Cauterization of Narrow Root Canals Untouched by Instruments by High-Frequency Current.

The mechanical removal of bacteria is fundamental to the treatment of infected root canals, but complete sterilization of biofilms tends not to extend to uninstrumented areas. However, during electrical conduction to a root canal filled with a conductor, the higher impedance where the root canal is narrower generates Joule heat that may result in a large temperature increase and sterilization. The effect of a high-frequency electric current on the wall of a simulated narrow root canal was investigated by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). Simulated root canals, 0.1 mm in diameter, were prepared in dentine blocks. The root canal wall was treated with Plank-Rychlo solution for 5 min to create a decalcified layer. The simulated root canal was filled with either saline or NaClO, and 150 or 225 V at 520 kHz was applied for 0 s, 1 s, or 5 s. As the conduction time increased, and when the saline was replaced with NaClO, the proportion with a flat decalcified surface decreased, dentinal tubules and a lava-like morphology were significantly more evident on SEM (p < 0.01), and EDS showed significant decreases in carbon and oxygen and increases in calcium (p < 0.01). It was concluded that filling uninstrumented root canals with NaClO and using electrical conduction for 5 s could incinerate and eliminate the organic material of the root canal wall. The application of high-frequency electric current may lead to the cure of many cases of persistent apical periodontitis.

High-throughput 3D Spheroid Formation and Effective Cardiomyocyte Differentiation from Human iPS Cells Using the Microfabric Vessels EZSPHERETM.

High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated. Graphic abstract: Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.

Greater consumption of noodle is associated with higher serum phosphorus levels: a cross-sectional study on healthy participants.

Higher serum phosphorus levels are associated with mortality and cardiovascular events, both in healthy individuals and those with chronic kidney disease. Owing to the increasing westernization of eating habits, a decrease in rice consumption and an increase in the intake of bread and noodle products were observed in Japan. This cross-sectional study investigated the influence of staple food (rice, bread, and noodle) consumption patterns on the serum levels of phosphorus and phosphate-regulating factors in 103 healthy young participants. Fasting blood and 24-h urine samples were collected; data about dietary habits were collected using a brief-type self-administered diet history questionnaire. Cluster analysis was conducted to assess subgroups classified according to staple food consumption patterns. Serum levels of phosphorus and phosphate-regulating factors did not significantly differ between subgroups classified based on the frequency of rice or bread consumption. However, the serum levels of phosphorus and fibroblast growth factor 23 were more significantly elevated in the higher than in the lower noodle consumption frequency subgroup. Cluster analysis defined three clusters, and the serum phosphorus levels in the high-noodle cluster were significantly higher than that in the high-bread and high-rice clusters. A high consumption of noodles was associated with elevated serum phosphorus levels. The trial was registered in the University Hospital Medical Information Network (UMIN) Center system (The name of the trial register: Hidekazu Arai, and UMIN accession number: UMIN000034352).

Collective cancer cell invasion in contact with fibroblasts through integrin-α5ß1/fibronectin interaction in collagen matrix.

Interaction of cancer cells with cancer-associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3-D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin-α5ß1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E-cadherin-expressing adenocarcinoma cell lines, DLD-1 (colon) and MCF-7 (breast). When hybrid spheroids of DLD-1 cells with CAFs were embedded into collagen gel, DLD-1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor-α promoted the collective invasion, possibly by reducing the E-cadherin junction, as did the transforming growth factor-ß inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor-ß itself inhibited the cancer cell invasion. Efficient collective invasion of DLD-1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function-blocking Abs and siRNAs confirmed that DLD-1 cells adhered to fibronectin fibrils on CAFs mainly through integrin-α5ß1. Anti-E-cadherin Ab promoted the single cell invasion of DLD-1 cells by dissociating the E-cadherin junction. Although the binding affinity of MCF-7 cells to CAFs was lower than DLD-1, they also collectively invaded the collagen matrix in a similar fashion to DLD-1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.

A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed.

Habitual confectionery intake is associated with serum phosphorus levels : A cross-sectional study on healthy subjects.

Hyperphosphatemia is associated with an increased risk of developing cardiovascular disease. Recently, it has been shown that high serum phosphorus levels are associated with increased cardiovascular events in healthy subjects, but the dietary factors determining serum phosphorus level have not been fully investigated. The study investigated the influence of habitual dietary factors on serum phosphorus levels in healthy young participants. This cross-sectional study conducted fasting blood sampling in 109 healthy young people and used a brief-type self-administered diet history questionnaire to evaluate the subject's habitual meals. Since the bioavailability of dietary phosphorus depends on the food sources, habitual phosphorus intakes from different food groups and intake frequency (times/month) of processed foods were calculated. The mean serum phosphorus level was 3.9 0.5 mg/dl ; 10.1% of the subjects had serum phosphorus levels that exceeded the reference levels of 4.5 mg/dl. Total phosphorus intake and phosphorus intake from animal-based food did not differ between serum phosphorus quartiles. Higher intake of confectionery was associated with increased serum phosphorus levels. This study showed that frequent consumption of confectionery was associated with elevated serum phosphorus levels. Additional studies are needed to determine whether this is a causal relationship. J. Med. Invest. 66 : 134-140, February, 2019.

Cancer cell migration on elongate protrusions of fibroblasts in collagen matrix.

Cancer-associated fibroblasts (CAFs) play critical roles in the tumor progression. However, it remains unclear how cancer cells migrate in the three-dimensional (3D) matrix of cancer tissues and how CAFs support the cancer invasion. Here we propose a novel mechanism of fibroblast-dependent cancer cell invasion in the 3D collagen matrix. Human cancer cell lines from the pancreas (Panc-1), lung (A549) and some other organs actively adhered to normal fibroblasts and primary lung CAFs in cultures. To show its significance in tumor invasion, we designed a new invasion assay in which homogeneous microspheroids consisting of cancer cells and fibroblasts were embedded into collagen gel. Time-lapse experiments showed that cancer cells adhered to and quickly migrated on the long protrusions of fibroblasts in the 3D collagen matrix. Fibroblast-free cancer cells poorly invaded the matrix. Experiments with function-blocking antibodies, siRNAs, and immunocytochemistry demonstrated that cancer cells adhered to fibroblasts through integrin α5ß1-mediated binding to fibronectin on the surface of fibroblasts. Immunochemical analyses of the co-cultures and lung cancers suggested that cancer cells could acquire the migratory force by the fibronectin/integrin signaling. Our results also revealed that the fibroblast-bound fibronectin was a preferential substrate for cancer cells to migrate in the collagen matrix.

Carbohydrate antigen 19-9 is significantly elevated in autosomal dominant polycystic kidney disease.

AIM: Liver cysts are the most common extrarenal manifestation in patients with autosomal dominant polycystic kidney disease (ADPKD). Carbohydrate antigen 19-9 (CA19-9) is generally used as a marker for biliopancreatic malignancies, although CA19-9 levels in patients with ADPKD are largely unknown. METHODS: A prospective observational study of 53 ADPKD patients and 83 non-ADPKD control subjects was performed. The serum levels of CA19-9 were studied to evaluate the association with clinical parameters and liver cysts. RESULTS: The serum CA19-9 levels were significantly higher in the ADPKD group than in the control group (32.9 U/mL vs. 9.8 U/mL, respectively, P < 0.001). The serum CA19-9 levels in the ADPKD group were positively correlated with the mean blood pressure (rho = 0.335, P < 0.05), gamma-glutamyl transferase (GTP) levels (rho = 0.541, P < 0.001), the largest cyst size (rho = 0.536, P < 0.001) and the liver cyst volume (rho = 0.682, P < 0.001). Multiple regression analyses showed that the gamma-GTP levels (P < 0.001) and the liver cyst volumes (P < 0.001) were independent predictors for serum CA19-9 levels. CONCLUSIONS: Serum CA19-9 levels are significantly elevated and appear to be dependent on the gamma-GTP levels and the volume of liver cysts in patients with ADPKD. Our findings indicate that the measurement of the baseline CA19-9 level in each patient with ADPKD may be useful for the interpretation of the value and the differential diagnosis of liver diseases, particularly the liver cyst infection.

Quantitative assessment of locomotive syndrome by the loco-check questionnaire in older Japanese females.

[Purpose] Locomotive syndrome (LS) is a condition by which older people may require care service because of problems with locomotive organs. This study examined whether the loco-check, a 7-item questionnaire, is useful for quantitatively assessing the severity of LS. [Subjects and Methods] Seventy-one community dwelling Japanese females aged 64-96 years (81.7 ± 8.0 years) participated in this study. The associations of the loco-check with thigh muscle mass measured by X-ray CT, physical performance, nutritional status, and quality of life (QOL) were investigated. [Results] The results showed that the number of times that "yes" was selected in the loco-check was significantly correlated with thigh muscle mass, major measures of physical performance, nutritional status, and QOL. This number was also significantly larger in the participants experiencing falling, fracture, and lumbar pain than in those without these episodes. [Conclusion] These results suggest that the loco-check might be useful for quantitatively evaluating LS.

Red Blood Cell Distribution Width Is Associated With All-Cause and Cardiovascular Mortality in Hemodialysis Patients.

Red blood cell distribution width (RDW) is an index of red blood cell variability that is usually used to differentiate the cause of anemia. However, clinical evidence for the relationship between RDW and mortality in hemodialysis patients is still lacking. We performed a single center, prospective longitudinal study. During more than 5 years of follow-up in 80 patients undergoing maintenance hemodialysis, 34 patients (42.5%) died. In the Kaplan-Meier curve analyses, higher RDW levels (≥ 14.9%) were significantly associated with all-cause and cardiovascular mortality (log-rank test, P < 0.05, each). In multivariate Cox proportional hazard models, each 1.0% increase in RDW value predicted an estimated 25% higher risk of mortality (P < 0.05) and a 40% higher risk of cardiovascular mortality (P < 0.05). In conclusion, higher RDW value was a significant predictor for all-cause and cardiovascular mortality in patients undergoing maintenance hemodialysis.

Effect of endurance training and branched-chain amino acids on the signaling for muscle protein synthesis in CKD model rats fed a low-protein diet.

A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched-chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately one-half of the animals in each group was subjected to treadmill exercise (15 m/min, 1 h/day, 5 days/wk). After 7 wk, renal function was measured, and soleus muscles were collected to evaluate muscle protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified.

Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells.

Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications.

[Chronic kidney disease and nutrition].

Abnormalities of mineral metabolism develop with decline of renal function in chronic kidney disease (CKD), and it is called as a CKD-mineral and bone disorder (CKD-MBD). The standard approach for management of CKD-MBD is to keep serum phosphorus, calcium, and parathyroid hormone in the reference range by dietary intervention and medications. It has been recently pointed out that starting the treatment from early CKD is important for suppressing CKD-MBD.

Circulating 20S proteasome is independently associated with abdominal muscle mass in hemodialysis patients.

UNLABELLED: Protein-energy wasting is highly prevalent in hemodialysis patients, and it contributes to patient morbidity and mortality. The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and it is involved in the regulation of basic cellular processes. However, the role of this system in the determination of nutritional status is largely unknown. To examine a relationship between protein-energy wasting and the ubiquitin-proteasome system, a cross-sectional study of 76 hemodialysis patients was performed. Plasma concentrations of 20S proteasome were studied to evaluate its association with muscle and fat mass, which were investigated by abdominal muscle and fat areas measured using computed tomography and by creatinine production estimated using the creatinine kinetic model. Plasma 20S proteasome concentrations significantly and negatively correlated with abdominal muscle areas and creatinine production (rho = -0.263, P < 0.05 and rho = -0.241, P < 0.05, respectively), but not abdominal subcutaneous and visceral fat areas. Multiple regression analyses showed that 20S proteasome was a significant independent predictor of abdominal muscle area (P < 0.05). In conclusion, plasma 20S proteasome concentrations were independently associated with abdominal muscle mass in hemodialysis patients. Our findings indicate a relationship between circulating 20S proteasomes and muscle metabolism in these patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000012341.

Impact of hyperuricaemia on the chronic kidney disease-associated risk factors in a community-based population.

AIM: Hyperuricaemia is a common finding in subjects with lifestyle related diseases. This study was performed to examine its association with chronic kidney disease (CKD) in relation to other risk factors in a community-based population. METHODS: Data from 187 914 participants, excepting CKD stage 5, of the health check-up were included in this analysis. The association between CKD and its risk factors were examined by a logistic analysis. The association of hyperuricaemia and CKD was also compared in the population without any lifestyle related diseases and the whole population. RESULTS: The prevalence of hyperuricaemia was significantly higher in the advanced stage of CKD. The odds ratio of hyperuricaemia was higher than that of other factors for the association with CKD. The odds ratio of many CKD-associated variables was increased in the advanced stage. Among them, the odds ratio of hyperuricaemia was markedly increased. The prevalence of hyperuricaemia was lower in the population without any lifestyle related diseases than in the whole population in the early stages, the difference of prevalence between the two populations becoming smaller in the advanced stage. CONCLUSIONS: The association of incident CKD with hyperuricaemia was stronger than with other chronic kidney disease-risk factors, this association becoming more significant in the advanced stage of chronic kidney disease. Although this result does not indicate the cause and result relationship, the data suggest that hyperuricaemia might not be appropriately treated in this population.

Caffeine intoxication successfully treated by hemoperfusion and hemodialysis.

Caffeine is an easily available drug which is generally regarded as being safe. However, it can lead to a lethal outcome in severe cases of caffeine intoxication. We herein report a case of a 32-year-old man who ingested approximately 15.6 g of caffeine in a suicide attempt. He suffered from sustained ventricular tachycardia despite conservative treatment. Therefore, we performed a combined therapy of hemoperfusion and hemodialysis which resulted in rapid improvement of the patient's ventricular tachycardia as well as a reduction of the patient's plasma caffeine level. To the best of our knowledge, this combination has not been previously reported to treat caffeine intoxication.

Protective effects of relaxin against cisplatin-induced nephrotoxicity in rats.

BACKGROUND: Cisplatin (CDDP)-induced acute kidney injury (AKI) involves pro-inflammatory responses, apoptosis of renal tubular epithelial cells and vascular damage. AKI increases the risk of chronic kidney disease. Relaxin (RLX) has anti-apoptotic and anti-fibrosis properties. The aim of this study was to investigate the effects of RLX on CDDP-induced nephrotoxicity. METHODS: We investigated the mitigating effects of RLX based on the etiopathology of AKI induced by CDDP, and also the anti-fibrotic effect of RLX on renal fibrosis after AKI. In the short-term experiments, rats were divided into the control group, CDDP group, and CDDP+RLX group. In the latter group, RLX was infused for 5 or 14 days using an implanted osmotic minipump. CDDP was injected intraperitoneally (6 mg/kg) after RLX or saline infusion. At 5 and 14 days post-CDDP, the kidneys were removed for analysis. The effect of RLX on renal fibrosis after AKI was evaluated at 6 weeks post-CDDP. RESULTS: In short-term experiments, CDDP transiently increased plasma creatinine and blood urea nitrogen with peaks at day 5, and RLX prevented such rises. Semiquantitative analysis of the histological lesions indicated marked structural damage and apoptotic cells in the CDDP group, with the lesions being reduced by RLX treatment. Overexpression of Bax, interleukin-6 and tumor necrosis factor-α observed in the kidneys of the CDDP group was reduced in the CDDP+RLX group. In the long-term experiments, RLX significantly reduced renal fibrosis compared with the CDDP group. CONCLUSIONS: The results suggested that RLX provided protection against CDDP-induced AKI and subsequent fibrosis by reducing apoptosis and inflammation.

Plasma levels of fibroblast growth factor-23 are associated with muscle mass in haemodialysis patients.

AIM: Malnutrition is highly prevalent in haemodialysis (HD) patients, and it contributes to morbidity and mortality. Fibroblast growth factor-23 (FGF-23) and Klotho contribute to chronic kidney disease-mineral and bone disorder (CKD-MBD) in HD patients, but the role that these molecules play in determining nutritional status is currently unknown. METHODS: A cross-sectional study examining 77 HD patients was performed. The plasma concentrations of FGF-23 and soluble Klotho (s-Klotho) were studied to evaluate their association with muscle mass, which was investigated by abdominal muscle areas measured using computed tomography and by creatinine (Cr) production estimated using the Cr kinetic model. RESULTS: Plasma FGF-23 concentrations were significantly and positively correlated with abdominal muscle areas and Cr production (rho = 0.301, P < 0.01 and rho = 0.345, P < 0.01, respectively). In contrast, s-Klotho was not significantly correlated with these muscle mass indices and plasma FGF-23 concentrations. Multiple regression analyses showed that FGF-23 was a significant independent predictor of both muscle mass indices (P < 0.01 and P < 0.05, respectively). CONCLUSION: Plasma FGF-23 concentrations were associated with muscle mass indices in HD patients. Our findings suggest that FGF-23 and nutritional status are linked and this link is most likely independent of s-Klotho.

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.