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Abstract Background Suicide rates in Japan have increased during the COVID-19 pandemic, and foreign residents may be more vulnerable to mental stress during such crises. Therefore, we aimed to compare the trends in suicide rates during the COVID-19 pandemic between foreign residents and Japanese citizens. Methods Vital statistics of Japan data from January 1, 2016 to December 31, 2021 were used to calculate quarterly sex-specific suicide rates for foreign residents and Japanese citizens. An event-study analysis was conducted to evaluate whether suicide rates during the COVID-19 pandemic increased compared to pre-pandemic estimates; foreign residents and Japanese citizens were compared using difference-in-difference-in-differences estimates. Results Between 2016 and 2021, 1,431 foreign residents and 121,610 Japanese citizens died from suicide in Japan. Although the suicide rate for foreign residents was lower than that for Japanese citizens, Korean residents, who comprise approximately half of the foreign decedents, had largely higher suicide rates than Japanese citizens. The event-study analysis indicated that suicide rates increased among foreign residents for both men and women, and continued for men by the end of 2021. In Japanese citizens, after a decline in suicide rates in the second quarter of 2020, suicide rates increased both among men and women, and lasted for women until the fourth quarter of 2021. The difference-in-difference-in-differences analyses confirmed the initial decline in the second quarter of 2020 in suicide rate only in Japanese men and women, and the persistent increase through 2021 in foreign men. Conclusions We found differential trends in suicide rates between foreign and Japanese men and women during the COVID-19 pandemic featuring a persistent increase in foreign men. Suicide prevention measures should be focused on these high-risk subpopulations.

Abstract The stratum corneum is the outermost skin layer with a vital role in skin barrier function. It is comprised of dead keratinocytes (corneocytes) and is known to maintain its thickness by shedding cells, although, the precise mechanisms that safeguard stratum corneum maturation and homeostasis remain unclear. Previous ex vivo studies have suggested a neutral-to-acidic pH gradient in the stratum corneum. Here, we use intravital pH imaging at single-corneocyte resolution to demonstrate that corneocytes actually undergo differentiation to develop three distinct zones in the stratum corneum, each with a distinct pH value. We identified a moderately acidic lower, an acidic middle, and a pH-neutral upper layer in the stratum corneum, with tight junctions playing a key role in their development. The upper pH neutral zone can adjust its pH according to the external environment and has a neutral pH under steady-state conditions owing to the influence of skin microbiota. The middle acidic pH zone provides a defensive barrier against pathogens. With mathematical modeling, we demonstrate the controlled protease activation of kallikrein-related peptidases on the stratum corneum surface that results in proper corneocyte shedding in desquamation. This work adds crucial information to our understanding of how stratum corneum homeostasis is maintained.

Abstract StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.

Objectives In April 2012, the Japanese government launched a new nursing service called the nursing small-scale multifunctional home care (NSMHC) to meet the nursing care demands of individuals with moderate-to-severe activities of daily living (ADLs) dysfunction and who require medical care, thereby allowing them to continue living in the community. We aimed to preliminarily analyse the characteristics of first-time users of NSMHC service.Design This pooled cross-sectional study used the Japanese long-term care insurance (LTCI) claims data from the users’ first use of NSMHC (from April 2012 to December 2019).Setting NSMHC includes nursing home visits, home care, daycare, overnight stays and medical treatment.Participants The study population included LTCI beneficiaries who received their first long-term care requirement certification in Japan from April 2012 onwards, died between April 2012 and December 2019, and used any LTCI service at least once.Results Among the 836 563 individuals who used any LTCI service at least once, 3957 (0.47%) used NSMHC. We analysed 3634 individuals without any missing data regarding long-term care requirement certification. Most individuals were aged 80 years or older, with 64.3% requiring care level 3 or above, indicating complete assistance with ADLs. Regarding ADLs in individuals with dementia, 70.6% were at level 2 or below, indicating they can live almost independently even with dementia. A large proportion of NSMHC users availed the service approximately 6 months before death, with no prior use of any LTCI services; they continued using the service for around 4 months, although some people continued to use NSMHC until their month of death.Conclusions Using individual data on nationwide LTCI, we described the characteristics of first-time users of NSMHC among those who died within 7.5 years from the first certification of care needs. Further studies are needed to investigate the effect of NSMHC use on user outcomes.

Abstract Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell–cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Abstract Phototherapy converts lipophilic unconjugated bilirubin to hydrophilic bilirubin photoisomers, such as lumirubin. We comparatively used a blue light-emitting diode (LED) and a green fluorescent lamp (FL) as light sources for phototherapy of hyperbilirubinemic preterm neonates with the aim of examining potential differences in urinary lumirubin excretion between these two wavelengths. Urinary lumirubin levels were measured using a fluorescence assay with blue light exposure in the presence of the unconjugated bilirubin-inducible fluorescent protein UnaG, and denoted as urinary UnaG-bound bilirubin (UUB)/creatinine (Cr) (μg/mg Cr). Preterm neonates born at ≤ 33 weeks gestational age and treated with phototherapy were subjected to this study. The maximum UUB/Cr level during phototherapy per device intensity was compared between neonates treated with the blue LED and the green FL. A total of 61 neonates were examined to determine the maximum UUB/Cr levels. The median of maximum UUB/Cr excretion per light intensity of each device (μg/mg Cr/μW/cm2/nm) was 0.83 for the blue LED and 1.29 for the green FL (p = 0.01). Green light was found to be more effective than blue one for bilirubin excretion via urinary lumirubin excretion. This is the first spectroscopic study to compare the efficacy of phototherapy at different wavelengths using fluorescence assay.

Abstract Reverse pH-dependent fluorescent protein, including dKeima, is a type of fluorescent protein in which the chromophore protonation state depends inversely on external pH. The dependence is maintained even when immobilized at the metal-solution interface. But, interestingly, its responses to the hydrogen evolution reaction (HER) at the interface are not reversed: HER rises the pH of the solution around the cathode, but, highly active HER induces chromophore deprotonation regardless of the reverse pH dependence, reflecting an interface-specific deprotonation effect by HER. Here, we exploit this phenomenon to perform scanning-less, real-time visualization of interfacial proton dynamics during HER at a wide field of view. By using dKeima, the HER-driven deprotonation effect was well discriminated from the solution pH effect. In the electrodes of composite structures with a catalyst, dKeima visualized keen dependence of the proton depletion pattern on the electrode configuration. In addition, propagations of optical signals were observed, which seemingly reflect long-range proton hopping confined to the metal-solution interface. Thus, reverse pH-dependent fluorescent proteins provide a unique tool for spatiotemporal analysis of interfacial proton dynamics, which is expected to contribute to a better understanding of the HER process and ultimately to the safe and efficient production of molecular hydrogen.

Objectives Although guidelines recommend antiviral therapy for outpatients with COVID-19 who are at high risk of progressing to severe conditions, such as older adults, many patients do not receive appropriate treatment. Little is known, however, about the physician factors associated with the prescription of guideline-recommended antiviral therapy for patients with COVID-19.Design A cross-sectional study.Setting Data including outpatient visits in primary care clinics in Japan from April to August 2023.Participants We analysed 30 953 outpatients aged ≥65 years treated with COVID-19 (mean (SD) age, 75.0 (7.6) years; 17 652 women (57.0%)) in 1394 primary care clinics.Outcome measures The primary outcome was the prescription of guideline-recommended antivirals (ie, nirmatrelvir–ritonavir or molnupiravir), adjusted for patient characteristics, months of visits and regions.Results Antiviral prescriptions were concentrated among a small proportion of physicians; for example, the top 10% of physicians that had the largest number of nirmatrelvir–ritonavir prescriptions accounted for 92.4% of all nirmatrelvir–ritonavir prescriptions. After adjusting for potential confounders, physicians with higher patient volumes were more likely to prescribe guideline-recommended antivirals to their patients (adjusted OR (aOR) for high vs low volume, 1.76; 95% CI 1.31 to 2.38; adjusted p

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively. Key words: fluorescent protein, cell cycle, time-lapse imaging, flow cytometry

Femtosecond laser direct write (fs-LDW), a three-dimensional (3D) printing technology, is a promising method for creating microstructures made of proteins that retain their original function, enabling the development of complex biomimetic 3D microenvironments and versatile enhancements of medical microdevices. Fabrication using pure proteins via photoactivator-free femtosecond laser multiphoton crosslinking has recently been demonstrated This approach avoids the undesirable effects caused by the leaching of photoactivator molecules, and is thus regarded as suitable for biological applications. Here, we present the 3D fabrication of microstructures made of pure fluorescent protein variants, namely mScarlet, enhanced green fluorescent protein (EGFP), and enhanced blue fluorescent protein (EBFP2). Multicolor fluorescent microstructures are created using a sequential procedure with various precursors. We evaluate the dependence of fluorescence retention on fabrication parameters such as the total accumulated fluence. We demonstrate that microstructures made of EGFP can be applied to the detection of physiological pH changes. The results show that fs-LDW fabrication can broaden the application scope of fluorescent protein variants.

Abstract Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9‐edited human‐induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor‐trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC‐NSCs was related to self‐repulsive action and pathotropism involved in EphB‐ephrinB and CXCL12‐CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC‐NSCs. Our results indicate the potential benefit of genome‐edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

Objectives: We aimed to describe patient characteristics, healthcare utilization, and in-hospital mortality among patients with COVID-19 in Japan across waves. Methods: Using a large-scale hospital-based database, we identified patients hospitalized for COVID-19 in the first (January–June 2020), second (June–October 2020), third (October 2020–February 2021), fourth (March–June 2021), and fifth (June–December 2021) waves. We summarized patient characteristics, healthcare utilization, and in-hospital mortality during each wave and performed multivariable logistic regression analyses for in-hospital mortality. Results: From the first to fifth waves, the number of patients (mean age ± standard deviation, years) was 2958 (61.2 ± 22.8), 7981 (55.6 ± 25.3), 18,788 (63.6 ± 22.9), 17,729 (60.6 ± 22.6), and 23,656 (51.2 ± 22.3), respectively. There were 190 (6.4%), 363 (4.5%), 1261 (6.7%), 1081 (6.1%), and 762 (3.2%) in-hospital deaths, respectively. The adjusted odds ratios for in-hospital deaths (95% confidence interval) were 0.78 (0.65–0.95), 0.94 (0.79–1.12), 0.99 (0.84–1.18), 0.77 (0.65–0.92), in the second to fifth waves, respectively, compared with the first wave. Conclusions: In-hospital COVID-19 mortality improved from the first to the second wave; however, during the third and fourth waves, mortality was as serious as in the first wave. Although in-hospital mortality during the fifth wave improved, careful monitoring is needed for upcoming waves, considering changing patient and viral characteristics.

Visualisation of the activity of neural grafts using engineered luciferase provides insights into the integration between the graft and host.

Abstract Lumirubin is the most prevalently excreted hydrophilic bilirubin photoisomer in phototherapy for neonatal jaundice caused by excess hydrophobic unconjugated bilirubin (ZZ-bilirubin). We developed a simple method to estimate the amount of lumirubin by monitoring the reverse photoisomerization of lumirubin to ZZ-bilirubin. Although lumirubin formation was long considered irreversible, exposure to blue light in the presence of the fluorescent protein UnaG, which binds specifically and tightly to ZZ-bilirubin, enables the reverse photoisomerization of lumirubin. This reaction was first detected using a fluorescence assay of neonatal urine sampled during phototherapy and purified lumirubin. The phenomenon of reverse photoisomerization of lumirubin was validated using liquid chromatography–mass spectrometry, which confirmed that lumirubin is reconverted to ZZ-bilirubin in the presence of UnaG. Analyses of 20 urine samples from 17 neonates revealed a significant correlation (correlation coefficient [r] = 0.978; 95% confidence interval 0.867–0.979; P

OBJECTIVES: Although higher occupational classes have been reported to be associated with better health, researchers do not fully understand whether such associations derive from the position or individual characteristics of the person in that position. We examined the association between being a manager and cardiovascular disease (CVD) risk factors using unique panel data in Japan that annually observed employees’ occupational class and health conditions. METHODS: We analyzed data for 45 888 observations from a Japanese company from 2013 through 2017. The association between being a manager and CVD risk factors (metabolic risks and health-related behaviors) were evaluated using simple pooled cross-sectional analyses with adjustment for age, sex, marital status, and overtime-working hours. We further incorporated employee-level fixed-effects into the models to examine whether the associations were subject to individual time-invariant factors. RESULTS: The pooled cross-sectional analyses showed that, compared to non-managers, managers had 2.0 mg/dl lower low density lipoprotein cholesterol (LDL-C) level, 1.4 mmHg-lower systolic blood pressure, and 0.2 kg/m^2 lower body mass index (BMI). After adjusting for employee-level fixed-effects, being a manager was associated with a significantly 2.2 mg/dl higher LDL-C level. However, the associations between an individual’s management status and blood pressure or BMI were not significant. Furthermore, managers were 5.5% less likely to exercise regularly and 6.1% less likely to report sufficient sleep in the fixed-effects models, although the pooled cross-sectional analyses did not demonstrate these significant associations. CONCLUSIONS: Our findings suggest the necessity of considering these unfavorable health risks associated with being promoted to a manager.

Objectives We aimed to examine the use and factors associated with the provision of low-value care in Japan.Design A multicentre observational study.Setting Routinely collected claims data that include all inpatient and outpatient visits in 242 large acute care hospitals (accounting for approximately 11% of all acute hospitalisations in Japan).Participants 345 564 patients (median age (IQR): 62 (40–75) years; 182 938 (52.9%) women) seeking care at least once in the hospitals in the fiscal year 2019.Primary and secondary outcome measures We identified 33 low-value services, as defined by clinical evidence, and developed two versions of claims-based measures of low-value services with different sensitivity and specificity (broader and narrower definitions). We examined the number of low-value services, the proportion of patients receiving these services and the proportion of total healthcare spending incurred by these services in 2019. We also evaluated the 2015–2019 trends in the number of low-value services.Results Services identified by broader low-value care definition occurred in 7.5% of patients and accounted for 0.5% of overall annual healthcare spending. Services identified by narrower low-value care definition occurred in 4.9% of patients and constituted 0.2% of overall annual healthcare spending. Overall, there was no clear trend in the prevalence of low-value services between 2015 and 2019. When focusing on each of the 17 services accounting for more than 99% of all low-value services identified (narrower definition), 6 showed decreasing trends from 2015 to 2019, while 4 showed increasing trends. Hospital size and patients’ age, sex and comorbidities were associated with the probability of receiving low-value service.Conclusions A substantial number of patients received low-value care in Japan. Several low-value services with high frequency, especially with increasing trends, require further investigation and policy interventions for better resource allocation.

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire+mTEC precursors during the Aire+mTEC differentiation process of the postnatal thymus.

Abstract Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.

Summary: Background: COVID-19 has worse mortality than influenza in American and European studies, but evidence from the Western Pacific region is scarce. Methods: Using a large-scale multicenter inpatient claims data in Japan, we identified individuals hospitalised with COVID-19 in 2020 or influenza in 2017–2020. We compared patient characteristics, supportive care, and in-hospital mortality, with multivariable logistic regression analyses for in-hospital mortality overall, by age group, and among patients with mechanical ventilation. Findings: We identified 16,790 COVID-19 patients and 27,870 influenza patients, with the different age distribution (peak at 70–89 years in COVID-19 vs. bimodal peaks at 0–9 and 80–89 years in influenza). On admission, the use of mechanical ventilation was similar in both groups (1·4% vs. 1·4%) but higher in the COVID-19 group (3·3% vs. 2·5%; p

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

Abstract The ability to identify specific cell–cell contact in the highly heterogeneous mammalian body is crucial to revealing precise control of the body plan and correct function. To visualize local connections, we previously developed a genetically encoded fluorescent indicator, GRAPHIC, which labels cell–cell contacts by restricting the reconstituted green fluorescent protein (GFP) signal to the contact site. Here, we modify GRAPHIC to give the reconstituted GFP motility within the membrane, to detect cells that make contact with other specific cells. Removal of leucine zipper domains, located between the split GFP fragment and glycophosphatidylinositol anchor domain, allowed GFP reconstituted at the contact site to diffuse throughout the entire plasma membrane, revealing cell morphology. Further, depending on the structural spacers employed, the reconstituted GFP could be selectively targeted to N terminal (NT)- or C terminal (CT)-probe-expressing cells. Using these novel constructs, we demonstrated that we can specifically label NT-probe-expressing cells that made contact with CT-probe-expressing cells in an epithelial cell culture and in Xenopus 8-cell-stage blastomeres. Moreover, we showed that diffusible GRAPHIC (dGRAPHIC) can be used in neuronal circuits to trace neurons that make contact to reveal a connection map. Finally, application in the developing brain demonstrated that the dGRAPHIC signal remained on neurons that had transient contacts during circuit development to reveal the contact history. Altogether, dGRAPHIC is a unique probe that can visualize cells that made specific cell–cell contact.

Abstract Background Many older people wish to die at home. However, there is still a huge gap between the place where older adults wish to die and the place where they, in fact, do die. We aimed to assess the association between each type of long-term care (LTC) services that home-dwelling older individuals utilized at their end of life and place of death. Methods A pooled cross-sectional study at the point of death was used for the analysis. Participants included beneficiaries of long-term care insurance in Japan, aged 65 years and above, who passed away between January 2008 and December 2013, excluding those who died due to external factors and those who were using residential services at their time of death. We conducted a multivariate Poisson regression analysis with robust standard errors adjusting for potential confounders and examined the association between the use of each type of LTC service for home-dwelling recipients, including in-home services, day services, and short-stay services, with the interaction terms being time of death (exposure) and home death (outcome). We calculated the adjusted probability of home deaths for each combination pattern of LTC services for home-dwelling recipients using standard marginalization. Results We analyzed 2,035,657 beneficiaries. The use of in-home services, day services, and short-stay services were associated with an increased probability of home deaths; the incident rate ratio (IRR) was 13.40 (with a 95% confidence interval (CI): 13.23–13.57) for in-home services, the IRR was 6.32 (6.19–6.45) for day services, and the IRR was 1.25 (1.16–1.34) for short-stay services. Those who used day or short-stay services with in-home services exhibited a higher probability of home deaths than those who used only day or short-stay services. Conclusions We demonstrated that home-dwelling older persons who used LTC services near end-of-life had a higher probability of home deaths as compared to those who did not. Our findings can clarify the importance of providing and integrating such services to support care recipients who wish to die at home as well as for the benefit of their informal caregivers.

Abstract Background It is unclear how formal long-term care (LTC) availability affects formal /informal caregiving patterns and caregiver health. We tested the impact of reduced formal LTC availability on formal LTC service use, intensity of informal caregiving, and caregiver health. Methods Using a representative, repeated cross-sectional sample of Japanese caregivers providing care to co-resident family members from 2001 to 2016, we applied a difference-in-differences approach by observing caregivers before and after the major reform of the public Japanese LTC insurance (LTCI) in 2006. The reform reduced coverage benefits for non-institutionalized older persons with low care needs, but not for those with high care needs. We analyzed 12,764 caregivers aged ≥30 years (mean age 64.3 ± 11.8 years, 73.5% women) and measured indicators of formal LTC use, hours of informal caregiving, and caregiver self-reported health outcomes after propensity score matching to balance caregivers’ background characteristics. Results We found the 2006 LTCI reform relatively reduced the use of formal LTC services and relatively increased the percentage of experiencing long hours of informal caregiving (> 3 h per day) among the caregivers for seniors with low care needs compared to those for seniors with high care needs. The effects of the LTCI reform for the caregivers for seniors with low care needs were 2.2 percentage point higher on caregivers’ experiencing poor self-rated health (95% confidence interval [CI]: 0.7–3.7; p = 0.01), 2.7 percentage point higher on experiencing symptoms of a depressive state (95%CI: 0.5–4.8; p = 0.03), and 4.7 percentage point higher on experiencing symptoms of musculoskeletal diseases (95%CI, 3.6–5.7; p

Background: The association of sensory loss with mortality remains unclear. We aimed to explore the associations of hearing loss (HL), visual loss (VL), and dual sensory loss (DSL) with survival. Methods: Data came from the Komo-Ise study cohort in Gunma Prefecture, Japan, where the community-dwelling residents aged 40–69 years were followed up from 1993 to 2010. We analyzed 9,522 individuals who answered the follow-up questionnaires in 2000 (average age 64 [range, 47 to 77] years in 2000). The primary exposures were “HL only,” “VL only,” or “DSL”, with “no HL/VL” as the reference. These sensory loss statuses were assessed by asking the difficulty in hearing conversation or reading newspaper even with aids in the follow-up questionnaires in 2000. All-cause and cause-specific mortality were ascertained from linkage to death certificate data. Cox proportional hazards models adjusting for confounders, including demographic factors, socioeconomic status, and health status, were used. Potential mediators (depression, walking disability, and social participation) were additionally adjusted for. Results: There were 1,105 deaths over the 10-year follow-up. After adjustment for the potential confounders, HL and DSL were associated with increased all-cause mortality (hazard ratios of 1.74 [95% CI, 1.18–2.57] and 1.63 [95% CI, 1.09–2.42], respectively). Potential mediators explained a modest portion of the association. As for cause-specific mortality, HL was associated with increased cancer mortality, while VL and DSL were associated with increased cardiovascular disease mortality. Conclusions: Self-reported HL and DSL may be risk factors of mortality among middle-aged or elderly Japanese populations.

Background: Physicians currently measure the pupil diameter and the pupillary light reflex with visual observations using a ruler and a traditional penlight, leading to possibly inaccurate and subjective assessments. Although a mobile pupillometer has been developed and is available in clinical settings, this device can only assess one pupil at a time. Hence, an indirect pupillary light reflex, including those under irradiation to the opposite side of pupil, cannot be evaluated. Consequently, we have developed a new automatic mobile pupilometer, the Hitomiru®, with Hitomiru Co., Ltd. (Tokyo, Japan). This device is a two-glass type pupilometer with a video recording system. The pupil diameter and light reflex of both pupils can be measured simultaneously; therefore, both indirect and direct light reflexes can be assessed.Purpose: To evaluate the clinical ability of the Hitomiru® pupilometer to assess the pupil diameter and the pupillary light reflex of healthy volunteers and patients with intracranial lesions in an intensive care unit (ICU).Methods: Twenty-five healthy volunteers and five ICU patients with intracranial lesions on only the left side were assessed using the Hitomiru® pupilometer. The protocol was as follows: infrared light was applied to both pupils, followed by visible light to the right pupil, infrared light to both pupils, visible light to the left pupil, and then infrared light to both pupils. All the intervals were 2 s, and the dynamics of pupil diameters on both sides were continuously recorded.Results: The healthy adults had approximately 0.5 mm anisocoria, miosis was harder, and mydriasis was less with increased age. There were several differences in miosis rates, miosis times, and mydriasis rates between the healthy adults and the patients with intracranial lesions with both direct irradiation and indirect irradiation.Conclusions: The initial trial estimated and digitally recorded direct and indirect light reflexes, including rapidity of miosis after direct and indirect lights on, and mydriasis after direct and indirect lights off. The Hitomiru® pupilometer was a useful device to digitally record and investigate the relationship between pupil reflexes and intracranial diseases.

BackgroundThe use of telemedicine outpatient visits has increased dramatically during the COVID-19 pandemic in many countries. Although disparities in access to telemedicine by age and socioeconomic status (SES) have been well-documented, evidence is limited as to how these disparities changed during the COVID-19 pandemic. Moreover, the equity of patient access to telemedicine has been scarcely reported in Japan, despite the huge potential for telemedicine expansion. ObjectiveWe aimed to investigate changes due to age and SES disparities in telemedicine use during the COVID-19 pandemic in Japan. MethodsUsing data from a large internet survey conducted between August 25 and September 30, 2020, in Japan, we examined the associations of participant age and SES (educational attainment, urbanicity of residence, and income level) with their telemedicine use in the following two time periods during the pandemic: April 2020 and August-September 2020. ResultsOf the 24,526 participants aged 18 to 79 years (50.8% [n=12,446] women), the proportion of individuals who reported using telemedicine increased from 2.0% (n=497) in April 2020 to 4.7% (n=1159) in August-September 2020. After adjusting for potential confounders, younger individuals were more likely to use telemedicine than older individuals in April 2020. Although this pattern persisted in August-September 2020, we also observed a substantial increase in telemedicine use among individuals aged 70 to 79 years (adjusted rates, 0.2% in April 2020 vs 3.8% in August-September 2020; P

Summary: Intercellular contacts are essential for precise organ morphogenesis, function, and maintenance; however, spatiotemporal information of cell-cell contacts or adhesions remains elusive in many systems. We developed a genetically encoded fluorescent indicator for intercellular contacts with optimized intercellular GFP reconstitution using glycosylphosphatidylinositol (GPI) anchor, GRAPHIC (GPI anchored reconstitution-activated proteins highlight intercellular connections), which can be used for an expanded number of cell types. We observed a robust GFP signal specifically at the interface between cultured cells, without disrupting natural cell contact. Application of GRAPHIC to the fish retina specifically delineated cone-bipolar connection sites. Moreover, we showed that GRAPHIC can be used in the mouse central nervous system to delineate synaptic sites in the thalamocortical circuit. Finally, we generated GRAPHIC color variants, enabling detection of multiple convergent contacts simultaneously in cell culture system. We demonstrated that GRAPHIC has high sensitivity and versatility, which will facilitate the analysis of the complex multicellular connections without previous limitations. : Biological Sciences; Molecular Biology; Cell Biology Subject Areas: Biological Sciences, Molecular Biology, Cell Biology

Abstract Background As society is aging, retirement takes on increasing importance for individuals in the later life. This study aimed to describe mortality before and after retirement in the Japanese middle-aged/elderly with special attention to socioeconomic position and social relationships. Methods We conducted a 10-year follow-up study (the Komo-Ise cohort study) and assessed mortality according to socioeconomic positions (relative poverty and occupation) and social relationships (e.g., marital status, living alone, and social support) in workers and the retired. Relative poverty was defined as a household equivalent income of 12,700 US dollars (1.37 million Japanese Yen) or less in 2000. Stratified analyses were conducted according to sex in two groups of employment status: the workers and the retired. Adjusted hazard ratios (HRs) were calculated using the Cox proportional hazard model. Results We included 5534 individuals. Of these, 3360 were men (working, 2499; retired, 861) and 2174 were women (working, 1306; retired, 868). We observed 610 deaths (475 in men and 135 in women) during the study period. Relative poverty was a significant risk factor for death (HR 1.52, 95% confidence interval [CI] 1.07–2.14) among retired men but not among working men (HR 1.20, 95% CI 0.79–1.83). Among workers, self-employed men showed a significantly higher hazard of death (HR 1.57, 95% CI 1.09–2.25) than white-collar employees. Retired men who lacked participation in social activities were more likely to die than those who did not (HR 1.44, 95% CI 1.06–1.94). All results, except marital status, indicated non-significant associations in women. Conclusions Relative poverty and lack of social engagement may be related to high mortality risk in retired men. Further studies are needed to assess the health status among the middle-aged/elderly population around retirement.

OBJECTIVES:In this paper, we aim to estimate the effect cancer diagnosis has on labour-force participation among middle-aged and older populations in Japan. We investigate the impact of cancer diagnosis on job cessation and the gap between gender or job types. METHODS:We sourced data from a nationwide, annual survey targeted population aged 51-70 featuring the same cohort throughout, and examined respondents' cancer diagnoses and whether they continued to work, while also considering differences between gender (observations: 53 373 for men and 44 027 for women) and occupation type (observations: 64 501 for cognitive worker and 20 921 for manual worker) in this regard. We also examined one-year lag effects, using propensity score matching to control for confounding characteristics. We also implement Logistic regression and derive the odds ratio to evaluate the relative risk of cancer diagnosis, which supplements the main result by propensity score matching. RESULTS:Overall, the diagnosis of cancer has a huge effect on labour-force participation among the population, but this effect varies across subpopulations. Male workers are more likely to quit their job in the year they are diagnosed with cancer (10.1 percentage points), and also in the following year (5.0 percentage points). Contrastingly, female workers are more likely to quit their job immediately after being diagnosed with cancer (18.6 percentage points); however, this effect totally disappears when considering likelihoods for the following year. Cognitive workers are more prone to quit their job in the year of diagnosis by 11.6 percentage points, and this effect remains significant, 3.8 percentage points, in the following year. On the other hand, for manual workers the effect during the year of diagnosis is huge. It amounts to 18.7 percentage points; however, the effect almost disappears in the following year. CONCLUSION:Our results indicate the huge effect of cancer on job cessation, and that there might be a degree of discrimination in workplaces between gender and job types.

Summary: Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. Video Abstract: : Kuroki et al. performed cell-type-specific, wide-field FRET-based calcium imaging to visualize cortical network activity induced by multisensory inputs. They observed phase-locking of cortical slow waves in excitatory neuronal hubs in association cortical areas that may underlie multisensory integration. Keywords: wide-field calcium imaging, multisensory integration, cortical slow waves, association cortex, phase locking, fluorescence resonance energy transfer, spontaneous activity, excitatory neuron, inhibitory neuron, mouse

The data presented here refer to a research article entitled “Self-Assembled Micellar Clusters Based on Triton-X-family Surfactants for Enhanced Solubilization, Encapsulation, Proteins Permeability Control, and Anticancer Drug Delivery” Solomonov et al., 2019. The present article provides the General Procedure for clusterization of Triton-X-based micelles and the effect of (i) metal ion, surfactant, and chelator concentration on the developed clusters formation, (ii) surfactant-chelator relation change, (iii) metal ion-micelles concertation ratio variation, (iv) metal ion replacement, (v) solvent replacement, (vi) kinetics of clusters formation, (vii) hydrophobic fluorescent dye (Coumarin 6) solubilization in aqueous MCs media, (viii) novel anticancer peptidyl drug synthesis and characterization and (ix) the viability of HeLa cells with and without the presence of drug-free Triton-X-based family MCs. These data provide additional insights useful for understanding all aspects of the micellar clusters formation, optimization, and control. Keywords: Micellar clusters, Surfactants, Triton-X, Self-assembly, Encapsulation, Anticancer drug synthesis, Fluorescence enhancement

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

Neural stem/progenitor cell (NSPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI). However, the efficacy of NSPC transplantation on severe SCI is poorly understood. We herein show that NSPC transplantation promotes functional recovery after mild and moderate SCI, but not after severe SCI. In severe SCI mice, there were few remaining host neurons within the range of NSPC engraftment; thus, we examined whether the co-distribution of transplant and host is a contributory factor for functional improvement. A cellular selective analysis using laser microdissection revealed that drug-induced host neuronal ablation considerably decreased the synaptogenic potential of the engrafted NSPCs. Furthermore, following host neuronal ablation, neuronal retrograde tracing showed less propriospinal relay connections bridging the lesion after NSPC transplantation. Our findings suggest that the interactive synaptic reorganization between engrafted NSPCs and spared host neurons is crucial for functional recovery, providing significant insight for establishing therapeutic strategies for severe SCI.

Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed Bacillus subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions, and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

AIMS:The purpose of this study was to examine whether periodontitis is associated with incident type 2 diabetes in a Japanese male worker cohort. METHODS:The study participants were Japanese men, aged 36-55 years, without diabetes. Data were extracted from the MY Health Up study, consisting of self-administered questionnaire surveys at baseline and following annual health examinations for an insurance company in Japan. The oral health status of the participants was classified by two self-reported indicators: (1) gingival hemorrhage and (2) tooth loosening. Type 2 diabetes incidence was determined by self-reporting or blood test data. Modified Poisson regression approach was used to estimate the relative risks and the 95% confidence intervals of incident diabetes with periodontitis. Covariates included age, body mass index, family history of diabetes, hypertension, current smoking habits, alcohol use, dyslipidemia, and exercise habits. RESULTS:Of the 2895 candidates identified at baseline in 2004, 2469 men were eligible for follow-up analysis, 133 of whom were diagnosed with diabetes during the 5-year follow-up period. Tooth loosening was associated with incident diabetes [adjusted relative risk = 1.73, 95% confidence interval = 1.14-2.64] after adjusting for other confounding factors. Gingival hemorrhage displayed a similar trend but was not significantly associated with incident diabetes [adjusted relative risk = 1.32, 95% confidence interval = 0.95-1.85]. CONCLUSIONS:Tooth loosening is an independent predictor of incident type 2 diabetes in Japanese men.

Microfluidic devices and biochips offer miniaturized laboratories for the separation, reaction, and analysis of biochemical materials with high sensitivity and low reagent consumption. The integration of functional or biomimetic elements further functionalizes microfluidic devices for more complex biological studies. The recently proposed ship-in-a-bottle integration based on laser direct writing allows the construction of microcomponents made of photosensitive polymer inside closed microfluidic structures. Here, we expand this technology to integrate proteinaceous two-dimensional (2D) and three-dimensional (3D) microstructures with the aid of photo-induced cross-linking into glass microchannels. The concept is demonstrated with bovine serum albumin and enhanced green fluorescent protein, each mixed with photoinitiator (Sodium 4-[2-(4-Morpholino) benzoyl-2-dimethylamino] butylbenzenesulfonate). Unlike the polymer integration, fabrication over the entire channel cross-section is challenging. Two proteins are integrated into the same channel to demonstrate multi-protein patterning. Using 50% w/w glycerol solvent instead of 100% water achieves almost the same fabrication resolution for in-channel fabrication as on-surface fabrication due to the improved refractive index matching, enabling the fabrication of 3D microstructures. A glycerol-water solvent also reduces the risk of drying samples. We believe this technology can integrate diverse proteins to contribute to the versatility of microfluidics.

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.

We previously demonstrated that degus (Octodon degus), which are a species of small caviomorph rodents, could be trained to use a T-shaped rake as a hand tool to expand accessible spaces. To elucidate the neurobiological underpinnings of this higher brain function, we compared this tool use learning task with a simple spatial (radial maze) memory task and investigated the changes that were induced in the hippocampal neural circuits known to subserve spatial perception and learning. With the exposure to an enriched environment in home cage, adult neurogenesis in the dentate gyrus of the hippocampus was augmented by tool use learning, but not radial maze learning, when compared to control conditions. Furthermore, the proportion of new synapses formed in the CA3 region of the hippocampus, the target area for projections of mossy fiber axons emanating from newborn neurons, was specifically increased by tool use learning. Thus, active tool use behavior by rodents, learned through multiple training sessions, requires the hippocampus to generate more novel neurons and synapses than spatial information processing in radial maze learning.

A transgenic mouse line expressing Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes allows us to monitor the cell cycle in the hematopoietic system. Two populations with high and low intensities of Fucci signals for Cdt1(30/120) accumulation were identified by FACS analysis, and these correspond to quiescent G0 and cycling G1 cells, respectively. We observed the transition of immune cells between quiescent and proliferative phases in lymphoid organs during differentiation and immune responses.

Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases--RhoA, Rac1, and Cdc42--and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC(CDH1). We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC(CDH1). Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.

BACKGROUND: Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and -negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and -negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3σ protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies. CONCLUSIONS: Our results show that the use of Fucci, and similar fluorescent cell cycle indicators, offers a convenient assay system with which to identify cell cycle events associated with cancer cell death. They also indicate genotype-specific cell death modes induced by deficient initiation of DNA replication in cancer cells and its potential exploitation for development of efficient cancer therapies.

BACKGROUND: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. CONCLUSIONS: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.

To investigate potential cures for spinal cord injury (SCI), several researchers have transplanted neural stem/progenitor cells (NS/PCs) into the injured spinal cord by different procedures, including intralesional (IL), intrathecal (IT), and intravenous (IV) injection. However, there are no reports quantifying or comparing the number of cells successfully transplanted to the lesion site by each procedure in vivo. The purpose of the present study was to determine the optimal method of cell transplantation to the SCI site in terms of grafted cell survival and safety. For this purpose, we developed mouse NS/PCs that expressed a novel Venus-luciferase fusion protein that enabled us to detect a minimum of 1,000 grafted cells in vivo by bioluminescence imaging (BLI). After inducing contusive SCI at the T10 level in mice, NS/PCs were transplanted into the injured animals three different ways: by IL, IT, or IV injection. Six weeks after the transplantation, BLI analysis showed that in the IL group, the luminescence intensity of the grafted cells had decreased to about 10% of its initial level, and appeared at the site of injury. In the IT group, the luminescence of the grafted cells, which was distributed throughout the entire subarachnoid space immediately after transplantation, was detected at the injured site 1 week later, and by 6 weeks had gradually decreased to about 0.3% of its initial level. In the IV group, no grafted cells were detected at the site of injury, but all of these mice showed luminescence in the bilateral chest, suggesting pulmonary embolism. In addition, one third of these mice died immediately after the IV injection. In terms of grafted cell survival and safety, we conclude that the IL application of NS/PCs is the most effective and feasible method for transplanting NS/PCs into the SCI site.

This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.

Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.