Ingenuity for enabling the habituation of pelvic floor muscle training.

[Purpose] To clarify factors contributing to habituation of pelvic floor muscle training (PFMT) for urinary incontinence. [Subjects and Methods] We included 13 healthy females and examined diurnal and nocturnal urination frequency at initial program participation and at 3 months. The survey used the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF), a 10-level self-assessment of anxiety associated with urinary incontinence, and a 10-level self-evaluation of PFMT understanding and skill acquisition. We evaluated PFMT practice at home and postures that facilitated PFMT. The practice of PFMT at home was surveyed during a 3-month period. [Results] Compared to baseline, the level of skill acquisition assessed by the ICIQ-SF and PFMT according to the 10-level self-evaluation improved significantly at 3 months. The rate of PFMT sessions performed at home per week was high. The number of times PFMT was performed per day was positively correlated with level of understanding and acquisition of skills pertaining to PFMT, according to the 10-level self-assessment. [Conclusion] By incorporating behavior modification techniques appropriate for urinary incontinence and by increasing the level of understanding regarding incontinence and PFMT, as well as the level of skill acquisition, self-efficacy increased. This may have motivated habituation of PFMT.

Angiotensin II-induced cardiac hypertrophy and fibrosis are promoted in mice lacking Fgf16.

Fibroblast growth factors (Fgfs) are pleiotropic proteins involved in development, repair and metabolism. Fgf16 is predominantly expressed in the heart. However, as the heart function is essentially normal in Fgf16 knockout mice, its role has remained unclear. To elucidate the pathophysiological role of Fgf16 in the heart, we examined angiotensin II-induced cardiac hypertrophy and fibrosis in Fgf16 knockout mice. Angiotensin II-induced cardiac hypertrophy and fibrosis were significantly promoted by enhancing Tgf-ß1 expression in Fgf16 knockout mice. Unexpectedly, the response to cardiac remodeling was apparently opposite to that in Fgf2 knockout mice. These results indicate that Fgf16 probably prevents cardiac remodeling, although Fgf2 promotes it. Cardiac Fgf16 expression was induced after the induction of Fgf2 expression by angiotensin II. In cultured cardiomyocytes, Fgf16 expression was promoted by Fgf2. In addition, Fgf16 antagonized Fgf2-induced Tgf-ß1 expression in cultured cardiomyocytes and noncardiomyocytes. These results suggest a possible mechanism whereby Fgf16 prevents angiotensin II-induced cardiac hypertrophy and fibrosis by antagonizing Fgf2. The present findings should provide new insights into the roles of Fgf signaling in cardiac remodeling.

Light and electron microscopic detection of inflammation-targeting liposomes encapsulating high-density colloidal gold in arthritic mice.

OBJECTIVE: We have previously demonstrated the efficient and time-dependent transvascular localization of Sialyl Lewis X (SLX)-liposomes to inflammatory sites, but the final target of the SLX-liposomes remained uncertain. The aim of this study was to identify the target cells of the liposomes within the inflamed joints of collagen antibody-induced arthritis (CAIA) model mice. METHODS: SLX-liposomes and unlabeled liposomes encapsulating high-density colloidal gold were administered intravenously into the caudal vein of CAIA mice on day 5 after induction of arthritis when the inflammatory score was maximal (n = 6 per group). Six hours or 24 h after liposome administration, animals were euthanized and hind limbs and ankles were excised without perfusion. After fixation, synovial tissues were examined by light microscopy after silver enhancement of colloidal gold or by transmission electron microscopy. RESULTS: Silver-enhanced signals were detected within the cells around E-selectin-positive blood vessels in the synovium of the SLX-liposome group. These cells were positive for the macrophage/monocyte marker F4/80 or neutrophil marker Ly-6G. Transmission electron microscopy detected the colloidal gold signals together with liposome-like structures within the phagosomes of synovial macrophages. Transmission electron microscopy and energy dispersive X-ray spectrometry could determine gold elements in the lysosomes of synovial macrophages. CONCLUSIONS: The results of the current study demonstrate that SLX-liposomes primarily targeting E-selectin in activated endothelial cells could potentially deliver their contents into inflammatory cells around synovial blood vessels in arthritic joints.

ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes.

Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy.

Antenatally diagnosed congenital orbital teratoma in which rupture was associated with intrauterine fetal death.

We report a case of a fetus with a congenital orbital teratoma (COT), in which rupture of the tumor was associated with an intrauterine fetal demise. An ultrasound scan at 27 weeks' revealed a solid and cystic, complex mass in the orbital region with extensive vascularization suggestive of an orbital cystic teratoma. Magnetic resonance imaging (MRI) supported this diagnosis and clarified tumor localization. At 32 weeks', the patient presented with fetal demise and rupture of the mass was noted. Fetal COTs, like sacrococcygeal teratomas, carry the risk of rupture. MRI in utero is useful for evaluating the extent of disease.

Pharmacological modulation of histone demethylase activity by a small molecule isolated from subcritical water extracts of Sasa senanensis leaves prolongs the lifespan of Drosophila melanogaster.

BACKGROUND: Extracts of Sasa senanensis Rehder are used in traditional Japanese medicine; however, little is known about the underlying mechanisms of their potential health benefits. METHODS: S. senanensis leaves were extracted with subcritical water. An active small-molecule was isolated using reversed-phase high-performance liquid chromatography (HPLC), and identified as 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA). The effects of PA on the activity of histone demethylase, the Drosophila melanogaster lifespan and gene expression in Drosophila S2 cells were investigated. RESULTS: PA inhibited the activity of Jumonji domain-containing protein 2A (JMJD2A) histone demethylase in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 11.6 µM. However, there was no effect on lysine-specific demethylase 1 (LSD1), histone deacetylase 1 (HDAC1) or HDAC8. PA significantly extended the lifespan of female, but not male, Drosophila. In Drosophila S2 cells, the eukaryotic translation initiation factor 4E binding protein (4E-BP) was up-regulated by PA exposure. CONCLUSIONS: Our findings provide insight into the possible relationship between the pharmacological modulation of histone demethylation and lifespan extension by PA; they might also be important in the development of alternative therapies for age-related disorders.

A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.

Molecular characterization of a TIA-1-like RNA-binding protein in cells derived from the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae).

A complementary DNA encoding a TIA-1-type RNA-binding protein (SfTRN-1) was isolated from cultured cells of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), to characterize its function. The deduced 388-amino acid sequence of SfTRN-1, which possessed three RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain, showed significant homology with mammalian TIA-1/TIAR and silkworm BmTRN-1, factors important in the metabolism of transcripts. It was found that inhibition of SfTRN-1 gene expression by a transfected oligonucleotide encoding the antisense sequence led to a marked increase in the production of a reporter protein and the amount of reporter transcript in the cultured cells. In addition, overexpression of the recombinant full-length SfTRN-1 open reading frame in the cultured cells led to a decrease in reporter protein production, but the truncated RRM1-3 domain lacking the C-terminal auxiliary domain lost its activity. Analysis using a GFP-fused recombinant protein revealed that, unlike mammalian TIA-1/TIAR, SfTRN-1, most likely shuttling between the nucleus and cytoplasm, had the characteristic of being largely distributed in the cytoplasm, where it perhaps acts to reduce the amount of transcripts, and that RRM1 and RRM3 were related to its nuclear accumulation, but RRM2 to its nuclear export. Furthermore, the posterior half of the auxiliary domain was also found to be related to its nuclear export. This study indicates that respective RRM subdomains of SfTRN-1 play distinct roles important to its subcellular distribution, and it identified unique systems for the distribution and functional regulation of the TIA-1 family in insect cells, ones which are clearly different from those in mammalian cells.

CSAHi study: Detection of drug-induced ion channel/receptor responses, QT prolongation, and arrhythmia using multi-electrode arrays in combination with human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: The use of multi-electrode arrays (MEA) in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides a promising method to predict comprehensive cardiotoxicity, including drug-induced QT prolongation and arrhythmia. We previously demonstrated that MEA in combination with hiPSC-CMs could provide a generalizable platform by using 7 reference drugs at 10 testing facilities. Using this approach, we evaluated responses to reference drugs that modulate a range of cardiac ion currents and have a range of arrhythmogenic effects. METHODS: We used the MEA system (MED64) and commercially available hiPSC-CMs (iCell cardiomyocytes) to evaluate drug effects on the beat rate, field potential duration (FPD), FPD corrected by Fridericia's formula (FPDc), and the incidence of arrhythmia-like waveforms. RESULTS: This assay detected the repolarization effects of Bay K8644, mibefradil, NS1643, levcromakalim, and ouabain; and the chronotropic effects of isoproterenol, ZD7288, and BaCl2. Chronotropy was also affected by K+ and Ca2+ current modulation. This system detected repolarization delays and the arrhythmogenic effects of quinidine, cisapride, thioridazine, astemizole, bepridil, and pimozide more sensitively than the established guinea pig papillary muscle action potential assay. It also predicted clinical QT prolongation by drugs with multiple ion channel effects (fluoxetine, amiodarone, tolterodine, vanoxerine, alfuzosin, and ranolazine). DISCUSSION: MEA in combination with hiPSC-CMs may provide a powerful method to detect various cardiac electrophysiological effects, QT prolongation, and arrhythmia during drug discovery. However, the data require careful interpretation to predict chronotropic effects and arrhythmogenic effects of candidate drugs with multiple ion channel effects.

Bite Force and Masticatory Performance Using Implant-supported Overdentures After Treatment of Mandibular Cancer.

AIM: To evaluate overdentures with regard to artificial restoration of oral function following mandibular cancer. MATERIALS AND METHODS: We examined 32 patients who had undergone mandibular bone resection as treatment for malignancy and were using implant-supported overdentures. The patients were aged 55-87 years (mean=68.6) with a male to female ratio of 23:9. Marginal resection was performed in 29 patients and segmentectomy in 3. RESULTS: Before and after using the attachment for overdenture, oral function differed significantly. After the setting of implant-retained overdentures, maximum bite force increased on average by 362% (average, from 16.2 N to 58.8 N; p<0.01). Xylitol gum examination showed a 363% increase in masticatory performance (average, 3.1 to 8.0 points; p<0.01). CONCLUSION: Implant-retained overdenture resulted in improved oral function, that was lost after treatment for mandibular cancer.

CSAHi study: Evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia--effects of 7 reference compounds at 10 facilities.

INTRODUCTION: Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia. METHODS: Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericia's formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated. RESULTS: The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms. DISCUSSION: MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans.

Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-ß-Smad2/3 pathway in chondrocytic cells.

Physiologic mechanical stress stimulates expression of chondrogenic genes, such as multifunctional growth factor CYR61/CTGF/NOV (CCN) 2 and α1(II) collagen (COL2A1), and maintains cartilage homeostasis. In our previous studies, cyclic tensile strain (CTS) induces nuclear translocation of transforming growth factor (TGF)-ß receptor-regulated Smad2/3 and the master chondrogenic transcription factor Sry-type HMG box (SOX) 9. However, the precise mechanism of stretch-mediated Smad activation remains unclear in transcriptional regulation of CCN2 and COL2A1. Here we hypothesized that CTS may induce TGF-ß1 release and stimulate Smad-dependent chondrogenic gene expression in human chondrocytic SW1353 cells. Uni-axial CTS (0.5Hz, 5% strain) stimulated gene expression of CCN2 and COL2A1 in SW1353 cells, and induced TGF-ß1 secretion. CCN2 synthesis and nuclear translocalization of Smad2/3 and SOX9 were stimulated by CTS. In addition, CTS increased the complex formation between phosphorylated Smad2/3 and SOX9. The CCN2 promoter activity was cooperatively enhanced by CTS and Smad3 in luciferase reporter assay. Chromatin immunoprecipitation revealed that CTS increased Smad2/3 interaction with the CCN2 promoter and the COL2A1 enhancer. Our results suggest that CTS epigenetically stimulates CCN2 transcription via TGF-ß1 release associated with Smad2/3 activation and enhances COL2A1 expression through the complex formation between SOX9 and Smad2/3.

trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli.

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

The use of opioids in a pregnant woman with lumbar disc herniation: a case report.

It is not easy to diagnose lumbar disc herniation during pregnancy due to the limitation of the examinations and it is also difficult to control the severe pain during this time. A pregnant woman with lumbar disc herniation was transferred to our hospital at the 23rd week of gestation. The pain was successfully controlled with opioids and epidural anesthesia. At the 35th week of gestation, she delivered a girl weighing 2316 g smoothly with an Apgar score of 8/9 without neonatal abstinence syndrome from morphine. In this case, opioid administration was found to be useful for perinatal care with lumbar disc herniation.

Stereoselective preparation, structures, and reactivities of phosphine-bridged mixed-metal trinuclear and pentanuclear complexes with tris[2-(diphenylphosphino)ethyl]phosphine.

The phosphine-bridged linear trinuclear and pentanuclear complexes with Pd(II)-Pt(II)-Pd(II), Ni(II)-Pt(II)-Ni(II), and Rh(III)-Pd(II)-Pt(II)-Pd(II)-Rh(III) metal-ion sequences were almost quantitatively formed by the stepwise phosphine-bridging reaction of the terminal phosphino groups of tris[2-(diphenylphosphino)ethyl]phosphine (pp3), which is the tetradentate bound ligand of the starting Pd(II) and Ni(II) complexes. The solid-state structures of the trinuclear complexes were determined by X-ray structural analyses, and the structures of the polynuclear complexes in solution were characterized by NMR spectroscopy. The trans and cis isomers of the trinuclear and pentanuclear complexes, which arise from the geometry around the Pt(II) center, were selectively obtained simply by changing the counteranion of the starting complexes: the tetrafluoroborate salts, [MX(pp3)](BF4) [M = Pd(II) or Ni(II), X = Cl- or 4-chlorothiophenolate (4-Cltp-)], gave only the trans isomers, and the chloride salt, [PdCl(pp3)]Cl, gave only the cis isomers. The formation of the trinuclear complex with the 4-Cltp- and chloro ligands, trans-[Pt(4-Cltp)2{PdCl(pp3)}2](BF4)2, proceeded with exchange between the thiolato ligand in the starting Pd(II) complex, [Pd(4-Cltp)(pp(3))](BF4), and the chloro ligands in the starting Pt(II) complex, trans-[PtCl2(NCC6H5)2], retaining the trans geometry around the Pt(II) center. In contrast, the formation reaction between [PdCl(pp3)]Cl and trans-[PtCl2(NCC6H5)2] was accompanied by the trans-to-cis geometrical change on the Pt(II) center to give the trinuclear complex, cis-[PtCl2{PdCl(pp3)}2]Cl2. The mechanisms of these structural conversions during the formation reactions were elucidated by the 31P NMR and absorption spectral changes. The differences in the catalytic activity for the Heck reaction were discussed in connection with the bridging structures of the polynuclear complexes in the catalytic cycle.