1-Kestose Supplementation Increases Levels of a 5α-Reductase Gene, a Key Isoallolithocholic Acid Biosynthetic Gene, in the Intestinal Microbiota.

1-Kestose (kestose) is the smallest fructooligosaccharide component and shows a particularly high prebiotic function. Both kestose and the bile acid metabolite isoallolithocholic acid (isoalloLCA) are known to be beneficial for human health, especially in terms of immune homeostasis in the gastrointestinal system; however, the effect of kestose on the levels of microbial isoalloLCA producers remains to be clarified. IsoalloLCA is known to be produced by several members of the phylum Bacteroidota that carry the 5α-reductase (5AR) gene, a key isoalloLCA biosynthetic gene. Thus, we designed a specific primer set to detect the 5AR gene based on the consensus sequences of the genes from several isoalloLCA producers. Using real-time quantitative PCR with this primer set and fecal DNA samples, we compared the 5AR gene level (5ar-level) in the intestinal microbiota of a kestose-supplemented group (n=20) and a placebo group (n=16) before and after intake for 12 wk. The 5ar-level was significantly increased in the kestose-supplemented group (p=0.015), but not in the placebo group (p=0.379), indicating that kestose supplementation increased the 5ar-level in human intestinal microbiota. Our findings suggest that targeting functional gene levels could potentially be used to predict and understand the beneficial prebiotic effects associated with changes in gut microbiota.

Supplementation of 1-Kestose Modulates the Gut Microbiota Composition to Ameliorate Glucose Metabolism in Obesity-Prone Hosts.

Insulin resistance leads to the onset of medical conditions such as type 2 diabetes, and its development is associated with the alteration in the gut microbiota. Although it has been demonstrated that supplementation with prebiotics modulates the gut microbiota, limited evidence is available for effects of prebiotics on insulin resistance, especially for humans. We investigated the prebiotic effect of 1-kestose supplementation on fasting insulin concentration in obesity-prone humans and rats. In the preliminary study using rats, the hyperinsulinemia induced by high-fat diet was suppressed by intake of water with 2% (w/v) 1-kestose. In the clinical study using obese-prone volunteers, the fasting serum insulin level was significantly reduced from 6.5 µU/mL (95% CI, 5.5-7.6) to 5.3 (4.6-6.0) by the 12-week intervention with supplementation of 10 g 1-kestose/day, whereas it was not changed by the intervention with placebo (6.2 µU/mL (5.4-7.1) and 6.5 (5.5-7.6) before and after intervention, respectively). The relative abundance of fecal Bifidobacterium was significantly increased to 0.3244 (SD, 0.1526) in 1-kestose-supplemented participants compared to that in control participants (0.1971 (0.1158)). These results suggest that prebiotic intervention using 1-kestose may potentially ameliorate insulin resistance in overweight humans via the modulation of the gut microbiota. UMIN 000028824.

Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

OBJECTIVE: Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. RESULTS: GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

Organ-confined plasmacytoid urothelial carcinoma: implication of the lack of expression of the receptor tyrosine kinase MET.

Plasmacytoid urothelial carcinoma (PUC), a morphological variant of urothelial carcinoma (UC), is composed of cancer cells that resemble plasma cells, monocytes, or both, and is clinicopathologically distinguished by its aggressive, non-organ-confined features. Here, we present a case of a patient with PUC with early invasion at diagnostic transurethral resection. Histopathologically, no residual cancer or lymph node metastasis was observed by total cystectomy. The patient remains disease-free after 18 months, without undergoing adjuvant chemotherapy. Interestingly, the immunoreactivity of MET, a receptor tyrosine kinase expressed in many invasive UCs, was minimal in the cancer cells. In contrast, archival pathological, non-organ-confined PUC cells exhibited strong MET immunoreactivity. The present case may imply a role for the MET protein in the aggressive behavior of PUCs. We propose the putative usefulness of MET inhibitors for the treatment of aggressive PUCs.

Shortening of the atrial-His bundle interval during atrial pacing as a predictor of successful ablation for typical atrioventricular nodal re-entrant tachycardia.

Aims: Shortening of the atrial-His bundle (AH) interval during the sinus rhythm is occasionally observed after slow pathway ablation for atrioventricular nodal re-entrant tachycardia (AVNRT). In addition, high-rate atrial pacing is useful for avoiding atrioventricular block. We hypothesized that shortening of the AH interval during slow pathway ablation under high-rate atrial pacing would lead to successful ablation of typical AVNRT. Methods and results: This retrospective study included 37 patients in whom successful ablation of typical AVNRT was performed under atrial pacing. The AH interval was measured immediately before the first radiofrequency (RF) application and immediately after the last RF application, prior to the first induction. Twenty-five of 37 patients achieved procedural success at the first induction (i.e. successful group). No patients developed a prolonged AH interval or atrioventricular block. The AH interval was shortened by an average of 14.6 ± 7.7 and 1.8 ± 1.2 ms in the successful and other patient groups, respectively (P < 0.01). An AH interval decrease of > 10 ms was observed in 23 of 27 (85%) patients in the successful group, whereas all other patients had an AH interval decrease of < 5 ms. Conclusion: Shortening of the AH interval during high-rate atrial pacing is a predictor of the successful ablation for typical AVNRT.

The efficacy of ablation based on the combined use of the dominant frequency and complex fractionated atrial electrograms for non-paroxysmal atrial fibrillation.

BACKGROUND: This study aimed to evaluate an approach for an endpoint of non-inducibility using a combined high-dominant frequency (DF) and continuous complex fractionated atrial electrogram (CFAE) ablation following circumferential pulmonary vein isolation (PVI) in a sequential fashion, including linear ablation as compared to PVI alone. METHODS AND RESULTS: A total of 84 non-paroxysmal patients with atrial fibrillation (AF) were investigated retrospectively. The AF patients were divided into two groups: patients with PVI following a combined high-DF and continuous CFAE ablation with linear ablation (substrate modification group, n=59) and those with PVI alone (n=25). DF sites of ≥8Hz and then continuous CFAE sites defined by fractionation intervals of ≤50ms were modified after PVI. The ablation endpoint was non-inducibility. Atrial tachyarrhythmias (ATs) could not be induced in 54 of 59 (92%) patients after a sequential ablation, and in 18 of 25 (64%) with PVI alone. The ATs freedom without antiarrhythmic drugs in the substrate modification group was significantly greater than that in those with PVI alone after 1 procedure during 12 months of follow-up (78.6% vs. 53.8%, log-rank test p=0.039). CONCLUSION: This sequential approach using a substrate based ablation was associated with a better clinical long-term outcome as compared to PVI alone.

[Three Patients with Acute Myocardial Infarction Associated with Targeted Therapy of Sorafenib for Metastatic Renal Cell Carcinoma : Case Report].

Sorafenib is a tyrosine kinase inhibitor (TKI) of the vascular endothelial growth factor receptor (VEGFR) used for advanced renal cell carcinoma. Treatment with sorafenib prolongs progression-free survival in patients with advanced clear-cell renal cell carcinoma. However, in spite of its therapeutic efficacy, sorafenib causes a wide range of adverse events. Cardiovascular adverse events have been observed when sorafenib was used with targeted agents. Although these adverse events like hypertension, reduced left ventricular ejection fraction, cardiac ischemia or infarction were manageable with standard medical therapies in most cases, some had a poor clinical outcome. We report three cases of acute myocardial infarction associated with sorafenib in patients with metastatic renal cell carcinoma.

Location of epicardial adipose tissue affects the efficacy of a combined dominant frequency and complex fractionated atrial electrogram ablation of atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) is located adjacent to high dominant frequency (DF) sites. OBJECTIVE: This study aimed to clarify the relationship between the EAT location and efficacy of a combined high DF site and continuous complex fractionated activation electrogram (CFAE) site ablation. METHODS: Fifty-five patients with nonparoxysmal atrial fibrillation (AF) (26 (47%) persistent and 29 (53%) long-standing persistent) underwent pulmonary vein isolation followed by high DF site and continuous CFAE site ablation. High DF sites (DF ≥8 Hz) and continuous CFAE sites (fractionated intervals ≤50 ms) were targeted. The patients were divided into an AF-free group and an AF-recurrent group. RESULTS: The AF freedom rate on antiarrhythmic drugs in patients with persistent and long-standing persistent AF was 88.5% and 75.9% over a 12-month follow-up period, respectively. The total EAT, left atrial (LA)-EAT, and right atrial (RA)-EAT volumes did not indicate significant differences between the AF-free and AF-recurrent groups. In the LA, the overlap between high DF sites and EAT was larger in the AF-free group than in the AF-recurrent group (57.0% ± 33.3% vs 22.6% ± 23.3%; P < .01). However, this overlap did not differ between the AF-free and AF-recurrent groups in the RA (20.4% ± 28.2% vs 19.0% ± 24.4%; P = .91). The overlap between continuous CFAE sites and EAT did not differ between the 2 groups in both the LA and the RA. CONCLUSION: High DF sites that overlap with EAT may be important sources of AF. However, the contribution of EAT to the AF substrate may differ between the LA and the RA.

Selection of Mycoplasma genitalium strains harbouring macrolide resistance-associated 23S rRNA mutations by treatment with a single 1 g dose of azithromycin.

OBJECTIVE: A single 1 g dose regimen of azithromycin has been recommended for the treatment of Mycoplasma genitalium infections. The authors evaluated whether this regimen could select M genitalium strains with macrolide resistance after treatment for M genitalium-positive non-gonococcal urethritis. METHODS: In seven men with non-gonococcal urethritis, who were infected with M genitalium without macrolide resistance-associated mutations but experienced microbiological azithromycin treatment failure, M genitalium DNAs in their post-treatment urine specimens were examined for mutations in the 23S rRNA gene and the ribosomal protein genes of L4 and L22. To assess the relatedness of M genitalium strains before and after treatment, their DNAs in pretreatment and post-treatment urine were genotyped by analysing short tandem repeats of an AGT/AAT unit in the MG309 gene and single nucleotide polymorphisms in the MG191 gene. RESULTS: In four of seven patients, M genitalium in post-treatment urine had an A-to-G transition at nucleotide position 2071 or 2072, corresponding to 2058 or 2059 in the 23S rRNA gene of Escherichia coli. In one of the four strains, Pro81Ser in the ribosomal protein L4 accompanied the mutation in the 23S rRNA gene. The genotyping of M genitalium DNAs suggested that these four post-treatment strains were selected from the respective closely related or identical pretreatment strains without macrolide resistance-associated mutations by the treatment. CONCLUSIONS: The single 1 g dose treatment of azithromycin could select M genitalium strains harbouring macrolide resistance-associated mutations. For M genitalium, this regimen might increase the risk of macrolide resistance selection after treatment.

Presurgical targeted therapy with tyrosine kinase inhibitors for advanced renal cell carcinoma: clinical results and histopathological therapeutic effects.

OBJECTIVES: We retrospectively analyzed our patients with advanced renal cell carcinoma who underwent presurgical targeted therapy with tyrosine kinase inhibitors to clarify the safety and clinical benefit. The histopathological effect of this treatment was also examined. METHODS: Between July 2005 and February 2010, nine patients with advanced renal cell carcinoma who were treated with tyrosine kinase inhibitors before surgery were the subjects of this study. Consolidative surgery was considered when these tumors showed clinical response or stable disease while on targeted therapy without evidence of disease progression at other sites. RESULTS: The agents used were sorafenib in seven patients and sunitinib in two. The median duration of presurgical therapy was 12.2 weeks, and seven patients had less than 4 months of treatment. Tumor reduction at 10-30% was obtained in all patients but one. Perioperative complications were observed in five of nine patients. Major complications occurred in two patients, including intraoperative excessive bleeding and delayed localized intraperitoneal abscess. Minor complications were found in three. The characteristics of the histopathological effect of tyrosine kinase inhibitors consisted of marked atrophy of the capillary sinus, confirming the pharmacological mechanisms of these agents. Other findings included nuclear pyknosis and degeneration of tumor cells. CONCLUSIONS: Presurgical targeted therapy with tyrosine kinase inhibitors appears to be feasible in most patients with advanced renal cell carcinoma. However, the indications, the clinical benefit and the standard protocol still remain to be determined. Therapeutic effects in the histology were compatible to their pharmacological effects.

Effect of vitamin E on learning and memory deficit in aged rats.

In order to verify whether vitamin E improves the cognitive impairment induced through aging, aged rats fed a vitamin E-supplemented diet had their learning and memory functions assessed in comparison with the aged rats fed a normal diet using a Morris water maze test. Although normal aged rats showed very poor learning ability concerning the place of a platform in the water maze apparatus, the aged rats fed the vitamin E-supplemented diet learned the place with a marked speed in only 5 trials. After old animals showed the maximum learning ability, they were kept in a normal atmosphere for 48 h without a trial followed by an assessment of their memory function using the same apparatus. The vitamin E-supplementation to aged rats resulted in marked retention of their maximum memory function, although normal aged rats showed a significant memory loss of about 60%. Pyrroloquinoline quinone (PQQ), which increases in the production of nerve growth factor, and protects neurons, had a similar effect on cognitive function to that of vitamin E in the aged rats. These results suggest that vitamin E may improve cognitive deficit caused through aging by not only its neuro-protecting effect but an antioxidant efficacy.

[Case of urachal cancer treated by neoadjuvant chemotherapy with FOLFOX4 (oxaliplatin, 5-FU and leukovolin)].

A 52-year-old woman was referred to our hospital for treatment of urachal cancer. She complained of supurapubic dull pain and gross hematuria. Computed tomography and magnetic resonance imaging showed a non-papillary sessile tumor, which was located on the dome of the bladder and invaded the small intestine. The tumor was diagnosed as Sheldon's stage IIIC urachal cancer. After three courses of neoadjuvant chemotherapy with FOLFOX4 (oxaliplatin, 5-FU and leukovolin), the tumor was reduced from 7 x 6 cm to 5.5 x 5 cm in size. Consequently, the patient underwent an en-bloc resection of the urachal tumor with the dome of the bladder and the parts of the ileum invaded by the tumor. One course of adjuvant chemotherapy (FOLFOX4) was performed. Surgical specimen revealed histologically well differentiated squamous carcinoma and invasion to the propria of the ileum. The surgical margins were negative for the cancer. For 1.5 years after the surgery, no local recurrence or distant metastasis has been observed.

Dehydroepiandrosterone supplementation improves endothelial function and insulin sensitivity in men.

The dehydroepiandrosterone (DHEA) concentration decreases with age. There is evidence that DHEA has a protective effect against age-related disorders, including cardiovascular disease. Accordingly, we examined the effect of DHEA supplementation (25 mg/d) on endothelial function, insulin sensitivity, and fibrinolytic activity in 24 men with hypercholesterolemia (mean age, 54 +/- 1 yr). All subjects were enrolled in a randomized, double-blind study. Flow-mediated dilation of brachial artery after transient occlusion, which was expressed as the percent change from the baseline value of the diameter, increased significantly with DHEA supplementation [DHEA: baseline, 3.9 +/- 0.5%; 4 wk, 6.9 +/- 0.7%; 8 wk, 7.9 +/- 0.6%; 12 wk, 8.4 +/- 0.7% (P < 0.01 vs. baseline for all, by ANOVA); placebo: 4.1 +/- 0.6%, 4.5 +/- 0.5%, 3.9 +/- 0.5%, and 4.4 +/- 0.6% (P < 0.01 for all, by ANOVA)]. There was a significant concurrent reduction in the plasma levels of plasminogen activator inhibitor type 1 during DHEA supplementation [DHEA: 9.1 +/- 2.2, 6.4 +/- 2.3, 5.5 +/- 2.8, and 5.1 +/- 2.0 IU/ml (P < 0.01 vs. baseline, by ANOVA); placebo: 9.0 +/- 2.1, 10.4 +/- 2.2, 9.5 +/- 2.2, and 9.6 +/- 2.1 IU/ml (P < 0.01, by ANOVA)]. DHEA supplementation also decreased steady state plasma glucose [DHEA: baseline, 178.9 +/- 12.2; 12 wk, 132.0 +/- 12.8 mg/dl (P < 0.01, by ANOVA); placebo: 181.0 +/- 13.8 and 179.6 +/- 12.4 mg/dl (P < 0.01, by ANOVA)]. In contrast, steady state plasma insulin did not change during the study in either group. The low dose DHEA supplementation improves vascular endothelial function and insulin sensitivity and decreases the plasminogen activator inhibitor type 1 concentration. These beneficial changes have the potential to attenuate the development of age-related disorders such as cardiovascular disease.