Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through a comprehensive search using a desolvating agent, trifluoroethanol (TFE), we identified 336 proteins, collectively dubbed "TFE-Dependent ReversiblY condensing Proteins (T-DRYPs)." Unexpectedly, we rediscovered CAHS proteins as highly enriched in T-DRYPs, 3 of which were major components of T-DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. Furthermore, CAHS proteins increased cell stiffness in a hyperosmotic stress-dependent manner and counteract the cell shrinkage caused by osmotic pressure, and even improved the survival against hyperosmotic stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeleton-like proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and could contribute to the exceptional physical stability in a dehydrated state.

Phase-Separated Giant Liposomes for Stable Elevation of α-Hemolysin Concentration in Lipid Membranes.

Staphylococcus aureus α-hemolysin (αHL) is one of the most popular proteins in nanopore experiments within lipid membranes. Higher concentrations of αHL within the lipid membrane are desirable to enhance the mass transport capacity through nanopores. However, the reconstitution of αHL at high concentrations is associated with the problem of membrane lytic disruption. In this study, we present a method that effectively increases αHL concentration while maintaining membrane stability. This method is achieved by using phase-separated giant liposomes, where coexisting liquid-disordered (Ld) and liquid-ordered phases (Lo) are enriched in unsaturated lipids and saturated lipids with cholesterol (Chol), respectively. Fluorescence observation of αHL in liposomes revealed that the presence of Chol facilitates αHL insertion into the membrane. Despite the preferential localization of αHL in the Ld phase rather than the Lo phase, the coexistence of both Lo and Ld phases prevents membrane disruption in the presence of concentrated αHL. We have explained this stabilization mechanism considering the lower membrane tension exhibited by phase-separated liposomes compared to homogeneous liposomes. Under hypertonic conditions, we have successfully increased the local concentration of αHL by invagination of the lipid-only region in the Ld phase, leaving αHL behind. This method exhibits potential for the reconstitution of various nanochannels and membrane proteins that prefer the Ld phase over the Lo phase, thus enabling the production of giant liposomes at high concentrations and the replication of the membrane-crowding condition observed in cells.

PepYLCIV and PepYLCAV resistance gene Pepy-2 encodes DFDGD-Class RNA-dependent RNA polymerase in Capsicum.

KEY MESSAGE: A begomovirus resistance gene Pepy-2 encoding the DFDGD-Class RNA-dependent RNA polymerase 3a was identified in pepper (C. annuum) through the forward and reverse genetic analyses. In several countries throughout the world, the whitefly-transmitted begomovirus causes massive yield losses in pepper (Capsicum spp.) production. Although introgression of the genetic resistance against begomovirus to commercial cultivars is strongly required, the recently discovered recessive resistance gene pepy-1, which encodes the messenger RNA surveillance factor Pelota, is the only begomovirus resistance gene identified in Capsicum so far. In this study, we fine-mapped another begomovirus resistance gene from PG1-1 (C. annuum), which is resistant to pepper yellow leaf curl Indonesia virus (PepYLCIV) and pepper yellow leaf curl Aceh virus (PepYLCAV), to further speed up the marker-assisted breeding of begomovirus resistance in peppers. A single dominant locus, Pepy-2, conferring resistance against PepYLCIV in PG1-1 was identified on chromosome 7 by screening recombinants from the F2 and F3 segregating populations derived from a cross between PG1-1 and begomovirus susceptible SCM334. In the target region spanning 722 kb, a strong candidate gene, the RNA-dependent RNA polymerase 3a (CaRDR3a), was identified. The whole-genome and transcriptome sequences of PG1-1 and SCM334 revealed a single Guanine (G) deletion in CaRDR3a first exon, causing a frameshift resulting in loss-of-function in SCM334. In addition, multiple loss-of-function alleles of CaRDR3a were identified in the reference sequences of C. annuum, C. chinense, and C. baccatum in the public database. Furthermore, virus-induced gene silencing of CaRDR3a in PG1-1 resulted in the loss of resistance against PepYLCIV. PG1-1 and the DNA marker developed in this study will be useful to breeders using Pepy-2 in their breeding programs.

Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology.

Polymer micromaterials in a liquid or gel phase covered with a surfactant membrane are widely used materials in pharmaceuticals, cosmetics, and foods. In particular, cell-sized micromaterials of biopolymer solutions covered with a lipid membrane have been studied as artificial cells to understand cells from a physicochemical perspective. The characteristics and phase transitions of polymers confined to a microscopic space often differ from those in bulk systems. The effect that causes this difference is referred to as the cell-size space effect (CSE), but the specific physicochemical factors remain unclear. This study introduces the analysis of CSE on molecular diffusion, nanostructure transition, and phase separation and presents their main factors, i.e., short- and long-range interactions with the membrane surface and small volume (finite element nature). This serves as a guide for determining the dominant factors of CSE. Furthermore, we also introduce other factors of CSE such as spatial closure and the relationships among space size, the characteristic length of periodicity, the structure size, and many others produced by biomolecular assemblies through the analysis of protein reaction-diffusion systems and biochemical reactions.

Dynamic ordering caused by a source-sink relation between two droplets.

Two droplets composed of different chemicals, 1-decanol and liquid paraffin, floating on the water surface show characteristic co-responsive behavior. The presence of two different types of droplets in the system imposes an asymmetry that would not be possible with single droplets alone. The self-propulsion and interactions between droplets appear because surface active 1-decanol molecules provided by the source are absorbed by the paraffin sink thus generating an asymmetric surface tension gradient. This source-sink relation between droplets stabilizes and enhances the self-propulsion, and leads to a variety of dynamic structures including oscillations in the inter-droplet distance. We found that the character of time evolution also depends on the concentration of dye, Sudan Black B, initially used just to stain the decanol droplet. A simple mathematical model explains the transition between the stationary state and the oscillations as a Hopf bifurcation.

Membrane Surface Modulates Slow Diffusion in Small Crowded Droplets.

Membranes are ubiquitous structures in cells. The effects of membranes on various functional molecules have been reported, but their behaviors under macromolecular crowding and cell-sized confinement have not fully been understood. In this study, we model an intracellular environment by crowding micrometer-sized droplets and investigate the effects of membrane properties on molecular diffusion. The molecular diffusion inside small droplets covered with a lipid layer of phosphatidylcholine (PC) becomes slower compared with that of the corresponding bulk solutions under a crowding condition of polysaccharide dextran but not of its monomer unit, glucose. The addition of a poly(ethylene glycol) conjugated lipid (PEGylated lipid) to the PC membrane significantly alters the degree of slow diffusion observed inside small droplets of concentrated dextran. Interestingly, the change is not monotonic against dextran concentration; that is, the PEGylated membrane increases and decreases the degree of slow diffusion with increasing dextran concentration. We explain the nonmonotonic alternation from the increase in effective dextran concentration and the hindered temporal adsorption of dextran to the membrane. Because diffusion alteration by adding PEGylated lipid is observed for condensed small droplets of linear polymer PEG and hydrophilic protein bovine serum albumin, the phenomenon is general for other polymer systems as well. Furthermore, our findings may facilitate the understanding of intracellular molecular behaviors based on membrane effects as well as the development of numerous applications using polymer droplets.

Perpendicular alignment of the phase-separated boundary in adhered polymer droplets.

We investigated the effect of the adhered interface on the phase separation pattern using two or three adhered droplets containing a binary solution of poly(ethylene glycol) and gelatin. Under the experimental conditions, single domains of the gelatin-rich phase exhibited partial wetting to the droplet adhered interface (DAI) and nonadhered droplet surface. In the case of isolated spherical droplets, the location of the phase separation interface (PSI) of the domains was completely random owing to spatial symmetry. In the adhered droplets, the random orientation of the PSI was observed when the PSI did not contact the DAI. On the other hand, when the PSI contacted the DAI, the PSI was aligned perpendicular to the DAI. Frequency analysis showed that whether the PSI contacts the DAI is purely stochastic. However, the PSI alignment perpendicular to the DAI increases significantly with three adhered droplets, suggesting that the probability increases with increasing DAI area ratio. We explain this perpendicular pattern by the minimization of the interfacial energy and kinetics with a change in the wetting contact angle. These findings will facilitate the research on the phase separation of polymer solutions inside nonspherical micrometric spaces.

Chronic stress and age-related pattern of blood pressure: A cross-sectional study in rural China.

OBJECTIVE: Chronic stress is a risk factor for hypertension in adults. However, there is conflicting evidence for older adults. We hypothesized that age-related arterial stiffening, which leads to a lower blood pressure (especially diastolic blood pressure [DBP]), is more pronounced among older adults with high vs low stress. The objectives of this study were (a) to investigate age-related trends in systolic and DBPs among adults in rural Fujian, China, and (b) to examine differences in age-related blood pressure trends according to levels of stress by using Epstein-Barr virus (EBV) antibody titer as a marker of chronic stress status. METHODS: We collected cross-sectional data from 764 rural community-dwelling adults in rural Fujian, China (mean age = 59.4). Participants were categorized into high and low stress groups by median split of EBV antibody titer. A least-squares regression analysis was used to investigate the association between age and blood pressures. RESULTS: We observed an inverted U-shaped association between age and DBP, while there was a linear association between age and systolic blood pressure in the overall sample. When stratified by stress, the inverted U-shaped associations with age (both systolic and DBPs) were seen only among those with high stress; DBP peaked at the age of ~68 years, and the declining trend later in life was more clearly observed among those with high chronic stress. DISCUSSION: Decrease of DBP was more pronounced among older adults with high vs low chronic stress in rural China.

Associations between urinary heavy metal concentrations and blood pressure in residents of Asian countries.

BACKGROUND: Previous studies have suggested that exposures to heavy metals (arsenic, cadmium, lead, and selenium) may be associated with differences in blood pressure. However, the findings of these studies have been inconsistent. This study was performed to examine the associations between urinary heavy metal concentrations and blood pressure among residents of four Asian countries (Bangladesh, Indonesia, Nepal, and Vietnam). METHODS: This cross-sectional study examined 1899 adults in four Asian countries. Urinary concentrations of heavy metals were measured by inductively coupled plasma mass spectrometry. A questionnaire survey was administered regarding individual characteristics. Anthropometric measurements (height and weight) were performed. Systolic and diastolic blood pressures were measured after a short rest. Multiple linear regression models were applied to investigate associations between urinary heavy metal concentrations and blood pressure after adjustments for age, sex, and body mass index. RESULTS: The geometric means of the urinary concentrations of arsenic, cadmium, lead, and selenium were 84.6, 0.885, 2.09, and 16.5 µg/g creatinine, respectively. The urinary arsenic concentrations were slightly higher than those typically reported in non-polluted populations, while urinary cadmium, lead, and selenium concentrations were equivalent or slightly lower. The urinary lead concentrations were positively associated with both systolic and diastolic blood pressure, but urinary selenium concentrations were negatively associated with them. CONCLUSIONS: Variations in the urinary concentrations of lead and selenium were associated with blood pressure at low levels of exposure/intake.

Liposomal adhesion via electrostatic interactions and osmotic deflation increase membrane tension and lipid diffusion coefficient.

Membrane adhesion is a ubiquitous phenomenon in cells and is related to various biological events such as migration, morphogenesis, and differentiation. To understand the physicochemical aspects of membrane adhesion, liposome-liposome adhesion and liposome-substrate adhesion have been studied. Although membrane adhesion has been shown to increase membrane tension and inhibit lipid diffusion, the relationship between these changes and the degree of membrane adhesion have not been quantified. Here, we analyzed the dependence of membrane tension and lipid diffusion on the degree of membrane adhesion, i.e., area fraction of the adherent region. For this purpose, we developed a simple method to prepare adhered liposomes by simple electrostatic interactions between the membranes and by osmotic deflation. We found that the membrane tension of the adhered liposomes increases slightly with an increase in the area fraction of the adherent region. In addition, the lipid diffusion coefficient of the adhered liposomes is larger than that of isolated liposomes, which is consistent with the theoretical prediction. The analysis provides a framework to understand the correlation between cell adhesion and bio-membrane properties such as membrane tension and molecular diffusion.

Protective role of selenium in the shortening of telomere length in newborns induced by in utero heavy metal exposure.

The effects of toxic heavy metals, such as arsenic (As), cadmium (Cd), and lead (Pb), on telomere length (TL) have been reported previously. Although selenium (Se) is considered as an anti-oxidant which may detoxify the effects, there are no data on whether Se could protect against the TL-shortening effects of heavy metals. Thus, the aim of this study was to evaluate the protective role of Se against heavy metal-induced TL shortening. A birth cohort study was conducted in Myanmar in 2016, including 408 mother-infant pairs. First, pregnant women in the third trimester were interviewed concerning their socioeconomic, and pregnancy and birth characteristics using a pre-validated questionnaire. Maternal spot urine samples were collected after the interview. During the follow-up period (1-3 months), blood samples were collected from the umbilical cord at birth by local health workers. Metal concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS). TL was measured by quantitative real-time polymerase chain reaction (PCR). Relative TL was calculated as the ratio of telomere genes to single-copy genes. To evaluate the effect of Se on TL shortening, molar ratios were calculated. Linear regression analyses were performed to examine the associations between heavy metals and TL, individually and after adjustment for Se level. The effects of As, Cd, and Pb exposure on TL were smaller after adjustment for the Se level, especially for Pb (unadjusted ß = -0.10; 95% CI: 0.18, -0.01; adjusted ß = -0.03; 95% CI: 0.13, 0.05). On stratifying the data by Se concentration, there was no significant association between Cd or Pb exposure and TL in the high-Se group. Our study indicated a protective effect of Se against the TL shortening induced by heavy metal exposure, where the effect sizes were smaller after adjusting for the Se level, compared to individual metal exposure.

The association between family members' migration and Epstein-Barr virus antibody titers among people left behind in rural Fujian, China.

OBJECTIVES: In contrast to the health of migrants, which has been extensively studied, little attention has been paid to the health of adults left behind by family members who out-migrated to other locations. This study was performed to investigate the association between being left behind and the Epstein-Barr virus (EBV) antibody titer, which is an indicator of cellular immune functioning previously shown to be associated with psychological stress. METHODS: A cross-sectional survey was conducted in rural communities in Fujian, China, in 2015. Questionnaire data and dried blood spot samples were collected from 748 adults. A mixed effect regression analysis was conducted to investigate the association. RESULTS: Results indicated that the EBV antibody titers were higher among people who had been left behind compared with those who had not. CONCLUSIONS: Our findings suggest that, in rural Fujian, China, family separation following the out-migration of family members is an important determinant of psychological stress.

In-utero arsenic exposure and growth of infants from birth to 6 months of age: a prospective cohort study in rural Bangladesh.

Arsenic exposure in postnatal life impacts the growth of children, but little is known about the effect of in-utero arsenic exposure on growth very early in childhood. The aim of this study was to examine the associations between in-utero arsenic exposure and the growth of infants from birth to 6 months of age using monthly follow-up data. A prospective cohort study was conducted in rural areas of Bangladesh with 108 mother-infant pairs. This study identified a negative association between in-utero arsenic exposure and head circumference of infants 1-6 months of age (coefficient = -1.20, 95% confidence interval [CI]: -1.97, -0.42), and the effect was more pronounced in the earlier ages of 1-3 months (coefficient = -0.88, 95% CI: -1.70, -0.05). Because head circumference is considered as a surrogate of brain size, our findings suggest that in-utero arsenic exposure influences brain growth during an important developmental period.

Effects of Short-term Exposure to Ambient Particulate Matter on the Lung Function of School Children in Dhaka, Bangladesh.

BACKGROUND: Ambient particulate pollution may adversely affect children's lung function. However, evidence on this association remains scarce in Asia despite this region having the greatest burden of disease due to air pollution. OBJECTIVES: To investigate the effect of short-term exposure to ambient particulate matter (PM) on the lung function of school children in Dhaka city, Bangladesh. The possible seasonal modification of this association was also examined. METHODS: A panel of 315 school children who were 9-16 years of age were recruited from three schools in and around Dhaka. Lung function was assessed using a spirometry test during the cool and warm seasons in 2013, yielding six measurements per child. Daily PM data were retrieved from nearby air monitoring stations. Linear mixed effects models were used to examine associations. Seasonal modification was examined by stratification. RESULTS: An inverse association was observed for the lung function parameters with PM2.5; peak expiratory flow (PEF) and forced expiratory volume within 1 second (FEV1) decreased with increasing PM2.5. The percent deviation from the personal median was -4.19% [95% confidence interval (CI): -5.72, -2.66] for PEF and -2.05% (95% CI: -2.92, -1.18) for FEV1 for a 20 µg/m increase in PM2.5 on the previous day. Results for PM10 were less consistent. The estimated effects of PM on lung functions were generally greater in the warm season. CONCLUSIONS: Short-term exposure to PM is associated with worse lung function in children living in highly polluted settings, with the strength of these adverse PM effects varying by season.

Association between early parental deprivation and cellular immune function among adults in rural Fujian, China.

This study investigated the role of childhood parental deprivation in the association between quality of life (QOL) and the Epstein-Barr virus (EBV) antibody titer, a marker of cellular immune functioning, using data from 734 adults living in seven communities in rural Fujian, China. Parental deprivation was defined if either of their parents had died, divorced, or left them for migratory work for more than 1 year before they turned (a) 16 years of age (childhood and adolescence), or (b) 3 years of age (early childhood). A mixed-effects regression analysis with a random effects model revealed that parental deprivation during early childhood was positively associated with the EBV antibody titer (coefficient = 0.33, p = 0.007), whereas parental deprivation during the first 16 years was not. Further analysis incorporating an interaction term between QOL and parental deprivation revealed a significant interaction between QOL and parental deprivation during early childhood; a significant association between QOL and the EBV antibody titer was found only among those who reported parental deprivation during early childhood. This study suggests that parental deprivation during the period of immune system development may be linked with physiological responses to stressors later in life.

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament.

Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

Cell-size confinement effect on protein diffusion in crowded poly(ethylene)glycol solution.

Micrometric membrane confinements and macromolecular crowding of cytoplasm are key factors that regulate molecular diffusion in live cells. Previous studies have shown that macromolecular crowding delays molecular diffusion. However, the effect of cell-size confinement on diffusion in the crowding environment is yet to be elucidated. Using fluorescence correlation spectroscopy (FCS), we analyzed protein diffusion in microdroplets containing polymer solution covered with lipid membranes that mimic cells. As a result, we found that a synergistic condition of crowding and micrometric confinement results in accelerated protein diffusion on a sub-millisecond time scale. This acceleration rate strongly depended on the size of the confined space and the degree of crowding. These findings indicate that cell-size confinement supports protein diffusion in highly crowded cytoplasm.

Epstein-Barr virus antibody titer as a stress biomarker and its association with social capital in rural Fujian communities, China.

OBJECTIVES: There has been little research on the association between social capital and psychological stress measured by a biomarker, particularly in developing countries. Our objective was to investigate the association between Epstein-Barr virus (EBV) antibody titer, an indicator of cellular immune function previously shown to be associated with psychological stress, and social capital among rural community dwellers in Fujian Province, China. METHODS: We conducted a cross-sectional survey in seven rural communities in Fujian in 2015. We used questionnaire data and dried blood spot samples for the measurement of biomarkers collected from 734 local residents for the analysis. We conducted a mixed effects regression analysis to investigate the association between EBV antibody titer and four social capital variables, which included cognitive and structural social capital assessed both at individual and community levels. RESULTS: Community-level structural social capital was inversely associated with psychological stress (coefficient = -0.96, 95% confidence interval [CI] = -1.91, -0.01) while individual-level structural social capital was positively associated with it (coefficient = 0.21, 95% CI = 0.07, 0.36). Neither individual- nor community-level cognitive social capital was associated with psychological stress status. CONCLUSIONS: In rural Fujian, China, social capital seemed to be an important determinant of psychological health. While living in a community with active social interaction may benefit the residents psychologically, social interaction in the form of strongly bonded relationships may be a source of psychological stress at the individual level due to the potential demands and obligations that may be associated with such connections.

Preterm birth rates in Japan from 1979 to 2014: Analysis of national vital statistics.

AIM: Secular trends of preterm birth in Japan between 1979 and 2014 were examined to determine whether changes could be explained by a shift in the distribution of maternal age at delivery and parity and/or by changes in age-specific preterm birth rates. METHODS: Live birth data for 1979 to 2014 were obtained from the Japanese Ministry of Health, Labour and Welfare. Analyses were limited to singleton children born in Japan (n = 43 632 786). Preterm birth was defined using two cut-offs at < 37 or < 34 weeks of gestation. Crude and standardized rates of preterm birth were calculated for firstborn and later-born singletons by maternal age at delivery for specific time periods. RESULTS: Throughout the study period, the rates of preterm birth (both at < 37 and < 34 weeks of gestation) were higher among mothers aged 20 and younger, and mid-30s and older, compared to mothers in their 20s or early 30s. The rates of preterm birth at < 37 (but not at < 34) weeks decreased for mothers aged in their late 30s and 40s, and increased for mothers in their 20s and early 30s. Standardized rates of preterm birth showed a secular increase for preterm births at < 37 but not < 34 weeks of gestation. CONCLUSION: The rates of preterm birth among mothers aged in their 20s and early 30s increased between 1979 and 2014, which contributed to the secular increase in rates of preterm birth at < 37 weeks.

Airborne pollen and suicide mortality in Tokyo, 2001-2011.

Prior research has indicated that pollen might be linked to suicide mortality although the few studies that have been undertaken to date have produced conflicting findings and been limited to Western settings. This study examined the association between the level of airborne pollen and suicide mortality in Tokyo, Japan in the period from 2001 to 2011. The daily number of suicide deaths was obtained from the Japanese Ministry of Health, Labour and Welfare, with pollen data being obtained from the Tokyo Metropolitan Institute of Public Health. A time-stratified case-crossover study was performed to examine the association between different levels of pollen concentration and suicide mortality. During the study period there were 5185 male and 2332 female suicides in the pollen season (February to April). For men there was no association between airborne pollen and suicide mortality. For women, compared to when there was no airborne pollen, the same-day (lag 0) pollen level of 30 to <100 grains per cm2 was associated with an approximately 50% increase in the odds for suicide (e.g. 30 to <50 grains per cm2: odds ratio 1.574, 95% confidence interval 1.076-2.303, p=0.020). The estimates remained fairly stable after adjusting for air pollutants and after varying the cut-points that defined the pollen levels. Our results indicate that pollen is associated with female suicide mortality in Tokyo.