CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms.

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

Lithium ion adduction enables UPLC-MS/MS-based analysis of multi-class 3-hydroxyl group-containing keto-steroids.

Steroids that contain a 3-hydroxyl group (3-OH steroids) are widely distributed in nature. During analysis with ESI-MS, they easily become dehydrated while in the protonated form, resulting in the production of several precursor ions and leading to low sensitivity of detection. To address this analytical challenge, here, we developed a method for the quantitation of 3-OH steroids by LC-MS/MS coupled with post-column addition of lithium (Li) ions to the mobile phase. The Li ion has a high affinity for the keto group of steroids, stabilizing their structures during ionization and permitting detection of analytes exclusively as the lithiated form. This not only improved the intensities of the precursor ions, but also promoted the formation of typical lithiated fragment ions. This improvement made the quantitation by multiple reaction monitoring more sensitive and reliable, as evidenced by 1.53-188 times enhanced detection sensitivity of 13 steroids that contained at least one keto and two hydroxyl groups or one keto and one 5-olefinic double bond, among 16 different 3-OH steroids. We deployed our newly developed method for profiling steroids in mouse brain tissue and identified six steroids in one tissue sample. Among these, 16-hydroxyestrone, tetrahydrocorticosterone, and 17α-hydroxypregnenolone were detected for the first time in the mouse brain. In summary, the method described here enables the detection of lithiated steroids by LC-MS/MS, including three 3-OH steroids not previously reported in the mouse brain. We anticipate that this new method may allow the determination of 3-OH steroids in different brain regions.

Correction to: The Japanese version of the questionnaire about the process of recovery: development and validity and reliability testing.

After publication of our article [1] we were notified that the 5-point Likert-type scale in Additional file 1 needs to change from "1 to 5" to "0 to 4". The updated Additional file 1 is included in this correction.

The usefulness of the retinal sensitivity measurement with a microperimetry for predicting the visual prognosis of branch retinal vein occlusion with macular edema.

PURPOSE: To evaluate the usefulness of the retinal sensitivity in branch retinal vein occlusion (BVO) with macular edema (ME) following the anti-vascular endothelial growth factor (anti-VEGF) treatment. METHODS: Best-corrected visual acuity (BCVA), microperimetry, and optical coherence tomography (OCT) measurements were carried out in 20 patients with BVO with ME, at baseline and 1 month after the anti-VEGF treatment. The relationships among BCVA, mean retinal sensitivity (MS), macular volume (MV), central retinal thickness (CRT), integrity of ellipsoid zone (EZ), mean retinal sensitivity in the most affected quadrant (qMS), and macular volume in the most affected quadrant (qMV) were investigated. In addition, the relationships among the change in BCVA at 1 month (ΔBCVA1m), mean sensitivity in the most affected quadrant at 1 month (ΔqMS1m), MV in the most affected quadrant at 1 month (ΔqMV1m), and CRT at 1 month (ΔCRT1m) were analyzed. The optimal model for BCVA at 3 months after the treatment (BCVA3m) was identified. RESULTS: There was not a significant difference in BCVA (paired Wilcoxon test, p = 0.058) between at baseline and after the treatment, but there were significant differences in MS, MV, CRT, qMS, and qMV (p < 0.05). There was a significant relationship between ΔqMS1m and ΔMV1m, ΔCRT1m, and ΔqMV1m, respectively. ΔMS1m or ΔqMS1m and BCVA at baseline and ΔBCVA1m were selected as explanatory variables in the optimal model for BCVA3m. CONCLUSION: Retinal sensitivity was related to retinal structure, whereas this was not the case with BCVA. In addition, retinal sensitivity was useful to predict BCVA after anti-VEGF therapy.

Early changes of vascular lesions and responses to combined photodynamic therapy in patients with polypoidal choroidal vasculopathy.

PURPOSE: To evaluate early changes of vascular lesions and their associations with the early responses to combined photodynamic therapy (PDT) in eyes with polypoidal choroidal vasculopathy (PCV). METHODS: This study evaluated 19 eyes of 19 patients with PCV who underwent PDT combined with anti-vascular endothelial growth factor injections and were followed for 3 months. All subjects were examined 1 week and 1, 2, and 3 months after combined PDT. "Active" cases were defined as recurrence or persistence of serous retinal detachment or subretinal hemorrhage detected within 3 months. Branching vascular networks (BVNs) were evaluated by optical coherence tomography angiography (OCTA) and polyps by indocyanine-green angiography. RESULTS: In total, 16%, 58%, 84%, and 89% of eyes displayed BVNs 1 week, 1, 2, and 3 months after PDT, respectively. BVNs were detected significantly more often 1 month after PDT in the "active" group than "inactive" group (89% vs. 30%, p = 0.020). There were significantly higher overall proportions of BVNs detected by OCTA in the "active" group than "inactive" group (p = 0.0058). CONCLUSION: In most cases, BVNs disappeared once and gradually appeared again within 3 months. Detecting BVNs using OCTA from early phases could be a helpful biomarker to assess the early responses to PDT in eyes with PCV.

The Japanese version of the questionnaire about the process of recovery: development and validity and reliability testing.

BACKGROUND: Personal recovery is increasingly recognised as an important outcome measure in mental health services. This study aimed to develop a Japanese version of the Questionnaire about the Process of Recovery (QPR-J) and test its validity and reliability. METHODS: The study comprised two stages that employed the cross-sectional and prospective cohort designs, respectively. We translated the questionnaire using a standard translation/back-translation method. Convergent validity was examined by calculating Pearson's correlation coefficients with scores on the Recovery Assessment Scale (RAS) and the Short-Form-8 Health Survey (SF-8). An exploratory factor analysis (EFA) was conducted to examine factorial validity. We used intraclass correlation and Cronbach's alpha to examine the test-retest and internal consistency reliability of the QPR-J's 22-item full scale, 17-item intrapersonal and 5-item interpersonal subscales. We conducted an EFA along with a confirmatory factor analysis (CFA). RESULTS: Data were obtained from 197 users of mental health services (mean age: 42.0 years; 61.9% female; 49.2% diagnosed with schizophrenia). The QPR-J showed adequate convergent validity, exhibiting significant, positive correlations with the RAS and SF-8 scores. The QPR-J's full version, subscales, showed excellent test-retest and internal consistency reliability, with the exception of acceptable but relatively low internal consistency reliability for the interpersonal subscale. Based on the results of the CFA and EFA, we adopted the factor structure extracted from the original 2-factor model based on the present CFA. CONCLUSION: The QPR-J is an adequately valid and reliable measure of the process of recovery among Japanese users with mental health services.

Roles of AML1/RUNX1 in T-cell malignancy induced by loss of p53.

AML1/RUNX1 is a frequent target of chromosome translocations and mutations in myeloid and B-cell leukemias, and upregulation of AML1 is also observed in some cases of T-cell leukemias and lymphomas. This study shows that the incidence of thymic lymphoma in p53-null mice is less frequent in the Aml1(+/-) than in the Aml1(+/+) background. AML1 is upregulated in p53-null mouse bone-marrow cells and embryonic fibroblasts. In the steady state, p53 binds to and inhibits the distal AML1 promoter. When the cells are exposed to stresses, p53 is released from the distal AML1 promoter, resulting in upregulation of AML1. Overexpression of AML1 stimulates T-lymphocyte proliferation. These results suggest that upregulation of AML1 induced by loss of p53 promotes lymphoid-cell proliferation, thereby inducing lymphoma development.

Dual role of mosapride citrate hydrate on the gastric emptying evaluated by the breath test in conscious rats.

Mosapride citrate hydrate (mosapride) has been known to act as a 5-HT4 agonist and to enhance gastric emptying. However, its mode of action, such as time course and dosage effect, on gastric emptying has not been clarified. This study aimed to clarify these points by the breath test using [1-(13)C]acetic acid in conscious rats. Mosapride significantly and dose-dependently enhanced the gastric emptying increased Cmax and AUC120 min at doses between 0.1 and 3 mg/kg. Pre-treatment with GR113808 (5-HT4 antagonist) significantly attenuated the enhancement of gastric emptying by mosapride. On the contrary, at a dose of 30 mg/kg, mosapride significantly inhibited the gastric emptying. The major metabolite (M1: 5-HT3 receptor antagonist) significantly inhibited gastric emptying at doses of 19.2 and 64.1 mg/kg (equimolar to 30 and 100 mg/kg of mosapride, respectively), suggesting that the inhibitory effect by mosapride may be caused at least in part by the 5-HT3 receptor antagonistic effect of M1. These findings show that mosapride has dual role on the gastric emptying and may support the usefulness of mosapride for the therapy of postprandial distress syndrome such as early satiation and postprandial fullness.

[About mental health outreach services in Japan].

Outreach services are very important in community mental health care. There are two types for outreach services. One is mental health activities, such as early intervention and consultation, and the other is intended to prevent recurrence and readmission by supporting the daily living activities of a patient in a community. We have 2.73 psychiatric care beds in hospitals per 1,000 population. So, it is just the beginning in changing from hospital centered psychiatry to community mental health care. Outreach services are being tried in several places in our country. In this essay, we describe mental health outreach services in Japan and we have illustrated vocational rehabilitation and outreach job support in our day treatment program.

The circadian clock in the piriform cortex intrinsically tunes daily changes of odor-evoked neural activity.

The daily activity in the brain is typically fine-tuned by the circadian clock in the local neurons as well as by the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. In the olfactory response, odor-evoked activity in the piriform cortex (PC) and olfactory behavior retain circadian rhythmicity in the absence of the SCN, yet how the circadian rhythm in the PC is achieved independently of the SCN remains elusive. Here, to define neurons regulating the circadian rhythm of the odor-evoked activity in the PC, we knocked out the clock gene Bmal1 in a host of specific neurons along the olfactory circuit. We discovered that Bmal1 knockout in the PC largely abolishes the circadian rhythm of the odor-evoked activity. We further showed that isolated PC exhibits sustained circadian rhythms of the clock gene Per2 expression. Quantitative PCR analysis revealed that expression patterns of multiple genes involved in neural activity and synaptic transmission exhibit circadian rhythm in the PC in a BMAL1-dependent manner. Our findings indicate that BMAL1 acts intrinsically in the PC to control the circadian rhythm of the odor-evoked activity in the PC, possibly through regulating expression patterns of multiple genes involved in neural activity and transmission.

Diurnal variation in declarative memory and the involvement of SCOP in cognitive functions in nonhuman primates.

Cognitive functions depend on the time of day in various organisms. Previously, we found that 24-h recognition memory performance of nocturnal mice changes diurnally through SCOP protein-dependent regulation. It remains unknown whether diurnal change and SCOP-dependent regulation of memory performance are conserved across species with diurnal/nocturnal habits. We tested whether the memory performance of diurnal Japanese macaques depends on the time of day. The memory association between bitter taste of drinking water and the nozzle color of the water bottle was established during the light period of the day to evaluate of memory performance for macaques. Here we found diurnal variation of declarative memory in Japanese macaques. The middle of the daytime is the most effective time for memory performance during the light period. To assess whether SCOP is involved in declarative memory performance, we interfered with SCOP expression by using lentiviral vector expressing shRNA against Scop in the hippocampus of Japanese macaques. Scop knockdown in the hippocampus abrogated the memory performance in the middle of the daytime. Our results implicate that SCOP in the hippocampus is necessary for the diurnal rhythm of the memory system and that the SCOP-dependent memory regulation system may be conserved in mammals.

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons.

Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.

Stress-activated protein kinase MKK7 regulates axon elongation in the developing cerebral cortex.

The c-Jun NH(2)-terminal protein kinase (JNK), which belongs to the mitogen-activated protein kinase family, plays important roles in a broad range of physiological processes. JNK is controlled by two upstream regulators, mitogen-activated protein kinase kinase (MKK) 7 and MKK4. To elucidate the physiological functions of MKK7, we used Nestin-Cre to generate a novel mouse model in which the mkk7 gene was specifically deleted in the nervous system (Mkk7(flox/flox) Nestin-Cre mice). These mice were indistinguishable from their control littermates in gross appearance during embryogenesis but died immediately after birth without breathing. Histological examination showed that the mutants had severe defects in brain development, including enlarged ventricles, reduced striatum, and minimal axon tracts. Electron microscopy revealed abnormal accumulations of filamentous structures and autophagic vacuoles in Mkk7(flox/flox) Nestin-Cre brain. Further analysis showed that MKK7 deletion decreased numbers of TAG-1-expressing axons and delayed neuronal migration in the cerebrum. Neuronal differentiation was not altered. In utero electroporation studies showed that contralateral projection of axons by layer 2/3 neurons was impaired in the absence of MKK7. Moreover, MKK7 regulated axon elongation in a cell-autonomous manner in vivo, a finding confirmed in vitro. Finally, phosphorylation levels of JNK substrates, including c-Jun, neurofilament heavy chain, microtubule-associated protein 1B, and doublecortin, were reduced in Mkk7(flox/flox) Nestin-Cre brain. Our findings demonstrate that the phenotype of Mkk7(flox/flox) Nestin-Cre mice differs substantially from that of Mkk4(flox/flox) Nestin-Cre mice, and establish that MKK7-mediated regulation of JNK is uniquely critical for both axon elongation and radial migration in the developing brain.

microRNA modulation of circadian-clock period and entrainment.

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY STUDY OF CHOROIDAL NEOVASCULARIZATION ASSOCIATED WITH EARLY-ONSET DRUSEN.

PURPOSE: To report three middle-aged cases with choroidal neovascularization (CNV) associated with early-onset drusen documented with optical coherence tomography angiography (OCTA). METHODS: Three patients with bilateral early-onset drusen were referred to our hospital. Fundus examination, fluorescein angiography, indocyanine green angiography, OCTA, and other multimodal imaging were performed. RESULTS: Case 1 involved a 47-year-old woman who presented with sudden unilateral anorthopia. She had no previous systemic pathologies. Funduscopic examination and fluorescein angiography revealed bilateral large colloid drusen accompanied by unilateral mild subretinal hemorrhage. Indocyanine green angiography revealed CNV, although it was unclear in fluorescein angiography. Optical coherence tomography angiography also showed interconnecting CNV beneath the retinal pigment epithelium. Case 2 involved a 40-year-old woman with membranoproliferative glomerulonephritis Type 3 who presented with unilateral anorthopia. On fluorescein angiography, cuticular drusen secondary to membranoproliferative glomerulonephritis were seen in both eyes. An interconnecting vascular network was revealed with OCTA and indocyanine green angiography indicating Type 1 CNV in the affected eye. Case 3 involved a 47-year-old man without any medical or family history. Predominant large colloid drusen associated with cuticular drusen were seen in both eyes. Unilateral mild serosanguinous changes were accompanied in the macula, where Type 1 CNV was detected with OCTA. CONCLUSION: All our cases with early-onset drusen showed Type 1 CNV that was detected by indocyanine green angiography or OCTA. Optical coherence tomography angiography has a potential to help noninvasively diagnose CNV in the cases of EOD.

Comparison of 12-month surgical outcomes of ab interno trabeculotomy with phacoemulsification between spatula-shaped and dual-blade microhooks.

PURPOSE: To compare 12-month clinical results of spatula-shaped and dual-blade microhooks ab interno trabeculotomy with phacoemulsification. STUDY DESIGN: Retrospective comparative study. METHODS: We conducted a retrospective chart review of Japanese open-angle glaucoma patients who underwent ab interno trabeculotomy with phacoemulsification with a 12-month follow-up. Two types of trabecular hook were used: the spatula-shaped Tanito Trabeculotomy ab interno Micro-hook® and the Kahook Dual Blade®. Changes in intraocular pressure (IOP) and medication scores comprised the main outcome metrics. We also analyzed and compared patient demographics and the occurrence of complications. RESULTS: Trabeculotomy was performed using a spatula-shaped hook in 17 eyes and a dual-blade hook in 15 eyes. Significant reductions in IOP (p < 0.001) and medication scores (p < 0.001) were noted in both groups after the 1-month time point. The percentage changes of IOP from baseline at each time point were not significantly different between groups, though there was a significant difference in medication scores at 12 months (p = 0.0192). Postoperative complications occurred similarly in both groups; one case in the dual-blade group required additional filtration surgery. CONCLUSIONS: Ab interno trabeculotomy with phacoemulsification was effective in lowering IOP both with spatula-shaped and with double-blade microhooks. At 12 months more medications were used postoperatively in the spatula-shaped microhook group; however, the reductions in the medication scores from baseline were statistically significant in both groups.

Twelve-month surgical outcome and prognostic factors of stand-alone ab interno trabeculotomy in Japanese patients with open-angle glaucoma.

The purpose of the study was to evaluate the 12-month surgical outcome and prognostic factors of stand-alone ab interno trabeculotomy. The changes in the intraocular pressure (IOP) and medication score and the success rate of the surgery were analyzed. Thirty-four eyes of 29 patients with primary open-angle glaucoma (POAG; n = 16) or pseudoexfoliation glaucoma (PEG; n = 18) with a 12-month follow-up period were included in the study. The decreases in IOP and medication score from the baseline to the all-time-point were statistically significant (P < 0.001). The surgical success rates were 97.1%, 76.5%, and 44.0% at 3 months (90 days), 6 months (180 days), and 12 months (365 days), respectively. A mixed effect Cox model revealed that the type of glaucoma (POAG) was significantly associated with surgical failure (P = 0.044). Furthermore, the surgical success rate was significantly higher in eyes with PEG than it was in those with POAG (P = 0.019). Stand-alone ab interno trabeculotomy significantly lowered both the IOP and the medication score in patients with glaucoma, although almost one quarter of the cases needed additional glaucoma surgeries. The surgical success rate was significantly higher in eyes with PEG than it was in those with POAG.

Yogurt containing Lactobacillus gasseri OLL 2716 (LG21 yogurt) accelerated the healing of acetic acid-induced gastric ulcer in rats.

We have reported that LG21 yogurt containing Lactobacillus gasseri OLL 2716 (LG21 yogurt) inhibits the formation of HCl-induced acute gastric lesions through the generation of prostaglandin E2. This study aimed to determine the role of viable Lactobacillus in the healing of acetic acid-induced chronic gastric ulcer. LG21 yogurt or γ-ray radiated LG21 yogurt was administered orally twice a day for 10 d at a dose of 5 ml/kg. LG21 yogurt significantly accelerated the healing of the ulcer, but γ-ray radiated LG21 yogurt did not. However, both yogurts significantly inhibited HCl-induced gastric erosive lesions and enhanced the generation of gastric mucosal prostaglandin E2. From the above results, it was found that viable bacteria are needed to accelerate the healing of chronic gastric ulcer, but not to inhibit gastric lesions.

Hippocampal 7α-Hydroxylated Neurosteroids Are Raised by Training and Bolster Remote Spatial Memory with Increase of the Spine Densities.

Neuroactive steroids, termed neurosteroids, are synthesized locally in the brain and influence biological functions including cognition and behavior. These neurosteroids are synthesized from cholesterol by a series of cytochrome P450 enzymes, among which a member of P450 hydroxylase, cytochrome P450-7b1 (CYP7B1), catalyzes the formation of 7α-hydroxylated neurosteroids, 7α-hydroxypregnenolone (7α-OH-Preg) and 7α-hydroxydehydroepiandrosterone (7α-OH-DHEA). Here we demonstrated the occurrence of these neurosteroids in the mouse hippocampus after spatial-learning tasks. Cyp7b1 deficiency impaired remote spatial memory with recent memory mostly unaffected. The hippocampal dendritic spine densities were reduced in Cyp7b1-deficient mice, and they were no more increased by the training. Furthermore, chronic intracerebroventricular administration of a mixture of 7α-OH-Preg and 7α-OH-DHEA rescued the deteriorated remote memory performance in Cyp7b1-deficient mice. It is concluded that the 7α-hydroxylated neurosteroids are required for long-term maintenance of spatial memory, and we suggest that these neurosteroids may induce synaptic remodeling to maintain the hippocampal function.

Bleb Plication for a Leaking Bleb After Trabeculectomy.

PURPOSE: The purpose of this work was to report a new method for the repair of ischemic bleb leaks. Bleb leakage is a serious complication of glaucoma filtering surgery with mitomycin C. Many surgical methods have been proposed to seal the leakage from a bleb; however, this novel plication method is effective, relatively easy to perform, and safe. METHODS: We describe the case reports of 2 patients with leakage from a bleb who were treated with the new method. The conjunctiva was lifted away from the sclera as extensively as possible around the ischemic conjunctiva toward the fornix using a bleb knife. Thereafter, 10-0 nylon sutures were applied between the nonischemic conjunctiva located just outside the ischemic conjunctiva and the corneal limbus. The ischemic conjunctiva was not removed, but covered with the nonischemic conjunctiva that was advanced toward the corneal limbus by these sutures. RESULTS: After treatment, no recurrence of bleb leakage was observed. Moreover, no ischemic changes were observed in the advanced nonischemic conjunctiva or plicated conjunctiva. CONCLUSION: This new method of bleb plication was effective for sealing bleb leakage.