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We recently reported that adeno-associated virus serotype 1-constitutively active Ras homolog enriched in brain [AAV1-Rheb(S16H)] transduction of hippocampal neurons could induce neuron-astroglia interactions in the rat hippocampus in vivo, resulting in neuroprotection. However, it remains uncertain whether AAV1-Rheb(S16H) transduction induces neurotrophic effects and preserves the cognitive memory in an animal model of Alzheimer&rsquo, s disease (AD) with characteristic phenotypic features, such as &beta, amyloid (A&beta, ) accumulation and cognitive impairments. To assess the therapeutic potential of Rheb(S16H) in AD, we have examined the beneficial effects of AAV1-Rheb(S16H) administration in the 5XFAD mouse model. Rheb(S16H) transduction of hippocampal neurons in the 5XFAD mice increased the levels of neurotrophic signaling molecules, including brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and their corresponding receptors, tropomyosin receptor kinase B (TrkB) and CNTF receptor &alpha, subunit (CNTFR&alpha, ), respectively. In addition, Rheb(S16H) transduction inhibited A&beta, production and accumulation in the hippocampus of 5XFAD mice and protected the decline of long-term potentiation (LTP), resulting in the prevention of cognitive impairments, which was demonstrated using novel object recognition testing. These results indicate that Rheb(S16H) transduction of hippocampal neurons may have therapeutic potential in AD by inhibiting A&beta, accumulation and preserving LTP associated with cognitive memory.

"Comfort set for palliative care"is narrowly defined as a set of drugs used to alleviate symptoms at the terminal stage. However, we think that the followingis important(1)The practice of advance care planning(ACP)is indispensable and it is done accordingto the situation after the start of the visit, but our clinic emphasizes careful consultation before the start as a means of team formation with the patient's family and the doctor.(2)Introduction of visitingmedicine management by pharmacist of dispensingpharmacy is preferable for the method of supply/inventory of drugs. 98% of cancer patients in our clinic use it. In addition, we have injection drugs, devices and emergency drugs in the clinic so that we can respond quickly.(3) Seamless collaboration with visitingnursingis necessary for understandingand implementingthe situation that medicines should be used. All cancer patients in our clinic are receivingvisitingnursingby in-clinic nurses. We mentioned the point that seems to be important points in past practice for "comfort of patient, family member and medical staff" in home palliative care.

Background and purpose We recently reported that AAV1-Rheb(S16H) transduction could protect hippocampal neurons through the induction of brain-derived neurotrophic factor (BDNF) in the rat hippocampus in vivo. It is still unclear how neuronal BDNF produced by AAV1-Rheb(S16H) transduction induces neuroprotective effects in the hippocampus and whether its up-regulation contributes to the enhance of a neuroprotective system in the adult brain. Experimental approach To determine the presence of a neuroprotective system in the hippocampus of patients with Alzheimer's disease (AD), we examined the levels of glial fibrillary acidic protein, BDNF and ciliary neurotrophic factor (CNTF) and their receptors, tropomyocin receptor kinase B (TrkB) and CNTF receptor α(CNTFRα), in the hippocampus of AD patients. We also determined whether AAV1-Rheb(S16H) transduction stimulates astroglial activation and whether reactive astrocytes contribute to neuroprotection in models of hippocampal neurotoxicity in vivo and in vitro. Key results AD patients may have a potential neuroprotective system, demonstrated by increased levels of full-length TrkB and CNTFRα in the hippocampus. Further AAV1-Rheb(S16H) transduction induced sustained increases in the levels of full-length TrkB and CNTFRα in reactive astrocytes and hippocampal neurons. Moreover, neuronal BDNF produced by Rheb(S16H) transduction of hippocampal neurons induced reactive astrocytes, resulting in CNTF production through the activation of astrocytic TrkB and the up-regulation of neuronal BDNF and astrocytic CNTF which had synergistic effects on the survival of hippocampal neurons in vivo. Conclusions and implications The results demonstrated that Rheb(S16H) transduction of hippocampal neurons could strengthen the neuroprotective system and this intensified system may have a therapeutic value against neurodegeneration in the adult brain.

New, more effective pharmacologic treatments for epilepsy are needed, as a substantial portion of patients (>30%) are refractory to currently available anti-epileptic drugs. Cenobamate (YKP3089) is an investigational anti-epileptic drug in clinical development. Two completed adequate and well-controlled studies demonstrated a significant reduction in focal seizures with cenobamate in patients with epilepsy. In this study, we characterized the effects of cenobamate on voltage-gated Na+ channels in acutely isolated rat hippocampal CA3 neurons using a whole-cell patch-clamp technique. While cenobamate had little effect on the peak component of transient Na+ current (INaT) induced by brief depolarizing step pulses, it potently inhibited the non-inactivating persistent component of INa (INaP). In addition, cenobamate potently inhibited the current by slow voltage-ramp stimuli. Cenobamate significantly shifted the steady-state fast inactivation relationship toward a hyperpolarizing range, indicating that cenobamate binds to voltage-gated Na+ channels at the inactivated state with a higher affinity. Cenobamate also accelerated the development of inactivation and retarded recovery from inactivation of voltage-gated Na+ channels. In current clamp experiments, cenobamate hyperpolarized membrane potentials in a concentration-dependent manner, and these effects were mediated by inhibiting the INaP. Cenobamate also increased the threshold for generation of action potentials, and decreased the number of action potentials elicited by depolarizing current injection. Given that the INaP plays a pivotal role in the repetitive and/or burst generation of action potentials, the cenobamate-mediated preferential blockade of INaP might contribute to anti-epileptic activity.

Voltage-gated Na+ channels in primary afferent neurons can be divided into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ channels. Although previous studies have shown the acid modulation of TTX-R Na+ channels, the effect of acidic pH on tetrodotoxin-sensitive (TTX-S) Na+ channels is still unknown. Here we report the effect of acidic pH on TTX-S Na+ channels expressed in large-sized trigeminal ganglion (TG) neurons using a whole-cell patch clamp technique. The application of acidic extracellular solution decreased the peak amplitude of TTX-S currents (INa) in a pH-dependent manner, but weak acid (≥pH 6.0) had no inhibitory effect on TTX-S INa. Acidic pH (pH 6.0) shifted both the activation and steady-state fast inactivation relationships of TTX-S Na+ channels toward depolarized potentials. However, acidic pH (pH 6.0) had no effect on use-dependent inhibition in response to high-frequency stimuli, development of inactivation, and accelerated the recovery from inactivation of TTX-S Na+ channels, suggesting that TTX-S Na+ channels in large-sized TG neurons are less sensitive to acidic pH. Given that voltage-gated Na+ channels play a pivotal role in the generation and conduction of action potentials in neural tissues, the insensitivity of TTX-S Na+ channels expressed in large-sized TG neurons to acidic pH would ensure transmission of innocuous tactile sensation from orofacial regions at acidic pH conditions.

Crude tea polysaccharide (crude TPS) was prepared from instant green tea by ethanol precipitation followed by ultrafiltration membrane treatment and its effects on blood lipid, liver lipid, and fecal lipid levels were examined with Sprague-Dawley rats fed a high-fat diet. Although crude TPS showed no effects on the serum lipid levels, it suppressed the liver lipid accumulation and increased the fecal excretion of dietary fat. Then, the structural features of crude TPS were investigated. After separation of crude TPS by DEAE-cellulose and gel-filtration column chromatography, two kinds of neutral tea polysaccharides (NTPS-LP and NTPS-HH) and an acidic polysaccharide (ATPS-MH) were obtained. According to monosaccharide composition, methylation, and NMR analyses, NTPS-LP, NPTS-HH, and ATPS-MH were presumed to be starch, arabinogalactan with β-1,3-linked galactosyl backbone blanched at position 6 and with 1,5-linked arabinofuranosyl residues, and α-1,4-linked galacturonic acid backbone with arabinogalactan region, respectively.

Phthalates and bisphenol A (BPA) are estrogenic endocrine disruptors. Polymorphisms in the gene encoding estrogen receptor 1 (ESR1) may contribute to the ratio of the lengths of the second and fourth digits (2D:4D), which is considered an index of prenatal exposure to sex hormones. Thus, we investigated whether ESR1 polymorphisms modify the effects of prenatal exposure to phthalates and BPA on 2D:4D in a birth cohort. Maternal serum in the first trimester was used to determine prenatal exposure to these compounds. Six hundred twenty-three children (7 years of age) provided mean 2D:4D from photocopies and were genotyped for single nucleotide polymorphisms in ESR1, particularly PvuII (T > C, dbSNP: rs2234693), XbaI (A > G, dbSNP: rs9340799), and rs2077647 (A > G). The associations among compound exposure, mean 2D:4D, and ESR1 polymorphisms were assessed by multiple linear regression adjusted for potential cofounding factors. Boys with the AG/GG genotype at rs2077647 in the group exposed to high levels of mono(2-ethylhexyl) phthalate (MEHP) or Σ Di(2-ethylhexyl) phthalate (DEHP) showed feminized 2D:4D compared with boys with the AA genotype at rs2077647 who had low exposure to MEHP or ΣDEHP (MEHP: increase in mean 2D:4D of 1.51%, 95% confidence interval [CI]: 0.40–2.63; ΣDEHP: increase in mean 2D:4D of 1.37%, 95% CI: 0.25–2.49). No significant differences were found among girls. There were no associations between mean 2D:4D and metabolites other than MEHP or BPA. These data suggest that ESR1 polymorphisms modify the effects of prenatal exposure to DEHP on mean 2D:4D among boys.

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABAA-mediated currents and GABAA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (IGABA) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC50, 164 μM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABAA currents (Itonic), which is defined as a holding current shift by the GABAA receptor antagonist bicuculline (EC50, 36.63 μM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated Itonic and Iphasic in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced IGABA mediated by GABAA receptors. This effect was similar for all tested hGABAA receptors containing six different alpha subunits (α1β2γ2 or α2-6β3γ2), with EC50 values ranging from 42 to 194 μM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABAA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABAA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances Itonic inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.

The ratio of the lengths of the 2nd and 4th digits (2D:4D) is considered an index of prenatal exposure to androgen. Indeed, androgen receptors have been linked to digit length, but estrogen receptors are rarely investigated in this context. Thus, we investigated the association between estrogen receptor 1 (ESR1) genetic polymorphisms and 2D:4D in school-aged children. The 2D:4D ratios were determined using Vernier calipers from photocopies of palms provided by 1800 children aged 7 years who were enrolled in an ongoing prospective cohort study in Hokkaido, Japan. The children were genotyped using cord blood collected at birth for single nucleotide polymorphisms in ESR1, specifically PvuII (T > C, dbSNP: rs2234693), XbaI (A > G, dbSNP: rs9340799), and rs2077647 (A > G). The association between ESR1 polymorphisms and 2D:4D was assessed by multiple linear regression adjusted for potential cofounding factors. Boys with the GG genotype at rs9340799 had a significantly lower 2D:4D in the right hand than boys with the AA/AG genotype (−0.96% lower, 95% confidence interval: −1.68 to −0.24). However, this association was detected only in boys born to non-smoking mothers. No significant differences were found between rs9340799 polymorphisms and 2D:4D among girls. There was also no link between 2D:4D and polymorphisms at rs2234693 and rs2077647. These data suggest that rs9340799 polymorphisms in ESR1 may contribute to digit length and 2D:4D.

Background It has been described that the incidence of testicular microlithiasis is high in several congenital disorders which may be associated with testicular impairment and infertility. Several reports have shown that a prepubertal or pubertal hormonal abnormality in the pituitary-gonadal axis was identified in some patients with hypospadias that is one of the most common disorders of sex development. However, exact prevalence or risk factors of testicular microlithiasis in patients with hypospadias have not reported so far. In the present study, to clarify the prevalence and risk factors of testicular microlithiasis in patients with hypospadias, a retrospective chart review was performed. Methods Children with hypospadias who underwent testicular ultrasonography between January 2010 and April 2016 were enrolled in the present study. Severity of hypospadias was divided into mild and severe. The prevalence and risk factors of testicular microlithiasis or classic testicular microlithiasis were examined. Results Of 121 children, mild and severe hypospadias were identified in 66 and 55, respectively. Sixteen children had undescended testis. Median age at ultrasonography evaluation was 1.7 years old. Testicular microlithiasis and classic testicular microlithiasis were documented in 17 children (14.0%) and 8 (6.6%), respectively. Logistic regression analysis revealed that presence of undescended testis was only a significant factor for testicular microlithiasis and classic testicular microlithiasis. The prevalence of testicular microlithiasis or classic testicular microlithiasis was significantly higher in children with undescended testis compared to those without undescended testis (testicular microlithiasis; 43.8% versus 9.5% (p = 0.002), classic testicular microlithiasis; 37.5% versus 1.9% (p

Dural afferent neurons are implicated in primary headaches including migraine. Although a significant portion of primary afferent neurons innervating the dura are myelinated A-type neurons, previous electrophysiological studies have primarily characterized the functional properties of small-sized C-type sensory neurons. Here we show the functional characterization of dural afferent neurons identified with the fluorescent dye DiI. DiI-positive neurons were divided into three types: small-, medium-, and large-sized neurons, based on their diameter, area, and membrane capacitance. The immunoreactivity of NF200, a marker of A-type myelinated neurons, was detected in most large-sized, but it was also present in a limited number of small- and medium-sized DiI-positive neurons. Capsaicin, a transient receptor potential vanilloid 1 agonist, induced the membrane currents in most small- and medium-sized neurons, but not in large-sized DiI-positive neurons. Tetrodotoxin-resistant Na+ channels were expressed in almost all types of DiI-positive neurons. Mechanosensitive currents were detected from a majority of large-sized, and to a lesser extent, small- and medium-sized DiI-positive neurons. The results suggest that most dural afferent neurons are nociceptive, e.g., polymodal C-type for small- and medium-sized neurons, and high-threshold nociceptive A-type mechanoreceptors for large-sized neurons. We also found that DiI-positive neurons differed with respect to passive and active membrane properties, and that sumatriptan, a representative drug used for the acute treatment of migraine attack, inhibited voltage-gated Ca2+ currents in all types of DiI-positive neurons. The present results showing the nociceptive properties of dural afferent neurons would contribute to understand the pathophysiology of primary headaches.

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes., ゲノム編集技術を用いたヒトiPS細胞での正確な一塩基置換技術（MhAX法）を開発. 京都大学プレスリリース. 2018-03-06.

Acid-sensing ion channels (ASICs) are widely expressed in both the peripheral and central nervous system, and contribute to the modulation of central nociceptive transmission under both physiological and pathophysiological conditions. In this study, we characterized the proton-induced membrane currents in acutely isolated rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using the whole cell patch-clamp technique. Exposure to acidic conditions (pH

Summary: Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC. : Rand et al. find that MYC promotes proliferation of human intermediate reprogrammed cells rather than initiation of reprogramming. MYC post-transcriptionally activates LIN41, resulting in post-transcriptional suppression of p21. Suppression of p21 results in reduction of RB activity, which is a negative regulator of reprogramming progression. Keywords: reprogramming, pluripotency, induced pluripotent stem cell, proliferation, senescence, immortalization, MYC, LIN41, post-transcriptional regulation

Objective To clarify the impact of endoscopic incision for ureterocele as an initial procedure, retrospective chart review was performed focusing on the prevalence and risk factors of symptomatic urinary tract infection after endoscopic incision. Materials and methods Among children with ureterocele who were managed between September 1994 and April 2016, patients who were observed conservatively without additional surgical management after endoscopic incision were included in this study. Type of ureterocele was divided into intravesical and ectopic. Symptomatic urinary tract infection was defined as either recurrent non-febrile or febrile urinary tract infection. Statistical analysis was performed using the Cox proportional Hazard model or Kaplan-Meier Curve with log-rank test for evaluation of the prevalence and risk factors. Results Thirty-six patients met the inclusion criteria. Median age at endoscopic incision was 8.9 months. Eleven children had symptomatic urinary tract infections (febrile in 9 and recurrent non-febrile in 2) during median follow-up of 75.5 months. Initial symptomatic urinary tract infection in each child occurred within 25 months after endoscopic incision. Symptomatic urinary tract infection-free rate after endoscopic incision was 65.6%. The risk factors for symptomatic urinary tract infection were female gender, duplex system, ectopic ureterocele, and unchanged hydronephrosis after EI. Conclusions The current study demonstrated the critical period and risk factors for symptomatic urinary tract infection after endoscopic incision for the treatment of ureterocele. These results suggest that when conservative management is indicated after endoscopic incision, patients, especially those with risk factors, should be followed carefully at least for 25 months after endoscopic incision for symptomatic urinary tract infection. This article is protected by copyright. All rights reserved.

Background Neurological sequelae occur in 40% of patients with acute encephalopathy (AE). The early prediction of poor outcomes is critical to the initiation of appropriate treatment. The aim of the present study was therefore to elucidate prognostic factors that can be quickly and feasibly evaluated on hospital admission in patients with AE. Methods We analyzed data from 51 AE patients admitted to Hirakata City Hospital between January 2005 and December 2014. Age at onset, sex, underlying disease, status epilepticus (SE), presence of benzodiazepine-resistant SE (BZD-resistant SE), and basic blood serum parameters on admission were evaluated in relation to each patient's outcome. Results On univariate analysis age at onset, BZD-resistant SE, and serum aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase, and platelet count varied significantly according to outcome. On multivariate analysis age at onset (≤21 months), presence of BZD-resistant SE, and AST (≥46 IU/L) were identified as independent variables associated with poor outcome. Conclusion Age at onset, presence of BZD-resistant SE, and AST are associated with a poor prognosis in AE.

In Japan the academic year starts in early April. Standard entrance exams are held each January and February, however some universities offer a limited number of students an opportunity to apply earlier. Depending on the university, provisional acceptance through this alternative is given up to ten months before lectures start. With no university entrance exams to prepare for, it is possible for students to experience a decline in study skills and knowledge during this interim period. To address this, it is common for universities to offer a pre-enrolment course, typically in the form of irregular on-campus events, paper-based correspondence courses or quiz-based online courses with little-to-no interaction with other students. However, the affordances of modern e-learning systems allow for the creation of a more engaging online environment. Students at the authors' university major in design and computer science-related fields. Computer literacy is essential; e-learning is part of their daily routine and they have to complete numerous tasks using computers. Three years ago, the authors designed a pre- enrolment English course with these students in mind. It has been created using the Moodle LMS for an annual cohort of 100 students who come from all over Japan. They generally have low technology skills, low English skills (CEFR A2 to B1), and no e-learning experience. The aims of the course are not only to improve their English, but to introduce them to e-learning, their future classmates, and university life. Analysis of activity logs and end-of-course surveys tells us what students thought of the course, and how they engaged with it, leading to suggestions for change. Generally, it proved to be a very positive experience for motivated students, but less so for those with lower motivation, the main target of the course.

Objective To review laparoscopy in patients with disorders of sex development (DSD) in order to clarify its usefulness in diagnosis, devising subsequent therapeutic strategies and managing patients with various conditions. Patients and methods Between April 1992 and December 2012, 29 laparoscopic surgeries were performed in 25 DSD patients. Among them, ten were diagnostic laparoscopy including gonadal biopsy, and 19 were therapeutic laparoscopy. Surgical procedures and complications were evaluated. Results For diagnostic laparoscopy, laparoscopic gonadal biopsy was performed in three patients. Inspection, with or without open gonadal biopsy, was performed on four out of seven patients with 46XY DSD or mixed gonadal dysgenesis (MGD). Additional surgery was planned and performed based on diagnostic laparoscopic findings in six out of seven patients. In the three patients with ovotesticular DSD, the gonadal pathology was diagnosed as: testis/ovary in one, testis/ovotestis in one and ovary/ovotestis in one – this was from the laparoscopic inspection and/or gonadal biopsy. However, the final diagnoses were bilateral ovotestis in two patients and ovary/ovotestis in one patient. For therapeutic laparoscopy, surgical procedures were: gonadectomy in 17 patients (bilateral in 13, unilateral in three, partial in two); hysterectomy in two patients; orchiopexy in one; and sigmoid vaginoplasty in one patient (included multiple procedures). There were no severe perioperative complications. In the four patients with a history of diagnostic laparoscopy, no severe intra-abdominal adhesions that would disturb therapeutic laparoscopic surgery were observed. Conclusion While diagnostic laparoscopy was helpful in devising a therapeutic surgical strategy in most of the patients with DSD who were suspected as having complex gonadal status or Mullerian duct derivatives, attention must be paid to precisely diagnosing the gonadal status in ovotesticular DSD. On the other hand, therapeutic laparoscopic surgeries were valuable procedures in treating DSD patients, even with a history of previous diagnostic laparoscopy.

Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s--the long-terminal repeats of HERV type-H (HERV-H)--to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H-driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.

Limited numbers of pediatric intratesticular cystic lesions have been reported. Although the majority of pediatric intratesticular cystic masses are benign, natural history of testicular cystic lesion in children has been rarely reported so far. We report a case of intratesticular cystic lesion in a prepubertal child who underwent testis sparing surgery after shrinkage during conservative follow-up. As an initial strategy for intratesticular cystic lesions in prepubertal children, observational approach with serial ultrasonographic evaluations may be a management of choice.

He was born with markedly dilated left megaureter. Since it disturbed his oral intake after birth, left temporary cutaneous ureterostomy was created at 3 days of age. Since his left kidney was dysplastic, laparoscopic left nephroureterectomy was performed using the stoma site of cutaneous ureterostomy as a multi-channel port site at 15 months of age. He had no complications after surgery, and the postoperative wound appearance was good.This is the first report of the stoma site being used as a multi-channel port site in the urology field. This surgical approach provides good cosmetic outcomes. Keywords: Laparoscopy, Stoma site, Multi-channel port, Children

Pluripotency can be induced in somatic cells by forced expression of POU domain, class 5, transcription factor 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), myelocytomatosis oncogene (c-MYC) (OSKM). However, factor-mediated direct reprogramming is generally regarded as an inefficient and stochastic event. Contrary to this notion, we herein demonstrate that most human adult dermal fibroblasts initiated the reprogramming process on receiving the OSKM transgenes. Within 7 d, ∼20% of these transduced cells became positive for the TRA-1-60 antigen, one of the most specific markers of human pluripotent stem cells. However, only a small portion (∼1%) of these nascent reprogrammed cells resulted in colonies of induced pluripotent stem cells after replating. We found that many of the TRA-1-60–positive cells turned back to be negative again during the subsequent culture. Among the factors that have previously been reported to enhance direct reprogramming, LIN28, but not Nanog homeobox (NANOG), Cyclin D1, or p53 shRNA, significantly inhibited the reversion of reprogramming. These data demonstrate that maturation, and not initiation, is the limiting step during the direct reprogramming of human fibroblasts toward pluripotency and that each proreprogramming factor has a different mode of action.

We report a fragment-completion questionnaire and a self-paced reading experiment investigating relative clauses in Japanese that contain two extraction sites in the same clause. The results indicate that in such double-gap relative clauses there is a preference to fill the object position before the subject position. Previous factors, especially those related to working memory resources, which have often been implicated in extraction preferences, do not predict the patterns observed. The results support the proposal that the accessibility of extraction sites in relative clauses is inversely correlated to the order in which semantic roles are preferentially assigned. We discuss ways of incorporating such a constraint in previous proposals such as expectation-based models.

Although mastermind-like domain containing 1 (MAMLD1) (CXORF6) on human chromosome Xq28 has been shown to be a causative gene for 46,XY disorders of sex development with hypospadias, the biological function of MAMLD1/Mamld1 remains to be elucidated. In this study, we first showed gradual and steady increase of testicular Mamld1 mRNA expression levels in wild-type male mice from 12.5 to 18.5 d postcoitum. We then generated Mamld1 knockout (KO) male mice and revealed mildly but significantly reduced testicular mRNA levels (65-80%) of genes exclusively expressed in Leydig cells (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3) as well as grossly normal testicular mRNA levels of genes expressed in other cell types or in Leydig and other cell types. However, no demonstrable abnormality was identified for cytochrome P450 17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B) protein expression levels, appearance of external and internal genitalia, anogenital distance, testis weight, Leydig cell number, intratesticular testosterone and other steroid metabolite concentrations, histological findings, in situ hybridization findings for sonic hedgehog (the key molecule for genital tubercle development), and immunohistochemical findings for anti-Müllerian hormone (Sertoli cell marker), HSD3B (Leydig cell marker), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (germ cell marker) in the KO male mice. Fertility was also normal. These findings imply that Mamld1 deficiency significantly reduces mRNA expression levels of multiple genes expressed in mouse fetal Leydig cells but permits normal genital and reproductive development. The contrastive phenotypic findings between Mamld1 KO male mice and MAMLD1 mutation positive patients would primarily be ascribed to species difference in the fetal sex development.

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo.

Voltage-gated Na

Sialylated glycans play a prominent role in the Drosophila nervous system where they are involved in the regulation of neural transmission. However, the functional pathway of sialylation in invertebrates, including Drosophila, remains largely unknown. Here we used a combination of genetic and behavioral approaches to shed light on the Drosophila sialylation pathway. We examined genetic interactions between Drosophila sialyltransferase (DSiaT) and β1,4-N-acetylgalactosaminyltransferase (β4GalNAcT) genes. Our results indicated that β4GalNAcTA and DSiaT cooperate within the same functional pathway that regulates neural transmission. We found that β4GalNAcTA is epistatic to DSiaT. Our data suggest an intriguing possibility that β4GalNAcTA may participate in the biosynthesis of sialylated glycans.

Assembly of the forespore membrane (FSM) initiates at the spindle pole body (SPB), and the leading edge of the FSM is a critical factor in the proper shaping of the FSM. We report a novel SPB component Spo7. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate FSM formation., Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM.

Reports of pubertal hormonal and gonadal status in patients with hypospadias are scarce. We evaluated the pituitary-gonadal axis and gonadal growth in patients with hypospadias at puberty.We retrospectively reviewed serum luteinizing hormone, follicle-stimulating hormone, testosterone and testicular volume at puberty (age 15 years or greater) in the medical charts of patients with hypospadias treated since 1986 and followed at our institution.Enrolled in this study were 43 patients with a mean age at evaluation of 17.6 years (range 15.1 to 22.8). Of these patients 14 and 29 were treated for mild and severe hypospadias, respectively. Six patients with severe hypospadias underwent bilateral orchiopexy for bilateral undescended testes. All patients were Tanner stage 4 to 5 at evaluation. Of 14 mild hypospadias cases we noted hypergonadotropic hypogonadism, hypogonadotropic hypogonadism, decreased luteinizing hormone and decreased testosterone in 1 each (7% each). Of 23 severe hypospadias cases without bilateral undescended testes we noted hypergonadotropic hypogonadism, partial androgen insensitivity syndrome and decreased testosterone in 2 (9%), 1 (4%) and 1 (4%), respectively. Of 6 patients with severe hypospadias and bilateral undescended testes we noted hypergonadotropic hypogonadism in 1 (17%) and increased luteinizing hormone with normal testosterone in 2 (33%). Testicular volume less than 10 ml with increased follicle-stimulating hormone was identified in 7 of 43 patients, including 1 of 14 (7%) with mild hypospadias, 3 of 23 (13%) with severe hypospadias without bilateral undescended testes and 3 of 6 (50%) with severe hypospadias and bilateral undescended testes.Our study revealed endocrine dysfunction in patients with mild and severe hypospadias at puberty even without an undescended testis. Of these patients those with severe hypospadias and an undescended testis may be at higher risk for impaired spermatogenesis.

Neurons within the periaqueductal gray (PAG) have been implicated in the central regulation of pain signals by affecting the descending inhibitory pathway. Here we report on the functional role of presynaptic kainate receptors within the PAG. Using a conventional whole-cell patch clamp technique, we recorded GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) from mechanically isolated rat PAG neurons in the presence of 300 nM tetrodotoxin and 20 µM dl -2-amino-5-phosphonovaleric acid under voltage-clamp conditions. Kainic acid at a 10 µM concentration significantly increased the frequency of GABAergic mIPSCs without affecting their amplitude, suggesting that kainic acid acts presynaptically to enhance spontaneous GABA release. The kainic acid-induced increase in mIPSC frequency was completely blocked by CNQX, a selective AMPA/kainate receptor antagonist. While neither AMPA nor NMDA affected GABAergic mIPSC frequency, ATPA, a selective agonist of GluR5-containing kainate receptors, increased GABAergic mIPSC frequency in a concentration-dependent manner. The kainic acid-induced increase in mIPSC frequency was completely suppressed either in the presence of 100 µM Cd 2+ , a general voltage-dependent Ca 2+ channel (VDCC) blocker, or in the Na + -free external solution. These results suggest that presynaptic kainate receptors have a low permeability to Ca 2+ , and that their activation elicits a presynaptic depolarization large enough to activate presynaptic VDCCs. Presynaptic kainate receptors on GABAergic nerve terminals appear to modulate GABAergic transmission, and in doing so may play an important role in the regulation of PAG neuron excitability.

In vertebrates, sialylated glycans participate in a wide range of biological processes and affect the development and function of the nervous system. While the complexity of glycosylation and the functional redundancy among sialyltransferases provide obstacles for revealing biological roles of sialylation in mammals,Drosophilapossesses a sole vertebrate-type sialyltransferase,Drosophilasialyltransferase (DSiaT), with significant homology to its mammalian counterparts, suggesting thatDrosophilacould be a suitable model to investigate the function of sialylation. To explore this possibility and investigate the role of sialylation inDrosophila, we inactivated DSiaTin vivoby gene targeting and analyzed phenotypes ofDSiaTmutants using a combination of behavioral, immunolabeling, electrophysiological, and pharmacological approaches. Our experiments demonstrated that DSiaT expression is restricted to a subset of CNS neurons throughout development. We found thatDSiaTmutations result in significantly decreased life span, locomotor abnormalities, temperature-sensitive paralysis, and defects of neuromuscular junctions. Our results indicate that DSiaT regulates neuronal excitability and affects the function of a voltage-gated sodium channel. Finally, we showed that sialyltransferase activity is required for DSiaT functionin vivo, which suggests thatDSiaTmutant phenotypes result from a defect in sialylation of N-glycans. This work provided the first evidence that sialylation has an important biological function in protostomes, while also revealing a novel, nervous system-specific function of α2,6-sialylation. Thus, our data shed light on one of the most ancient functions of sialic acids in metazoan organisms and suggest a possibility that this function is evolutionarily conserved between flies and mammals.

In this study, we have examined the possible roles of a glycine transporter type 2 (GlyT-2) in the forskolin-induced increase of the amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in acutely isolated rat substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, increased the amplitude of glycinergic mIPSCs in the presence, but not in the absence, of a low concentration of extracellular glycine. This effect disappeared by the addition of ALX1393 (a GlyT-2 antagonist). These results suggest that both extracellular glycine and GlyT-2 are essential for the forskolin-induced increase in the amplitude of glycinergic mIPSCs. These mechanisms might contribute, at least in part, to the maturation of inhibitory synaptic transmission, including the developmental neurotransmitter switch from GABA to glycine within the spinal dorsal horn during postnatal development.

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na+ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na+ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na+ currents, it strongly inhibited steady-state currents of transient as well as persistent Na+ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na+ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na+ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na+ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

We report the long-term outcome of vaginoplasty with the bilateral labioscrotal flap with special emphasis on vaginal stenosis.Of 23 children with ambiguous genitalia and low vaginal entry who underwent vaginoplasty between January 1985 and July 2003 with the bilateral labioscrotal flap 13 followed more than 5 years after surgery who were 10 years old or older at the most recent evaluation were included in this long-term outcome study. Vaginal caliber was estimated according to a previously described assessment system adopted for vaginoplasty results.The underlying disease was congenital adrenal hyperplasia in 11 cases, mixed gonadal dysgenesis in 1 and ovotesticular sexual development disorder in 1. Mean age at vaginoplasty and at the most recent evaluation was 3.8 (range 2.0 to 12.9) and 14.6 years (range 10.9 to 21.5), respectively. Vaginal caliber at the most recent evaluation was adequate in 6 patients (46%), stenotic in 5 (39%) and strictured in 2 (15%). Three of the 7 patients diagnosed with stricture or stenosis were diagnosed at age less than 12 years. One of these patients diagnosed with stricture was treated with dilation and the other 2 patients were observed. These patients had no trouble with menstruation. Four patients diagnosed with stricture or stenosis at age 14 years or older were treated surgically with dilation in 1 and perineal flap vaginoplasty in 3. They showed adequate vaginal caliber at 3 to 31 months of followup. In 7 patients evaluated at the beginning of puberty and several years later vaginal caliber had enlarged in 5 but remained unchanged in 2.To our knowledge this is the first report of the long-term outcome of vaginoplasty with the bilateral labioscrotal flap. Although vaginal stenosis/stricture was observed at puberty in about half of the patients, severe stricture was uncommon. Serial evaluation for vaginal stenosis/stricture at the beginning of puberty for menstruation and several years later for vaginal intercourse is recommended in patients treated with vaginal reconstruction.

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca(2+) concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.

Although it has been well established that GABA(A) receptors are molecular targets of a variety of allosteric modulators, such as benzodiazepines, the pharmacological properties of presynaptic GABA(A) receptors are poorly understood. In this study, the effects of diazepam and Zn(2+) on presynaptic GABA(A) receptors have been investigated by measuring the GABA(A) receptor-mediated facilitation of spontaneous glutamate release in mechanically dissociated rat CA3 pyramidal neurons. Diazepam significantly enhanced the muscimol-induced facilitation (particularly at submicromolar concentrations) of spontaneous glutamate release and shifted the concentration-response relationship for muscimol toward the left, whereas Zn(2+) (OR= 100 muM) had little effect on the muscimol-induced facilitation of spontaneous glutamate release. In contrast, Zn(2+) significantly suppressed the muscimol-induced currents mediated by GABA(A) receptors expressed on dentate gyrus granule cells, which are parent neurons of mossy fibers, whereas the effect of diazepam on GABA(A) receptors expressed on dentate gyrus granule cells was lesser than that on presynaptic GABA(A) receptors. The results suggest that the pharmacological properties of GABA(A) receptors differ considerably between presynaptic (axon terminals) and somatic regions in the same granule cell and that presynaptic GABA(A) receptors should be considered as one of the important pharmacological targets of many drugs affecting GABA(A) receptors.

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl − currents in a concentration-dependent manner. The glycine-induced current ( I Gly ) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01–1 µM), ICS-205,930 potentiated I Gly , but at higher concentrations (> 10 µM) it inhibited I Gly . These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing α2 subunits. Furthermore, at lower concentrations (1–30 µM), Zn 2+ potentiated I Gly , but at higher concentrations (> 100 µM) it inhibited I Gly . Considering that Zn 2+ is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn 2+ modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.

α-Lipoic acid (ALA) is an essential cofactor in mitochondrial multi-enzyme complexes related to energy production. However, it is unstable under light or heat, and its decomposition is accompanied by an unpleasant odor. Therefore, its stabilization by complex formation with the cationic polymer chitosan (CS) was investigated. The ALA dissolved in demineralized water was efficiently adsorbed on the precipitated insoluble CS particles, and an ALA–CS complex was obtained. The amount of ALA adsorbed on CS was affected by the CS species and the quantity ratio of ALA to CS. The ALA from the ALA–CS complex was released immediately by changing the pH. When ALA was incubated at 65 °C, it melted and polymerized. In addition, some decomposition of ALA was also observed in the physical mixture of ALA with CS. However, the ALA–CS complex did not decompose at all under the same conditions. Thus, the stabilization of ALA was achieved by complex formation with CS. CS is useful as a material for the stabilization of ALA, leading to its clinical use.

We present an emergentist alternative to a classic phenomenon from the literature on Universal Grammar – the contrast in the acceptability of contraction in sentences such Who do you want to/wanna see? and Who do you want to/*wanna go? Earlier experimental work suggests that although this contrast is quite robust for native speakers, including children, it is frequently not respected by adult second language learners. We present the results of a psycholinguistic experiment to support the idea that the deficit manifested by second language learners is due to their propensity to resolve wh dependencies at the matrix verb, regardless of its properties, thereby suppressing the potential for a contrast between the two want patterns.

The musculature of the hip and thigh in the orangutan has been described previously. Anatomically, there are various descriptions among primates in those structures, in particular, the relationship between M. biceps femoris and M. gluteus maximus, their derivatives, and the muscle segment. However, a detailed innervation system to this ischiofemoral part has not been described, thus there is still uncertainty as to with which muscle it is associated. In this analysis, we examined the gross anatomy of the hip and thigh muscles of the orangutan and chimpanzee, including their innervation. Also, a comparison was made with documented data of other primates. As a result of these observations, it was found that the ischiofemoral part in the orangutan is innervated by the same sciatic nerve branch (the common peroneal nerve) as the long head of M. biceps femoris, but not by the same nerve as M. gluteus maximus. Therefore, the ischiofemoral part is appropriately considered as a part of the long head of M. biceps femoris. It appears that this morphologic feature is an adjustment to the arboreal life of the orangutan. The development of the flexor complex of the thigh is necessary for this arboreal adaptation, resulting in a unique musculature of M. biceps femoris in the orangutan.

Non-steroidal anti-inflammatory drugs (NSAIDs) are well-known inhibitors of cyclooxygenases (COXs) and are widely used for the treatment of inflammatory pain; however several NSAIDs display COX-independent analgesic action including the inhibition of voltage-gated Na(+) channels expressed in primary afferent neurons. In the present study, we examined whether NSAIDs modulate tetrodotoxin-resistant (TTX-R) Na(+) channels and if this modulation depends on the extracellular pH. The TTX-R Na(+) currents were recorded from small-sized trigeminal ganglion neurons by using a whole-cell patch clamp technique. Among eight NSAIDs tested in this study, several drugs, including aspirin and ibuprofen, did not affect TTX-R Na(+) channels either at pH 7.4 or at pH 6.0. However, we found that indomethacin, and, to a lesser extent, ibuprofen and naproxen potently inhibited the peak amplitude of TTX-R Na(+) currents at pH 6.0. The indomethacin-induced inhibition of TTX-R Na(+) channels was more potent at depolarized membrane potentials. Indomethacin significantly shifted both the voltage-activation and voltage-inactivation relationships to depolarizing potentials at pH 6.0. Indomethacin accelerated the development of inactivation and retarded the recovery from inactivation of TTX-R Na(+) channels at pH 6.0. Given that indomethacin and several other NSAIDs could further suppress local nociceptive signals by inhibiting TTX-R Na(+) channels at an acidic pH in addition to the classical COX inhibition, these drugs could be particularly useful for the treatment of inflammatory pain.