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The COVID-19 pandemic has affected the mental health of health care workers. This study aimed to investigate the stress factors that cause burnout in Japanese physicians and their coping methods during the COVID-19 pandemic.We conducted a sequential explanatory mixed-method study to investigate the psychological responses of physicians in the early stages of the pandemic. A cross-sectional, web-based, anonymous survey was conducted among members of the American College of Physicians Japan Chapter to quantitatively investigate the stress factors and prevalence of burnout. An open-ended questionnaire with questions about stress factors and coping methods was additionally administered. The qualitative data were analyzed using qualitative content analysis.Among the 1173 physicians surveyed, 214 (18.2%) responded. Among the participants, 107 (50.0%) responded "yes" to the question "I feel or have felt very stressed at work during the COVID-19 pandemic," and 68 (31.8%) reported burnout symptoms. Those who reported feeling stress (117 respondents) were asked to select 12 items of the stress factors related to COVID-19. The most significant stress factor related to COVID-19 was "Perceived risk of spreading COVID-19 to family members" (Several distinct stressors existed during the COVID-19 pandemic, which might be related to burnout among physicians. Practicing stress-coping strategies, as identified in the present study, may help reduce work-related stress and prevent burnout.

Objective: People with psychiatric disorders are one of the most vulnerable populations in disasters, and the 2011 Great East Japan Earthquake reported higher post-evacuation mortality rates among psychiatric inpatients. A psychiatric hospital evacuated after the nuclear accident was surveyed to gain valuable insights for future disaster preparedness. Methods: The authors interviewed two Odaka Akasaka Hospital (a private psychiatric hospital) staff responsible for evacuation due to the nuclear accident. Results: At the time of the earthquake, 104 patients had been admitted to the hospital. They were instructed to evacuate on the grounds that they existed within a 20 km radius of Fukushima Daiichi Nuclear Power Station. Although the evacuation process was extraordinarily demanding, the staff acted professionally, and no patient experienced a significant deterioration in health during the evacuation. Conclusion: It was reasonable to follow the evacuation order because of the difficulty of obtaining accurate information about radiation exposure and staff availability in high-risk situations. The staff’s knowledgeable and attentive care of the patients was one of the factors that enabled them to successfully carry out this severe evacuation. However, this may be related to the high mortality rate after the evacuation of patients who were separated from such caregivers.

Recent studies have provided insights into the effect of vaccine boosters on recall immunity. Given the limited global supply of COVID-19 vaccines, identifying vulnerable populations with lower sustained vaccine-elicited antibody titers is important for targeting individuals for booster vaccinations. Here we investigated longitudinal data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to December 2021. Antibody titers following two doses of a COVID-19 vaccine were repeatedly monitored and information on lifestyle habits, comorbidities, adverse reactions, and medication use was collected. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers and identified vulnerable populations with low sustained antibody titers. Moreover, we showed that only 5.7% of the participants in our cohort were part of the “durable” population with high sustained antibody titers, which suggests that this durable population might be overlooked in small cohorts. We also found large variation in antibody waning within our cohort. There is a potential usefulness of our approach for identifying the neglected vulnerable population.

ObjectivesThis study aimed to identify factors that delayed emergency medical services (EMS) in evacuation order zones after the 2011 Great East Japan Earthquake and Fukushima Daiichi Nuclear Power Plant accident and to investigate how the lifting of the evacuation affected these factors over time.DesignThis research was a retrospective observational study. The primary outcome measure was onsite EMS time. A gradient boosting model and a decision tree were used to find the boundary values for factors that reduce EMS.SettingThe target area was Minamisoma City, Fukushima, Japan that was partly designated as an evacuation order zone after the 2011 Fukushima disaster, which was lifted due to decreased radiation.ParticipantsThis study included patients transferred by EMS from 1 January 2013 through 31 October 2018. Patients who were not transported and those transported for community events, interhospital patient transfer and natural disasters were excluded.Outcome measuresThis study evaluated the total EMS time using on-site time which is the time from arrival at the scene to departure to the destination, and other independent factors.ResultsThe total number of transports was 12 043. The decision tree revealed that the major factors that prolonged onsite time were time of day and latitude, except for differences by year. While latitude was a major factor in extending on-site time until 2016, the effect of latitude decreased and that of time of day became more significant since 2017. The boundary was located at N37.695° latitude.ConclusionsThe onsite time delay in EMS in evacuation order zones is largely due to regional factors from north to south and the time of day. However, the north-south regional factor decreased with the lifting of evacuation orders.

Disaster deaths can be classified into direct and indirect deaths. Direct deaths are those caused by the direct physical effects of disasters, such as earthquakes, tsunamis, and radiation exposure. Indirect deaths are those caused by secondary health effects such as emergency evacuation, relocation, evacuation environment, disruption of health care delivery services, and psychosocial effects. In addition, in Japan, the term disaster-related deaths refers to indirect deaths in accordance with the disaster condolence payments system, which provides relief for bereaved families. On 11 March 2011, the Great East Japan Earthquake exposed several issues related to disaster-related deaths in Japan. Therefore, on 1 February 2022, a symposium on disaster-related deaths hosted by this study was held on the website. The symposium discussed the issues and challenges associated with disaster-related deaths for future disaster preparedness. The authors introduced the concept of ‘shaking’ at the symposium by defining ‘shaking’ as ‘the repeated changes in the social and living environment that worsen health conditions, regardless of the disaster’. It was also pointed out that vulnerable populations are more likely to experience more pronounced health effects. This generalised concept of ‘shaking’ associated with disaster-related deaths suggests that it is important to anticipate disasters before they occur to take specific preventive measures, targeted at vulnerable populations. This study found that disaster-related deaths in Japan create several problems in terms of future radiation disaster preparedness and medical countermeasures. In the future, there will be a need to examine the relevance of the issues of disaster-related deaths identified as a result of this symposium for future radiation disaster preparedness.

Many efforts have been made to develop therapeutic agents for Alzheimer’s Disease (AD) based on the amyloid cascade hypothesis, but there is no effective therapeutic agent at present. Now, much attention has been paid to infiltrate pathogens in the brain as a trigger of AD. These pathogens, or their virulence factors, may directly cross a weakened blood-brain barrier, reach the brain and cause neurological damage by eliciting neuroinflammation. Moreover, there is growing clinical evidence of a correlation between periodontitis and cognitive decline in AD patients. Recent studies have revealed that microglial cathepsin B is increasingly induced by lipopolysaccharide of Porphylomonas gingivalis, a major pathogen of periodontal disease. Moreover, gingipains produced by P. gingivalis play critical roles in neuroinflammation mediated by microglia and cognitive decline in mice. Furthermore, an orally bioavailable and brain-permeable inhibitor of gingipain is now being tested in AD patients. It is largely expected that clinical studies countering bacterial virulence factors may pave the way to establish the prevention and early treatment of AD.

Objective To evaluate the change in the prevalence of burnout during the COVID-19 pandemic among internists and primary care physicians in Japan, and to identify factors associated with the exacerbation of burnout among these populations during this period. Methods This was a cross-sectional study based on two web-based surveys conducted in January 2020 (before the declaration of the COVID-19 pandemic) and June 2020 (during the pandemic). The participants were internists and primary care physicians of the Japanese Chapter of the American College of Physicians. The main outcome was the change in the prevalence of burnout between before and during the "first wave" of the pandemic. We also examined factors associated with the exacerbation of burnout during this period. Results Among the 283 respondents in the first survey and 322 in the second survey, 98 (34.6%) and 111 (34.5%) reported symptoms of burnout, respectively. In June 2020, 82 respondents (25.5%) reported that their level of burnout exacerbated compared to January 2020. Only the experience of self-quarantine was associated with the exacerbation of burnout [odds ratio (OR) 3.12; 95% confidence interval (CI) 1.49-6.50; p=0.002], while being a woman, being a resident physician, and an experience of having worked in a prefecture under a state of emergency were not. Conclusions No marked change in the prevalence of burnout among internists and primary care physicians in Japan was observed during the COVID-19 pandemic as a whole. However, self-quarantine was associated with the exacerbation of the burnout level.

Despite a clear correlation between the infiltration of periodontal pathogens in the brain and cognitive decline in Alzheimer's disease (AD), the precise mechanism underlying bacteria crossing the blood-brain barrier (BBB) remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteases termed gingipains. Gingipains appear to be key virulence factors that exacerbate sporadic AD. We herein report that gingipains are involved in increasing permeability of hCMEC/D3 cell monolayer, human cerebral microvascular endothelial cell lines, through degradation of tight junction proteins including Zonula occludens-1 (ZO-1) and occludin. There was a significant decrease in the mean protein levels of ZO-1 and occludin after infection of hCMEC/D3 cells with wild-type (WT) P. gingivalis. However, infection of these cells with a gingipain-deficient P. gingivalis strain showed significantly lower reduction of the mean protein levels of either ZO-1 and occludin, compared to the WT strain. Similar results were obtained after treatment with culture supernatant from WT and gingipain-deficient P. gingivalis strains. In vitro digestion of human recombinant ZO-1 and occludin by WT P. gingivalis culture supernatant in the absence or presence of gingipain inhibitors indicated that gingipains directly degraded these tight junction proteins. A close immunohistochemical examination using anti-gingipain antibody further revealed that gingipains localized in the cytosol and nuclei of hCMEC/D3 cells after infection with WT P. gingivalis and treatment with its culture supernatant. Furthermore, intracellular localization of outer membrane vesicles (OMVs) bound gingipains from WT P. gingivalis and OMV-induced degradation of ZO-1 and occludin were also observed in hCMEC/D3 cells. Thus, the delivery of gingipains into the cerebral microvascular endothelial cells, probably through OMV, may be responsible for the BBB damage through intracellular degradation of ZO-1 and occludin.

The initial health impact caused by radiation disasters can be broadly classified into direct and indirect effects. Though no direct health hazards caused by radiation, such as acute radiation injury, were observed following the Fukushima Daiichi nuclear power plant accident, indirect deaths have been reported, including those caused by initial emergency evacuation and relocation, medical disruption, and psychological and social health effects. However, these indirect health effects have not been prioritised for addressal. We evaluated the radiation disaster experience with that of the coronavirus disease (COVID-19) pandemic that emerged while facing the challenges from the radiation disaster. Most of the health effects of COVID-19 are directly associated with infection, but indirect health effects of various scales and entities have been reported. The two disasters have similarities in terms of the strain on community healthcare and the large number of deaths. Adapting the measures implemented in the acute to subacute phases of the COVID-19 disaster to radiation disasters may help improve management following future radiation disasters. Based on the experience and findings during the COVID-19 pandemic, the pattern of deaths in radiation disasters can be divided into five major groups: direct deaths, and four indirect patterns of deaths due to a deteriorating supply-demand balance (a hospital-level problem), collapse of the healthcare system (a community-level problem), death due to neglect alongside underlying disease, and diseases other than direct invasion. From the similarities between the two disasters, three main issues should be prioritised as initial emergency evacuation measures in a radiation disaster: emergency exposure medicine, the establishment of a medical system, and protection of death with dignity. The validity of these priority issues needs to be verified in future research.

Objective The Mini-Z 2.0 is a new, simple, and nonproprietary tool for assessing physician well-being and burnout. To date, a non-English version of the Mini-Z 2.0 survey has not been validated. Therefore, we aimed to develop a Japanese version of the Mini-Z 2.0 and to evaluate its validity and reliability using survey data from physicians affiliated with an internal medicine academic society. Methods The Mini-Z 2.0 survey was translated into Japanese using a forward-backward translation method. The participants belonged to the American College of Physicians' Japan Chapter. The translated version of the Mini-Z 2.0 survey was distributed to participants using an electronic mailing list. Convergent validity was assessed between burnout and other items using Pearson's product-moment statistic. Structural validity was evaluated using an exploratory factor analysis and confirmatory factor analysis, and reliability was assessed using internal consistency. Results Of the 1,255 physicians and medical residents contacted, 283 responded (22.5%). Burnout was present in 34.6% of the participants, with 48.8% reporting high stress levels. Convergent validity was demonstrated, with satisfactory correlations between burnout and satisfaction, value alignment, work control, and stress. An exploratory factor analysis identified two factors (i.e., Well-Being and Relationships and Work-Related Stressors); however, the three models evaluated using the confirmatory factor analysis revealed a poor fit. Cronbach's alpha for the sample was 0.80. Conclusion The Japanese version of the Mini-Z 2.0 demonstrated good internal consistency and convergent validity. Despite its inadequate structural validity, it can be used to measure physician well-being and related workplace conditions in Japan.

Cortical microglia exhibit a ramified shape during sleep, while they have a hyper-ramified shape during wakefulness, which is characterized by their longer processes with increased branching points. The microglial molecular circadian clock regulates expressions of both cathepsin S (CatS) and P2Y12 receptors in the brain with a peak at zeitgeber time 14 (2 h after beginning of the dark phase). We postulated that these two microglia-specific molecules contribute to diurnal alterations of microglial shapes and neuronal activities in the cerebral cortex. During wakefulness, CatS secreted from cortical microglia may be involved in P2Y12 receptor-dependent process extension. Secreted CatS subsequently degrades the perineuronal nets, initiating the downscaling of both spine density and synaptic strength of cortical neurons toward the beginning of sleep. The downscaling of both spine density and synaptic strength of cortical neurons during sleep could improve signal-to-noise, which would benefit memory consolidation, or allow for new learning to occur during subsequent waking. Furthermore, disruption of CatS induces the sleep disturbance and impaired social interaction in mice. Moreover, the microglial clock system disruption may also play a role in the early pathogenesis of Alzheimer's disease. The reduced expression of BMAL1 in cortical microglia caused by oligomeric amyloid β may induce the increased presence of inflammatory phenotype through a reduction in RORα, which in turn reduced IκBα and enhanced NF-κB activation. These observations suggest that the microglial clock system disruption contribute to pathogeneses of sleep disturbance, impaired social interaction and cognitive impairment. Therefore, the growing understanding of the microglial circadian molecular clock might aid in the development of novel pharmacological interventions against both neuropsychiatric and neurodegenerative disorders.

Objectives A previous report indicated that evacuation owing to the government order after the Fukushima disaster impacted the health status of older people; however, the association between living in evacuation areas and independence in their daily lives was unclear. This study examined the hypothesis that people who were forced to evacuate their homes were more likely to need long-term care (LTC). Design Historical cohort study. Setting and Participants Older individuals (n = 13,934) in Minamisoma, Fukushima Prefecture, Japan, part of which was designated as an evacuation area. Methods Background and certification data from April 2012 to December 2016 were extracted. Logistic regression analysis with sex stratification was performed to examine the association between certification of care levels 1-5 and living in evacuation areas. Results In total, 18,178 Minamisoma residents aged ≥65 years who had not received LTC certification as of March 11, 2011, were eligible for follow-up. Of these, 4244 residents without a certificate of residence by June 1, 2016, were excluded. Of 13,934 residents followed up, 1553 (11.1%) were newly certified as care levels 1-5. Certification of care levels 1-5 was associated with living in evacuation areas [odds ratio (OR) 1.61, 95% confidence interval (CI) 1.20-2.18] and living alone at the time of the disaster (OR 2.58, 95% CI 1.92-3.47) in men and living alone at the time of the disaster (OR 1.35, 95% CI 1.15-1.59) in women. Conclusions and Implications Living in evacuation areas and living alone were associated with LTC certification. In evacuation areas, individuals with family members were more likely to be certified compared with those in nonevacuation areas. Among residents in evacuation areas, several individuals with family members experiencing family structure changes might need LTC. Preventive measures (including group relocation) may be beneficial for those being evacuated following a disaster.

Many efforts have been made to develop therapeutic agents for Alzheimer's Disease (AD) based on the amyloid cascade hypothesis, but there is no effective therapeutic agent at present. Now, much attention has been paid to infiltrate pathogens in the brain as a trigger of AD. These pathogens, or their virulence factors, may directly cross a weakened blood-brain barrier, reach the brain and cause neurological damage by eliciting neuroinflammation. Moreover, there is growing clinical evidence of a correlation between periodontitis and cognitive decline in AD patients. Recent studies have revealed that microglial cathepsin B is increasingly induced by lipopolysaccharide of Porphylomonas gingivalis, a major pathogen of periodontal disease. Moreover, gingipains produced by P. gingivalis play critical roles in neuroinflammation mediated by microglia and cognitive decline in mice. Furthermore, an orally bioavailable and brain-permeable inhibitor of gingipain is now being tested in AD patients. It is largely expected that clinical studies countering bacterial virulence factors may pave the way to establish the prevention and early treatment of AD.

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocytespecific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc., Embargo Period 12 months

Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.

We previously reported that Drosophila phagocytes enhance their phagocytic activity after apoptotic cell engulfment accompanied by the activation of the transcription repressor Tailless and an increase in the levels of engulfment receptors. We herein investigated the underlying mechanisms. We found that Tailless phosphorylation levels decreased in Drosophila phagocytes following the stimulation with apoptotic cells. Anticipating the involvement of another transcription repressor, we examined the possible involvement of Kruppel, a bibliographically identified repressor whose expression is controlled by Tailless. The level of Kruppel in phagocytes decreased after the stimulation in a Tailless-dependent manner. The RNAi knockdown of Kruppel abrogated increases in the levels of engulfment receptors and phagocytic activity in stimulated phagocytes. The binding of Kruppel to the 5'-upstream regions of genes coding for engulfment receptors was demonstrated. These results suggest the following pathway: Tailless is activated by de-phosphorylation; Kruppel expression is inhibited by Tailless; the transcription of engulfment receptors-encoding genes is augmented due to a decrease of inhibition by Kruppel; and finally phagocytic activity is enhanced.

Much attention has been paid to the connection between periodontitis and Alzheimer's disease (AD). We previously showed that infection of P. gingivalis, one of major periodontal pathogen causing periodontitis, induced migration and inflammatory responses in murine microglia through gingipain-induced activation of protease-activated receptor 2 (PAR2). In this study, we have attempted to clarify effects of secreted gingipains on cell migration of human microglial cell line, cleavage sites of PAR2 by gingipains and subsequent signaling pathways. P. gingivalis culture supernatant induced migration and membrane ruffling, which is necessary for microglia migration, in human microglial cell line HMC3 cells through PAR2. These effects were mainly mediated by gingipains, because cell migration and membrane ruffling were dramatically inhibited by treatment with gingipain inhibitors. Furthermore, pharmacological and genetic inhibition of Src kinase and β-arrestin, which are important for the internalization of G protein-coupled receptors, significantly inhibited P. gingivavlis culture supernatant-induced membrane ruffling in HMC3 cells. After treatment with P. gingivalis culture supernatant in Flag-PAR2-HA transfected HEK293T cells, Flag was removed from the cell surface, and HA was detected in the cytosol, indicating the internalization of PAR2. Furthermore, the phosphorylation level of ERK1/2 increased in PAR2-transfected HEK293T cells after treatment with P. gingivalis culture supernatant. The gingipain inhibitors, Src kinase inhibitor and β-arrestin knockdown suppressed PAR2 internalization and ERK1/2 phosphorylation. These observations suggest that secreted gingipains from P. gingivalis induce Src- and β-arrestin-dependent internalization of PAR2 and further activate the ERK1/2 pathway to promote migration of microglia. PAR2 are activated by the tethered ligands exposed by cleavage of extracellular N-terminal of PAR2. We also estimated potential gingipain cleavage sites in PAR2 and exposed tethered ligands, which are required for PAR2 internalization and membrane ruffling. The identified mechanism in this study might contribute to the retrogression of sporadic AD in patients after infection with P. gingivalis.

Apoptotic cells are rarely detected in vivo because of their rapid clearance by phagocytes, suggesting that apoptosis is the cell clearance rather than cell death machinery. In consistent with this, biochemical changes associated with apoptosis including caspase-3 activation are engaged locally in synaptic compartments without neuronal death. This phenomenon is referred to as synaptic apoptosis or synaptosis, which triggers a rapid clearance of localized synaptic compartments by microglia. In this clearance system, integrin ανβ3/TAM receptors with bridging molecules and CR3 on microglia are essential for recognition of phosphatidylserine (PS)-exposed and C1q/C3-tagged focal synaptic compartments, respectively. Caspase-3 plays pivotal roles in both synaptic PS exposure and C1q deposition. Therefore, the accumulating knowledge concerning with focal apoptotic synapses will contribute to better understanding of synapse loss and subsequent neuronal death in neurodegenerative diseases such as Alzheimer's disease.

The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spatzle (Spz) has been identified and characterized. We previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. Here, we demonstrate that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, we further showed that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, we found a genetic interaction between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Autobiographical memories can be recalled from either a field (first-person) or an observer (third-person) perspective. Previous studies have reported that field-to-observer perspective change reduced the emotional intensity of recalled events. In the present study, we examined whether this effect has a long duration by employing follow-up measurements. The participants were asked to recall the same events repeatedly across three sessions (S1, S2, and S3): S2 was conducted about three days after S1, and S3 was conducted about four weeks after S2. The results showed a reduction in the emotional intensity of the recalled events when the perspective was changed from field to observer at S2. More importantly, this reduction in emotional intensity persisted until S3. These effects were not observed under observer-to-field perspective change at S2. These results suggest that observer perspective taking can cause plastic change in the autobiographical memory system.

Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and β. Integrin βν, a β-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with βν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and βν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and βν. These results collectively indicated that integrin αPS3/βν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin βν, one of the two β subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin βν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin βν in hemocytes of an integrin βν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin βν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin βν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.