCX entry mode is effective in the prevention of LV fibrosis and in the development of HFPEF., Methods and Results: (i) Dahl salt-sensitive rats fed 8% NaCl diet from age 6 weeks served as hypertensive HFPEF model. In this model, 24 h urine excretion of DLFs was greater than that in the age-matched control at compensatory hypertrophic and heart failure stages. (ii) Continuous administration of ouabain for 14 weeks developed LV fibrosis without affecting blood pressure in Sprague-Dawley rats. (iii) Ouabain elevated intracellular Ca(2+) concentration through the entry of extracellular Ca(2+), increased the phosphorylation level of p42/44 mitogen-activated protein kinases, and enhanced (3)H-proline incorporation in cardiac fibroblast; and SEA0400, the inhibitor of the NCX entry mode, suppressed these effects. (iv) In the HFPEF model, administration of SEA0400 at subdepressor dose improved the survival rate in association with the attenuation of LV fibrosis and stiffening., Conclusion: Digitalis-like factors and the subsequently activated NCX entry mode may play an important role in the development of hypertensive HFPEF, and the blockade of the NCX entry mode may be a new therapeutic strategy for this phenotype of heart failure.