Mouse REC114 is essential for meiotic DNA double-strand break formation and forms a complex with IHO1. Its N-terminal region forms a Pleckstrin homology domain, while its C-terminal region is interacting with MEI4. Saccharomyces cerevisiae . These three S. cerevisiae proteins and their mouse orthologs  co-localize on the axes of meiotic chromosomes, and mouse IHO1 and MEI4 are essential for meiotic DSB formation. Here, we show that mouse Rec114 is required for meiotic DSB formation. Moreover, MEI4 forms a complex with REC114 and IHO1 in mouse spermatocytes, consistent with cytological observations. We then demonstrated in vitro the formation of a stable complex between REC114 C-terminal domain and MEI4 N-terminal domain. We further determine the structure of the REC114 N-terminal domain that revealed similarity with Pleckstrin homology domains. These analyses provide direct insights into the architecture of these essential components of the meiotic DSB machinery.Programmed formation of DNA double-strand breaks (DSBs) initiates the meiotic homologous recombination pathway. This pathway is essential for proper chromosome segregation at the first meiotic division and fertility. Meiotic DSBs are catalyzed by Spo11. Several other proteins are essential for meiotic DSB formation, including three evolutionarily conserved proteins first identified in  Saccharomyces cerevisiae, several genes are essential for their formation and at least five of them are evolutionarily conserved. Spo11, Top6bl, Iho1, Mei4, and Rec114 are the mouse orthologs of these five genes  shares only one domain of similarity with TOPOVIBL  are separated through a process called reductional segregation. In most species, reductional segregation requires the establishment of connections between homologues. To achieve this, homologous recombination is induced during meiotic prophase to allow homologues to find each other and to be connected by reciprocal products of recombination  . This hove genes  and are ve genes . SPO11 ave genes . TOPOVIBTOPOVIBL . Based oTOPOVIBL , which aTOPOVIBL .S. cerevisiae , Schizosaccharomyces pombe , Arabidopsis thaliana , Sordaria macrospora , Caenorhabditis elegans , and Mus musculus. Several important properties of these proteins suggest that they act as a complex. Indeed, in S. cerevisiae  at DSB sites. Interactions between Rec114 and Rec102 and Rec104 have been detected by yeast two-hybrid assays  seems not to be required for DSB formation Wtsi), exon 3 and 4 were deleted, and a lacZ-neomycin cassette with a splice acceptor site was inserted upstream of this deletion. This deletion includes the conserved motifs SSM3, 4, 5, and 6  and homozygous (Rec114−/−) mutant mice were viable. We also generated from this allele, another mutant allele (named and registered as Rec114del) without the insertion cassette. We performed all subsequent analyses using mice with the Rec114− allele unless otherwise stated, and confirmed several phenotypes in mice carrying the Rec114del allele.We analyzed mice carrying a null allele of 5, and 6   mice . In these tubules, the most advanced cells were spermatocytes, although some were also depleted of spermatocytes. Testis weight was significantly lower in Rec114−/− than wild-type mice  crossed for 4 months yielded no progeny.To monitor the consequences of REC114 absence on gametogenesis, we performed histological analysis of testes and ovaries. Spermatogenesis was altered in /+) mice . Specifiype mice . In ovar at 8 wk . In ovarent mice . ConsistRec114−/− mice, meiotic DSBs were absent or strongly reduced in both spermatocytes and oocytes, whereas chromosome axes formed normally, based on SYCP3 detection  of the single-strand DNA-binding protein complex RPA. Chromosome axes and assembly of the synaptonemal complex were monitored by detection of SYCP3 and SYCP1, respectively. Detection of γH2AX revealed that in etection . Quantifetocytes . Consistpe cells , and S3.etocytes  and S5A.of γH2AX . The detof SPO11 . Meioticof SPO11 . Analysiormation  and S6. Spo11−/− , Mei1−/− and with DSB repair (MEI4 foci are excluded from DMC1 foci) . At lept oocytes , and S9A oocytes . The MEIatocytes . This suMei4−/− spermatocytes, where their number was reduced by more than 10-fold compared with wild-type cells  that was suitable for structural analysis. We determined the crystal structure of this REC114 N-terminal region at a resolution of 2.5 Å by SAD using the selenomethionine (SeMet)-substituted protein. The final model, refined to an s 15–150 .2OQB) was the closest homologue  forms a PH domain, with two perpendicular antiparallel β-sheets followed by a C-terminal helix . Severalomologue .2 and included several salt bridges and hydrogen bonds formed by the well-conserved Arg98 of β6, and Glu130 and Gln137 of α1  suggests that, in yeast, this complex may play a direct role in promoting DSB activity  are structural elements (β sheets and α helices) within this PH domain and are readily identified in many eukaryotes . At the S. cerevisiae for the recruitment of Rec114 and Mei4 by Mer2  with IHO1 or with axis proteins independently from REC114 at least in an Rec114−/− genetic background as weak MEI4 axis-associated foci were observed in Rec114−/− spermatocytes and oocytes and because IHO1 protein was detected upon immunoprecipitation of MEI4 in Rec114−/− spermatocyte extracts. The details of these interactions and their dynamics during early meiotic prophase remain to be analyzed.A previous study showed that IHO1 is required for MEI4 and REC114 focus formation on axis and that it directly interacts with REC114 by two-hybrid assay . As IHO1 by Mer2 . In agrerevisiae . TherefoS. cerevisiae, it has been proposed that the local control of meiotic DSB formation is constrained by the chromatin loop organization and involves Tel1 (ATM)   and possactivity . The IHOactivity  could beRec114 mutant allele is referenced as 2410076I21Riktm1(KOMP)Wtsi and named Rec114−/− in this study. The Rec114del allele, in which the inserted lacZ-Neo cassette was deleted, was obtained by expression of Flp in mice carrying Rec114−/−. These mice are in the C57BL/6 background. The Mei4−/−, Spo11−/−, and Spo11YF/YF strains were previously described  according to the manufacturer's instructions. One μg of total RNA was treated with RQ1 RNAse-free DNase (Promega) 30 min at 37°C to degrade genomic DNA. 1 μg of total RNA was used for cDNA synthesis, using random primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Each PCR was performed using 0.05 μl of cDNA and KAPA Taq PCR kit (Sigma-Aldrich). Oligonucleotides sequences are provided in the Table S1. PCR cycling conditions were 3 min at 95°C, 35 cycles for 16U22/232L22 and 695U22/806L25 or 40 cycles for 16U21/644L22 and 16U21/688L24 with 15 s at 95°C, annealing for 15 s at 53°C, 51°C, 64°C, and 59°C for 16U22/232L22, 695U22/806L25, 16U21/644L22, and 16U21/688L24, respectively, and extension for 30 s at 72°C, followed by 5 min at 72°C at the end of cycles. Amplified products were separated on 2% agarose gels. PCR products obtained from the oligonucleotide combinations 16U21/644L22 or 16U21/688L24 were run in a gel and the most abundant product purified and sequenced.Total RNA was extracted from two testes of young (14 dpp) wild type or Rec114List of oligonucleotides used for RT-PCR.Table S1 Chicken anti-REC114 antibodies were generated against mouse REC114 and affinity-purified. The anti-SYCP3, anti-MEI4, and anti-IHO1 antibodies were previously described . Other aTestes and ovaries were fixed in Bouin's solution (Sigma-Aldrich) at room temperature overnight and for 5 h, respectively. After dehydration and embedding in paraffin, 3-μm sections were prepared and stained with periodic acid-Schiff for testes and with hematoxylin and eosin for ovaries. Image processing and analysis were carried out with the NDP.view2 software (Hamamatsu).Spermatocyte and oocyte chromosome spreads were prepared by the dry-down method .γH2AX was quantified using Cell profiler 2.2.0. The total pixel intensity per nucleus was quantified. The intensity of MEI4 foci was the mean pixel value within a focus. Axis-associated MEI4 foci were determined by co-labelling with SYCP3.Whole testis protein extracts were prepared as described in For REC114 immunoprecipitation, 1.5 mg of extract (from 14 dpp mice) was diluted in IP buffer  and incubated with 2 μg of affinity-purified chicken anti-REC114 antibody at 4°C overnight. Then, 50 μl of agarose-immobilized anti-chicken IgY Fc (goat)  was added at 4°C for 1 h. Beads were washed five times with washing buffer . Immunoprecipitated material was eluted and incubated with 2× Laemmli loading buffer (with 20 mM DTT) at 95°C for 5 min.For MEI4 immunoprecipitation, 3 μg of guinea pig anti-MEI4 antibody  was crosSPO11 immunoprecipitations on protein extracts from adult mice were performed as described . Rabbit Immunoprecipitates and inputs were separated on 10% Mini-PROTEAN TGX precast gels (Bio-Rad) and then transferred onto nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The following primary antibodies were used: rabbit anti-MEI4 1/500; , chickenE. coli BL21-Gold (DE3) (Agilent) from the pProEXHTb expression vector (Invitrogen). The protein was first purified by affinity chromatography using Ni2+ resin. After His-tag cleavage with the TEV protease, the protein was further purified through a second Ni2+ column and by size-exclusion chromatography. Pure protein was concentrated to 10 mg·ml−1 in buffer . The best-diffracting crystals grew within 1 wk at 20°C in a solution containing 0.25 M ammonium sulfate, 0.1 M MES, pH 6.5, and 28% PEG 5000 MME. For data collection at 100 K, crystals were snap-frozen in liquid nitrogen with a solution containing mother liquor and 25% (vol/vol) glycerol. SeMet-substituted REC114 was produced in E. coli BL21-Gold (DE3) and a defined medium containing 50 mg·l−1 of SeMet. SeMet REC114 was purified and crystallized as for the native protein.Mouse REC114 (15-159) fused to His-tag was expressed in P6122 with the unit cell dimensions a, b = 107.5 Å and c = 82.8 Å. The asymmetric unit contains one molecule and has a solvent content of 71%. A complete native dataset was collected to a resolution of 2.5 Å by the autonomous ESRF beamline MASSIF-1  belong to the space group MASSIF-1 . The SeMsing XDS . The strsing XDS . The modsing XDS , completsing XDS  and refising XDS . The modsing XDS . The nate of 30%  with alle of 30% .2+ column. The complex was then purified by size-exclusion chromatography.MEI4 (1-127) was cloned in the pProEXHTb expression vector to produce a His-tag fusion protein. REC114 and its deletion mutants were cloned in the pRSFDuet-1 vector as Strep-tag fusion proteins. REC114 variants alone or co-expressed with MEI4 were purified using a Strep-Tactin XT resin (IBA). The resin was extensively washed with a buffer containing 20 mM Tris, pH 7.0, 200 mM NaCl, and 5 mM mercaptoethanol, and bound proteins were eluted with the same buffer containing 50 mM biotin and analyzed by 15% SDS–PAGE. The minimal REC114-MEI4 complex was then purified using the Strep-Tactin XT resin. The His-tag of MEI4 was removed with the TEV protease and a passage through a Ni


It was applied to PAM data generated from the 2003–2004 National Health and Nutrition Examination Survey (NHANES 2003–2004). We discuss two corner case conditions that can produce unexpected, and perhaps unintended results when the algorithm is applied. We show, using simulated data of two special cases, how this algorithm classifies a 24-hour period with only 72 total counts as 100% wear in one case, and classifies a 24-hour period with 96,000 counts as 0.1% wear in another. The prevalence of like scenarios in the NHANES 2003–2004 PAM dataset is presented with corresponding summary statistics for varying degrees of the algorithm’s nonwear classification threshold (T). The number of participants with valid days, defined as 10 or more hours classified as wear time in a 24-hour day, increased while the mean counts-per-minute (CPM) decreased as the threshold for excluding non-wear was reduced from the allowed 4,000 counts in an hour. The number of participants with four or more valid days increased 2.29% (n = 113) and mean CPM dropped 2.45% (9.5 CPM) when adjusting the nonwear classification threshold to 50 counts an hour. Applying the most liberal criteria, only excluding hours as nonwear which contained 1 count or less, resulted in a 397 more participants (7.83% increase) and 26.5 fewer CPM (6.98% decrease) in NHANES 2003–2004 participants with four or more valid days. The algorithm should be used with caution due to the potential influence of these corner cases.The National Cancer Institute’s (NCI) wear time classification algorithm uses a rule based on the occurrence of physical activity data  Physical activity monitoring (PAM) devices, such as those produced by ActiGraph , often measure acceleration, in units of gravity, along one to three axes. These measures are commonly converted to and reported for each axis as  methods , 2. Muchactivity –13. A chwear and nonwear, in order to improve accuracy of these activity estimates [This poses difficulty in estimating quantities of interest related to physical activity and sedentary behavior. Thus, a number of classification algorithms have been developed for the purpose of distinguishing between periods of stimates , 14–20.accelerometry package (RAP) [This manuscript focuses on the nonwear classification algorithm developed by the National Cancer Institute (NCI) that we refer to as NCINW. NCINW was first implemented as part of a suite of SAS programs provided by NCI . It is age (RAP) , 23. We ge (RAP) –26. Thesge (RAP) .allowance that is key in differentiating non-wear from wear . Matthews et al, described the same classifier this way, “Periods of nonwear were defined as ending when count levels exceeded 100 counts/minute or when 3 consecutive minutes of observation were between 1 and 100 counts/minute.” [Troiano et al. were first to publish their findings on physical activity in the U.S. using NHANES 2003–2004 with NCINW. The authors rejected participant data deemed problematic  and focused on those with one or more valid days, which they defined as a 24-hour period with at least 10 hours of wear time classified by NCINW . Mean CPminute.”  InterpreAn important question is how this may impact some of the findings based on analyzing the NHANES 2003–2004 with the NCINW. For example, if the algorithm misclassifies wear time as nonwear time, it may be that days that are valid for inclusion in an analysis by the author’s definition are incorrectly excluded from analysis and do not contribute to these findings. Matthews et al. , in a foWe investigate the most extreme form of these corner cases using simulated data, and examine the implication of similar patterns on the analysis of NHANES 2003–2004. Our aim is to raise awareness of NCI’s nonwear classification algorithm’s (NCINW) behavior to certain patterns of activity as implemented, which may not be clear otherwise, and how this may impact PAM studies of free-living participants. We examine output from implementing NCINW through ActiLife and R in two scenarios that highlight potential classification problems. Our expectation is that there are conditions in which a large number of counts can accrue over a 24-hour day and still be considered nonwear, while a much smaller number of counts can accrue over a day and still be considered wear according to NCINW. We additionally examine NHANES 2003–2004 to determine the frequency with which these type of scenarios occur, and how the number of valid days, average CPM, and minutes spent in moderate intensity levels change when NCINW’s definition of wear is revised to include nonwear times with a minimum number of pre-classification counts.To test our proposition, we created two 24-hour day scenarios, Scenario A and Scenario B, by repeating two hour-long patterns (Patterns A and B) of vertical-axis counts (1-min epoch), from midnight to 11:59PM  to determine wear and nonwear time, as well as total counts accrued and total counts discarded as nonwear in each 24 hour simulation. This helped verify that the implementations were consistent with one another, and allowed us to more easily extend our analysis of the NHANES 2003–2004 data set next. Information concerning the specific configuration and setup of these software implementations and the necessary input data format is available in the supplement.Next, we examined the prevalence of A and B type patterns in the NHANES 2003–2004 dataset, which includes 7,176 participants. The participants represent a diverse demographic of both youth and adults (6–85+ years in age), male and female. These differences do not affect NCINW’s behavior, which only considers the occurrence and quantity of counts in its classifications, and so we did not differentiate youth and adults in this analysis. The National Center for Health Statistics (NCHS) Ethics Review Board (ERB) obtained written consent from all participants and approved the NHANES 2003–2004 release of deidentified information to the public domain –31. StanAccelerometry package (see supplement for specific configuration). Only patterns that occurred during hourly clock intervals were considered ; keeping with NCINW behavior.Scenarios A and B were simulated to demonstrate NCINW’s most extreme behavior in a 24-hour day. Patterns like A and B may also occur naturally with similar, though less extreme levels of behavior. We use the number of counts in an hour, defined as T, to quantify these types of patterns. The specific ordering and magnitude of counts of A-type patterns consist of hours classified as nonwear with ≥ T counts for each hour, prior to classification . B-type patterns represent hours classified as wear with ≤ T counts for each hour. Prevalence of A-type patterns was determined for T, evaluated on an interval from 10 to 500 counts per hour, and prevalence of B-type patterns was determined for T from 1 to 60 counts per hour. NCINW classification of wear and nonwear was done using the R valid and considered for further. Participants with ≥ 1 and ≥ 4 valid days were tallied, and their average CPM calculated during valid day wear time, for each case. The number of minutes spent above ≥ 2,020 counts–the cutoff used by Troiano et al for moderate and vigorous physical activity in adults [Lastly, we explored how using the NCINW algorithm may impact the analysis of NHANES 2003–2004 using an approach similar to Troiano et al [The NCINW is vulnerable to poor performance under two corner case conditions. In examining the NHANES 2003–2004 dataset, with which the NCINW algorithm was provided in order to replicate its analysis, we found A-type patterns to be much more prevalent than B-type patterns. The maximum number of counts any NCINW classified nonwear hour can have is 4,000 . SettingThe most noticeable difference occurred when there was no effort to account for nonwear (i.e. T = 0) and all data were included . In this case, mean CPM was 75.80% less for participants with ≥ 1 valid days, and 73.41% less for participants with ≥ 4 valid days when compared to NCINW’s standard nonwear criteria. The number of minutes-per-day with counts at or above 2,020 was -29.00% in participants with ≥ 1 valid days, and -25.98% in participants with ≥ 4 valid days. Interestingly, in the case of number-of-minutes spent above 2,020 counts for participants with ≥ 4 valid days, the maximum value (34.48 minutes) was reached at T = 75 and T = 80 counts per hour. It then dipped slightly before increasing again to the value of 34.46 minutes. The maximum number of counts allowed per minute during nonwear is capped at 100 counts by definition , which mThere are several reasons the NHANES 2003–2004 analysis data set is protected from the potential misclassification observed in these corner case conditions. First, because these corner cases are less common, the large number of participants examined continuously for up to one week helps account for and generalize much of the results as intended. Second, the first cut-point for physical activity was set at 2,020 counts per-minute, well above the 100 count maximum allowed by NCINW to still be considered, and possibly excluded, as nonwear. Finally, the 10 hour, valid day criteria imposed on NHANES 2003–2004 bounds the examined data to a range of activity, most likely captured during daytime. Therefore, studies using smaller datasets, with shorter periods of observation, less restrictive inclusion criteria, or using lower count-per-minute cut-points, may be more susceptible to these corner case effects and need to exercise caution when using NCINW. In addition, studies of individual-level relationships between physical activity and/or sedentary behavior with other factors may be more sensitive to the effects of these corner cases than estimates of sample summary statistics. Further research is needed to clarify these points. We recommend using the NCINW with caution in the meantime.Our study showed specific, simulated conditions where NCI’s nonwear classification algorithm produces counterintuitive results. In Scenario A, hours with 4,000 counts each were classified as nonwear, while in Scenario B, hours with only 3 counts each were classified as wear. We examined the likelihood of finding similar patterns, A-type and B-type, within the NHANES 2003–2004 dataset and demonstrated how analysis of NHANES 2003–2004 is impacted when different levels of A-type patterns are reconsidered as wear. The uncertainty here is incorrectly classifying these times as nonwear when they may truly represent sedentary or sleeping behavior. The NCINW shows a bias towards classifying such activity as nonwear instead of low level, sedentary behavior. Despite these dangers and the prevalence of A-type patterns in NHANES 2003–2004, there was little change to the results for overall summary estimates of population levels of physical activity when accounting for these patterns. Still, the NCINW algorithm should be used with caution for smaller datasets, shorter periods of observation, and analysis with lower activity cut-points, and/or more lenient criteria for valid days of wear, or studies with different aims than NHANES 2003–2004, particularly including those that describe individual-level relationships between physical activity and an outcome, as opposed to summary statistics.S1 Text(DOCX)Click here for additional data file.S1 TableSoftware that implement the same wear/nonwear classification algorithm developed by the National Cancer Institute (NCI) to analyze the physical activity monitoring portion of the 2003–2004 National Health and Nutrition Examination Survey (NHANES 2003–2004).(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S2 Tableaccel.weartime (RAP-2) method configured to match NCI’s SAS script (nci = TRUE) with default or unset values (parameter set 1) and non-default values (parameter set 2) set to match the original nonwear definition. The default parameters must be changed (parameter set 2) to mimic the NCISAS script.The classification results for scenario A according to the R accelerometry package (DOCX)Click here for additional data file.S3 Tablenonwear according to NCINW, are shown in (A). The number of participants with one or more hours containing, inclusively between, 1 and T counts and classified as wear, are shown in (B). The percent of participants compared to the number evaluated  is shown for each case.The number of NHANES 2003–2004 participants with one or more hours with ≥ T counts, but classified as (DOCX)Click here for additional data file.


Prunus mume; ume) is a traditional food in Japan that has been shown to have various beneficial health effects. There is some evidence to suggest that ume is also effective against allergic disease. Here, we conducted a cross-sectional epidemiological pilot study to examine the association between ume intake frequency and allergic symptoms including rhinitis in 563 adults (288 men and 275 women) who resided in Wakayama, Japan. After adjusting for age, present illness and medication, women with high ume intake had significantly lower odds ratio (OR) for the presence of symptoms of allergy [OR: 0.49 with 95% confidence interval (CI): 0.25–0.97]. Therefore, we investigated the anti-allergic effect of ume on passive cutaneous anaphylaxis (PCA) reaction in immunoglobulin E (IgE)-sensitized mice. The animal study demonstrated that oral administration of ume extract attenuated the PCA reaction and mast cell degranulation. Furthermore, RBL-2H3 mast cells were used to identify anti-allergic ume compounds. The following ume compounds inhibited IgE-mediated mast cell degranulation: vanillin, syringic acid, protocatechuic aldehyde, lyoniresinol and p-coumaric acid. These results suggested that ume has the potential to inhibit mast cell degranulation and may be associated with reduced risk of allergic symptoms in women.Japanese apricot ( Because allergy symptoms can result not only in a decline in quality of life but also in life-threatening reactions, allergies have become a social problem. Development of Japanese cedar or Japanese cypress pollen allergy (pollinosis) has recently increased in Japan. The most common cause of pollinosis in Japan is Japanese cedar. A nationwide survey found that the prevalence of Japanese cedar pollinosis increased from 16.2% in 1998 to 26.5% in 20084. Functional foods, defined as foods that can provide additional health benefits beyond that of traditional nutrients they contain, have attracted attention as a potential solution, and some studies have focused on elucidating anti-allergic functions of food components. For example, catechin derived from Japanese green tea6 or flavonoid derived from citrus fruits were demonstrated to have potential anti-allergic effects9. Identification and adoption of anti-allergic foods may be one strategy to decrease the severity of some allergic symptoms, such as those associated with pollinosis.The number of people suffering from an immunoglobulin E (IgE)-mediated (type I) response to an allergen has increased worldwide. Allergic reactions including hay fever, food allergy and bronchial asthma occur due to environmental antigens  such as pollenin vitro. Mast cells play an important role in immediate allergic reactions10. Antigen-specific IgE antibody binds to high-affinity IgE receptor (FcεRI) on the mast cell membrane11. In IgE-dependent mast cell activation, antigen-mediated cross-linking of cell surface FcεRI induces degranulation and the release of allergic chemical mediators such as histamine, cytokines and proteases12. Histamine not only regulates several essential events in the allergic response but also plays an important role both physiologically and pathologically13. Because β-hexosaminidase is also released along with histamine upon mast cell degranulation, this enzyme is typically used as a marker for mast cell degranulation in in vitro studies. Therefore, anti-allergic compounds are identified using inhibition of mast cell degranulation as an indicator. In in vivo studies, inhibitory testing of natural compounds or food extracts is usually performed by the passive cutaneous anaphylaxis (PCA) test as an animal model of IgE-mediated allergic response16. Anaphylaxis is triggered in response to allergen exposure following IgE sensitization17. When mast cells are exposed to an antigen, IgE binding brings FcεRI receptors on mast cells in close proximity, allowing cross-linking between receptors. Receptor cross-linking then triggers the release of chemical mediators from mast cells and basophils18. Therefore, the PCA test is widely used as in vivo model of type I allergy induced by the release of chemical mediators.In research on the anti-allergic effect of functional foods, mast cells are frequently used to detect an active compound from foods or to clarify the mechanism of anti-allergic effects Prunus mume is considered a traditional food and medicine in Asian countries such as Japan and China. In Japan, P. mume, called ‘ume’ in Japanese, is consumed dried or pickled with salt and in ume-containing liquor or soft drinks. Recently, studies have suggested that ume has the potential to prevent osteoporosis20, atherosclerosis21 and Helicobacter pylori infection22. Ume seed extract is known to have various functions including a protective effect in human ovarian granulosa cells against oxidative stress23 and inhibition of adult T cell leukaemia proliferation24. Research on the medicinal properties of ume extract is as important as those of processed ume because ume seed components are transferred into pulp, liquor or soft drinks during processing. Here, to understand the effect of ume intake, we conducted a pilot study targeting apparently healthy community-dwelling people to investigate the association between frequency of ume intake and allergic symptoms in a specific area in Japan. Then, to clarify the mechanism, the effect of ume seed extract was studied by the PCA test in mice. Bioactive ume compounds were detected by guided isolation based on the inhibitory effect of ume seed extract on allergen-mediated β-hexosaminidase release from mast cells and the mechanisms of compounds were discussed.ume intake and description of allergy symptoms. The median of age was 51 years (mean ± SD 50.8 ± 13.5) in men and 63 years (mean ± SD 60.7 ± 12.4) in women. The proportions of men and women with allergic symptoms were 21.2% (61/288) and 26.9% (74/275), respectively. Among allergic symptoms of men and women, pollinosis accounted for 70.5% (43/61) and 82.4% (61/74), respectively. A linear tendency was observed in the proportion of women with allergic symptoms among the 3 categories of ume intake: the higher the frequency of ume intake, the lower the proportion of women with symptoms of allergy  -specific IgE and intravenously challenged with the antigen 2,4-dinitrophenylated bovine serum albumin (DNP-BSA), PCA reaction was concomitantly induced with rapid capillary dilatation and increased vascular permeability in mouse ears. The PCA reaction was visualized by leakage of Evans blue dye into the reaction site of ears. However, in mice treated with oral administration of methanol extract of ume seed, vascular permeability of the ears was attenuated, as judged from the extent of blue staining in the ear , ear tissue contained intact toluidine blue-positive mast cells Fig. . In the ume seed, methanol extracts were further separated into hexane, dichloromethane, ethyl acetate and water fractions. These extracts were used to screen anti-allergic fractions guided by an inhibitory effect on β-hexosaminidase release of RBL-2H3 mast cells induced by antigen-antibody reaction. Because dichloromethane and ethyl acetate fractions exhibited an inhibitory effect on β-hexosaminidase release, these fractions were further separated and purified by column chromatography. Finally, we identified the following five active compounds from ume seeds by high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) and proton nuclear magnetic resonance (1H-NMR): vanillin (VA), syringic acid (SA), protocatechuic aldehyde (PA), lyoniresinol (LR) and p-coumaric acid (CA)  and all compounds were contained in pickled ume. No cytotoxicity was observed in the sample concentration range adopted in this study  of these compounds on degranulation was calculated from the β-hexosaminidase release inhibition rate. The IC50 values of VA, SA, PA, LR, CA and TL were 0.88 mM, 1.63 mM, 0.60 mM, 1.48 mM, 0.43 mM and 0.44 mM, respectively. We further examined the inhibitory effect of these ume compounds on IgE-mediated degranulation of mouse bone marrow-derived mast cells (BMMCs) as normal mast cells. All compounds showed inhibitory effects on BMMC degranulation, similar to their effects on RBL-2H3 cells  was measured in an area measuring 1024 × 1024 pixels monitored by confocal laser scanning microscopy (CLSM)   Fig. . Intraceume compounds on a non-IgE-mediated stimulus, calcium ionophore A23187-induced degranulation   Fig.  and actiEM) Fig. . Membranume intake and allergic symptoms in a specific area in Japan using a questionnaire survey. The proportion of women with allergic symptoms tended to be low in the high-intake group. Although it is the result of preliminary research, this result is consistent with the belief that ume is effective against allergic disease. Thus, to clarify the mechanism of ume on allergic reaction, we performed subsequent animal and in vitro studies.In the cross-sectional epidemiological pilot study, we investigated the association between ume seed extract on PCA reaction in IgE-sensitized mice. Antigen challenge induced vascular permeability of mouse ears and mast cell degranulation was attenuated by oral administration of ume seed extract. These results indicated that ume seed extract inhibits mast cell-mediated allergic reactions.We next demonstrated the effect of ume compounds, which were fractionated by guiding as an inhibitory effect on IgE-mediated degranulation of RBL-2H3 cells. The five isolated active compounds were VA, SA, PA, LR and CA. A few functions of these compounds are already known. For example, VA has been shown to decrease fat accumulation by increasing fatty acid oxidation and decreasing adipogenesis in 3T3-L1 adipocytes28. VA is also known to inhibit mast cell degranulation29. However, the inhibitory mechanism of VA is not clearly understood. The present study showed that VA inhibits IgE-mediated degranulation, antigen-induced intracellular Ca2+ mobilization and membrane ruffling but not non-IgE-mediated degranulation. Therefore, VA affects a part of the event from antigen stimulation to intracellular Ca2+ mobilization.We then identified the active et al. reported that LR derived from whisky congeners inhibits IgE-mediated degranulation of RBL-2H3 cells and reduces elevated intracellular Ca2+ concentrations29. In our study, LR hardly attenuated intracellular Ca2+ elevation in RBL-2H3 cells. A potential explanation for the discrepancy between the two studies may be the LR treatment period. In the previous study, the pretreatment period of LR was 30 min, whereas in our study it was 10 min. This result is consistent with the finding that LR had the lowest inhibitory effect of both β-hexosaminidase release and membrane ruffling among the five active compounds identified. In addition, LR did not inhibit non-IgE-mediated degranulation. Therefore, because LR does not inhibit non-IgE-mediated degranulation, it is expected to suppress the Ca2+-independent pathway, such as translocation of granules to the cell membrane30.Itoh 2+ ionophore-induced mast cell degranulation is inhibited by CA but not SA31. However, the present study showed that both CA and SA inhibited IgE-/non-IgE-mediated mast cell degranulation. These compounds also inhibited antigen-stimulated Ca2+ mobilization. Therefore, CA and SA may suppress not only Ca2+ mobilization due to IgE-mediated stimulation but also events occurring after elevated Ca2+ concentration. To our knowledge, this is the first report to demonstrate an anti-allergic effect for SA.It has been reported that Ca32, neuroprotective effects against oxidative stress33 and anti-cancer activities34. We also reported that PA has the potential to attenuate oxidative stress in human granulosa cells23. PA also inhibited both antigen-induced intracellular Ca2+ mobilization and membrane ruffling but not non-IgE-mediated degranulation, similar to VA. Therefore, PA likely affects a part of the event from antigen stimulation to intracellular Ca2+ mobilization. From the above findings, the five active ume compounds appeared to exhibit inhibitory effects through multiple mechanisms.PA has been demonstrated to have anti-inflammatory activities50 calculated from the β-hexosaminidase release inhibition rate revealed the degree of degranulation inhibitory effect of these active compounds (in decreasing order): CA, PA, VA, LR and SA. Furthermore, the IC50 of CA (0.43 mM) was equivalent to that of TL (0.44 mM), an anti-allergic drug. Therefore, although the degree of inhibitory effect of these compounds on IgE-mediated degranulation is variable, synergistic effects of multiple compounds are expected as various compounds are actually contained in the edible part of ume. Thus, we next investigated the combined effect of the five active compounds. From the effect on β-hexosaminidase release and analysis of the combined effect, we found that the combination of the five active compounds maintained the ability to inhibit mast cell degranulation  and nitric oxide (NO)38. The effect of ume and ume-related products suggested by the results of our study using human ovarian granulosa cell lines have been considered to be a regulation of sex hormone secretion23. From these views, ume intake may not only inhibit mast cell degranulation but also may influence the regulation of oestradiol or affect the regulation of oestradiol-mediated eNOS and NO, thereby exerting an anti-allergic effect in women. These views also may be related to the fact that the proportion of women with allergic symptoms in the high-intake group was lower than those in the middle- and low-intake groups, which was more prominent than in men.In this study, the frequency of et al. indicated ≥3 ume daily intake inhibits H. pylori infection, and they speculated the mechanism is related to the inhibitory effect of (+)-syringaresinol contained in ume on H. pylori motility39. Maekita et al. showed that ≥1 ume daily intake improves digestive motility symptoms40. The authors considered that increased alimentary fibres due to ume ingestion could also reduce digestive motility because ume fibres increase faecal output and improve gastrointestinal motility by promoting gastrointestinal tract emptying. Indeed, in experimental mouse studies, it has been reported that ume fibres possess faecal lipid excretion effects and faeces bulking effects, which affects the intestinal flora composition41. Recently, the correlation between allergic symptoms and intestinal microflora has been studied. Inhibition of an increase in bacterial species related to development of allergic symptoms may effectively prevent pollinosis42. Therefore, ume influences intestinal microflora and microorganisms, and daily ume intake may prevent allergy by maintaining the balance of intestinal microflora.In previous epidemiological studies, Enomoto ume inhibits allergic reactions from degranulation to inflammation because we only examined the effect of ume on mast cell degranulation. Furthermore, ume may influence processes from antigen exposure to antibody production. At least five compounds derived from ume were found to inhibit IgE-mediated degranulation from studies using RBL-2H3 cells and BMMCs. The PCA reaction was also inhibited by ume extract. These findings suggested that ume intake suppresses mast cell degranulation. However, the concentration of active compounds contained in the ume extract administered in the animal experiment was about 10–100 times higher than that contained in one piece of ume ingested by humans. Because the animal experiment in this study only examined the presence or absence of an anti-allergic effect by single administration, the amount of ume active compounds does not necessarily need to be consistent with amounts ingested by humans. However, future studies should also investigate the inhibitory effect of low-dose and long-term administration of ume on the PCA reaction similar to the effect of ume on the regulation of oestradiol and oestradiol-mediated eNOS and NO. In addition, we have not studied the inhibitory effect of degranulation due to intake of ume active compounds in humans. Therefore, it is also necessary to conduct detailed clinical investigations including not only the association between ume intake and allergic symptoms but also the effect of ume on sex hormones and eNOS. It is important to clarify these points in future work.There remains the possibility that ume intake may be associated with reduced risk of allergic symptoms in women. One of the mechanisms involved in the anti-allergic effect of ume is inhibition of mast cell degranulation, which was revealed by the PCA test using IgE-sensitized mice as an in vivo model of type I allergy. In addition, we isolated active ume compounds that exhibited inhibitory effects on IgE-mediated degranulation by using RBL-2H3 cells and BMMCs, which were confirmed as VA, SA, PA, LR and CA. The in vitro study suggested that these compounds inhibited IgE-sensitized mast cell degranulation by attenuating intracellular Ca2+-dependent or -independent pathways. These results suggested that ume has the potential to inhibit mast cell degranulation and may be associated with reduced risk of allergic symptoms.This cross-sectional epidemiological pilot study suggested that ume-growing region in Wakayama Prefecture. In December 2014, questionnaires were mailed to the 689 people who participated in Japan agricultural cooperatives-held community activities conducted in the target area in 2013. A stamped envelope was provided to encourage return of the questionnaire. We received the questionnaires from 586 participants  by March 2015. They were asked to answer the self-administered questionnaire including the following items about frequency of ume intake and allergy symptoms: ‘How often do you intake umeboshi?’ and ‘Do you have any allergies (symptoms including rhinitis during specific pollen seasons)?This study was performed in accordance with the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of Wakayama Medical University (Reference No. 1005). All participants provided written informed consent to participate in the study. The survey target was apparently healthy community-dwelling individuals living in the Kinan area, a well-known ume-related products, 95% of them ate pickled ume (‘umeboshi’ in Japanese). Other ume-containing products such as liquor, juice, jam and minor products were noted, however, the number of subjects who consumed these products and their intake frequency were low. In addition, these products were usually consumed with umeboshi. Thus, we judged that the intake frequency of other ume products was not necessary for analysis. Therefore, ume here means umeboshi.Among our participants who typically consumed ume intake frequency: ≥1 ume daily , <1 ume daily  and ≤2 ume weekly or none . In this study, we conducted all analyses stratified by sex, as prior studies have indicated sex differences in some allergic reactions in human46. First, descriptive analyses were performed. Next, the odds ratios (ORs) and 95% confidence intervals (95% CI) of ume intake frequency on presence of allergy symptoms were calculated using multivariate logistic regression analyses adjusted for age , presence/absence of illness  and presence/absence of medication . All statistical analyses were performed by using SPSS version 25.0 for Windows . P values < 0.05 were considered significant.In this study, after excluding participants with missing information (n = 23), the data of 563 participants (288 men and 275 women) were analysed. We divided 563 participants into 3 groups according to p-Nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG) was purchased from Merck Millipore .Mouse monoclonal anti-DNP antibody was purchased from Sigma-Aldrich . DNP-BSA was purchased from Biosearch Technologies . ad libitum. The experimental protocols of this study were approved by Wakayama Medical University Animal Care and Use Committee (Reference No. 679). All animal experiments were performed according to the Regulations for Animal Experiments at Wakayama Medical University.Male ICR mice (4-week-old) and male BALB/c mice (7-week-old) were purchased from CLEA Japan  and housed at 23 ± 1 °C and 55 ± 10% humidity under 12-h light/12-h dark cycles. Mice had access to laboratory rodent feed  and water Ume extract was dissolved in water and TL was suspended in 0.5% methyl cellulose solution . Mice were sacrificed under anaesthesia 30 min after antigen challenge, and their ears were removed.ICR mice (5- to 6-week-old) were sensitized with 20 μL of anti-DNP-IgE/PBS (25 μg/mL) by intradermal injection in the ears with a 0.5-mL insulin syringe . After 24 h, passive cutaneous anaphylaxis (PCA) reactions were induced by intravenous injection of 200 μL of DNP-BSA/PBS (0.5 mg/mL) containing 1% Evans blue. Samples or vehicle (water) were orally administered by feeding needle 1 h before antigen challenge. For measurement of Evans blue extravasation, mouse ears were punched by an 8-mm biopsy punch  and punched ears containing extravasated dye were dissolved in 800 μL of 1 N KOH at 37 °C for 24 h. An equal amount of phosphoric acid/acetone  was mixed with ears dissolved in KOH and filtered by a 0.45-μm filter. The absorbance of the extract was measured at 620 nm. For histological observation, remaining ears were fixed in 10% formalin and stained with toluidine blue .47 was obtained from the Japanese Collection of Research Bioresources Cell Bank . Cells were grown at 37 °C in 5% CO2 atmosphere in minimum essential medium , which was supplemented with 10% (v/v) fetal bovine serum , 100 U/mL penicillin and 100 µg/mL streptomycin .The rat mast cell line RBL-2H3, which has been well-documented to release IgE-mediated histamine, established from peripheral blood with basophilic leukaemia2 atmosphere in Dulbecco’s modified Eagle medium  supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin (Thermo Fisher Scientific), 5 ng/mL interleukin-3 , 5 ng/mL recombinant murine stem cell factor  and non-essential amino acid (Life Technologies). After 4 weeks of culture, ≥95% of cells were confirmed to be mast cells, as assessed by flow cytometric analysis of cellular staining with anti-FcεRIα conjugated FITC antibodies  and anti-c-kit conjugated PE antibodies (Miltenyi Biotec GmbH) using FACSVerse .BMMCs were obtained by culturing bone marrow cells (BMCs) from 7-week-old male BALB/c mice (CLEA Japan). BMCs, derived from femur, were cultured at 37 °C in 5% CO4 cells/well in 96-well plates and cultured for 24 h. Then, cells were treated with 100 μL of anti-DNP IgE at a concentration of 100 ng/mL in culture medium for 18 h. Cells were then pretreated with 50 μL of samples or vehicle  in HEPES-buffered Tyrode’s solution  for 10 min. Following pretreatment, cells were stimulated by addition of 50 µL of DNP-BSA (2 μg/mL) in HT buffer for 1 h at 37 °C.For antigen-induced β-hexosaminidase release assay, RBL-2H3 cells were seeded at a density of 7 × 104 cells in 96-well plates and cultured for 48 h. Cells were then pretreated with 50 μL of samples or vehicle (0.1% DMSO) in HT buffer for 10 min. Following pretreatment, cells were stimulated by addition of 50 µL of A23187 (10 μM) in HT buffer for 30 min at 37 °C.For calcium ionophore-induced β-hexosaminidase release assay, RBL-2H3 cells were seeded at a density of 7 × 10p-NAG in 50 mM citrate buffer (pH 4.5) was added to each well and plates were incubated at 37 °C for 30 min. The reaction was stopped by adding 100 μL of carbonate buffer (pH 10.6). The absorbance due to p-nitrophenol from hydrolysis of p-NAG by β-hexosaminidase was measured at 405 nm by a microplate reader . The absorbance at 650 nm was measured as a reference wavelength. The β-hexosaminidase release inhibition rate was calculated as follows:To determine β-hexosaminidase release, supernatants were moved to new plates and remaining cells were lysed in 100 μL of HT buffer with 0.1% Triton X-100. Then, 25 μL of 3.3 mM 5 cells/well in 96-well plates and cultured with anti-DNP IgE at a concentration of 100 ng/mL in medium for 18 h. Cells were then centrifuged at 1,000 rpm for 5 min and medium was removed. Cells were then pretreated with 50 μL of samples or vehicle (0.1% DMSO) in HT buffer. After 10 min, cells were stimulated by addition of 50 µL of DNP-BSA (2 μg/mL) in HT buffer for 1 h at 37 °C. For determination of β-hexosaminidase release, cells were centrifuged and supernatant and cells were separated. Subsequent operations and rate determination were carried out as described above for RBL-2H3 cells.BMMCs were seeded at a density of 1.0 × 10Ume seeds were a gift from Okahata Farm Co., Ltd. . The seeds were crushed, and kernels were removed. The kernel-removed seeds (described hereafter as ‘seeds’) were used for the experiment. The seeds (300 g) were extracted three times (for 3 h each) with methanol (900 mL). The methanol extract was filtered through a Whatman #1 filter paper and concentrated under reduced pressure.The resultant methanol extract (46 g) was suspended in distilled water (1 L) and extracted with hexane (3 L), dichloromethane (3 L) and ethyl acetate (3 L) to obtain hexane, dichloromethane and ethyl acetate fractions. Each fraction was concentrated under reduced pressure to yield hexane (3049 mg), dichloromethane (670 mg) and ethyl acetate (1130 mg) fractions. Each fraction was dissolved in DMSO and used to measure the inhibitory effect on β-hexosaminidase release. Active fractions, based on inhibitory effects on β-hexosaminidase release, were further separated by column chromatography as described below.The dichloromethane fraction (670 mg) was subjected to silica gel column chromatography and eluted with dichloromethane/acetone to separate six fractions (Fr. 1−6). Fr. 2 was further subjected to silica gel column chromatography and eluted with hexane/ethyl acetate to separate six subfractions (Fr. 7−12). Fr. 8 was further separated by preparative thin layer chromatography (TLC) with dichloromethane/acetone to yield compound 1 (0.6 mg). Fr. 4 was also further subjected to silica gel column chromatography and eluted with hexane/ethyl acetate to separate five subfractions (Fr. 13–17). Fr. 16 was separated by preparative TLC with dichloromethane/acetone to yield compound 2 (0.6 mg).2O/MeOH to separate eleven subfractions (Fr. 24–34). Fr. 27, 33 and 34 were subjected to preparative high-performance liquid chromatography (HPLC) to yield compounds 3 (1.5 mg), 4 (2.8 mg) and 5 (0.5 mg), respectively.The ethyl acetate extract (129 mg) was subjected to silica gel column chromatography and eluted with a gradient of dichloromethane/MeOH to separate six fractions (Fr. 18−23). Fr. 20 was further subjected to C18 column chromatography  and eluted with a gradient of Hp-coumaric acid by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), proton nuclear magnetic resonance (1H-NMR) and direct comparison with an authentic sample. The HR-ESI-MS spectrum was obtained using a micrOTOF mass spectrometer . The 1H-NMR spectrum was acquired using a Bruker AVANCE400 instrument .The chemical structures of compounds 1−5 were identified as vanillin, syringic acid, protocatechuic aldehyde, lyoniresinol and 4 cells/well in 96-well plates and cultured for 48 h. Then, cells were treated with 100 µL of samples or vehicle (0.1% DMSO) in culture medium. After 70 min , the medium was replaced with 100 µl of fresh medium, and 25 µl of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt  solution was added to each well. Cells were then incubated for 1 h at 37 °C in 5% CO2 atmosphere. The absorbance of each well was recorded at 490 nm using a microplate spectrometer because MTS is converted to a formazan product by dehydrogenase enzymes found in metabolically active cells. Cell viability was expressed as a percentage relative to the vehicle. Data are expressed as the mean ± SD of triplicate wells (n = 3).RBL-2H3 cells were seeded at a density of 7 × 1048. The CI and 50% inhibitory concentration (IC50) were calculated based on the β-hexosaminidase release inhibition rate using Compusyn software . The combined effect was defined as follows: CI < 0.7, synergism; 0.7 ≤ CI ≤ 0.9, moderate synergism; 0.9 < CI < 1.1, additive effect; 1.1 ≤ CI ≤ 1.45, moderate antagonism; 1.45 < CI < 3.3, antagonism; and 3.3 ≤ CI, strong antagonism.The combined effects of active compounds were evaluated by using the combination index (CI) estimated from the median-effect theorem2+ was monitored by CLSM using fluo-3 acetoxymethyl ester  as an indicator. RBL-2H3 cells (2.5 × 105 cells/well) were subcultured on 8-well chamber slides  and sensitized with 100 ng/mL of anti-DNP IgE for 18 h. Cells were then incubated at 37 °C for 30 min with HT buffer in the presence of 3 μM fluo 3-AM, 0.04% cremophor (Wako) and 1.25 mM probenecid (Sigma). Before antigen challenge, cells were preincubated with 250 µL of HT buffer containing bioactive compounds from ume seed extract or DMSO for 10 min.Intracellular Ca2+ images were recorded using CLSM. Successive images were collected at 6-s intervals with a convert 20×/0.8 NA  objective lens. Fluo 3-AM fluorescence was excited with a laser at 488 nm and an LP490 emission filter was chosen. After starting the time-lapse image scan, DNP-BSA was added to the well  at the indicated time point. Quantification of the fluorescence intensity of the scan area was performed using ZEN2009 software (Carl Zeiss).Ca4 cells/well in 96-well cover glass bottom plates and cultured for 24 h. Then, cells were treated with 100 μL of anti-DNP IgE at a concentration of 100 ng/mL for 18 h. Cells were then pretreated with 50 μL of samples or vehicle (0.1% DMSO) in HT buffer for 10 min. After pretreatment, cells were stimulated by addition of 50 µL of DNP-BSA (2 μg/mL) in HT buffer for 1 h at 37 °C.RBL-2H3 cells were seeded at a density of 7 × 10For CLSM imaging, cells were washed with PBS twice and fixed with 4% (w/v) paraformaldehyde for 10 min at room temperature. Following two washes with PBS, cells were permeabilized in 0.5% Triton X-100 in PBS for 5 min at room temperature. For actin cytoskeleton staining, cells were treated with Acti-stain 488 phalloidin (Wako) for 30 min. Cells were visualized by CLSM . Acti-stain 488 phalloidin was excited by a 488-nm wavelength argon-ion laser. Optical fluorescence signals of Acti-stain 488 phalloidin were observed using an emission filter . Confocal images were acquired using Plan-Apochromat  objective lens. On taking confocal images , Z-directional movement for optical sectioning of the entire specimen was controlled by the focus motor unit on axial scanning at 0.7–1.0 μm focus steps. CLSM image resolution was set at 1024 × 1024 pixels. The images taken using CLSM can be made into computer-assisted three-dimensional reconstructions using ZEN2009 software . The three-dimensional images were converted to one-dimensional stacked images using ZEN2009 software.23. Briefly, cells were fixed with 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4) for 2 h at 4 °C. Cells were washed with PBS and dehydrated using a graded series of ethanol , 50% tert-butyl alcohol (in ethanol) and 100% tert-butyl alcohol. Then, dehydrated cells were lyophilized. Samples were coated with palladium and platinum using an E102 ion sputter coater , followed by SEM observation .For SEM, cells were prepared by a general method as previously describedt-test was used to analyse the results of animal and in vitro studies. P values < 0.05 were considered significant. All statistical analyses were performed by using JMP Pro 12 software .Student’s


Arctic and Antarctic polynyas are crucial sites for deep-water formation, which helps sustain global ocean circulation. During glacial times, the occurrence of polynyas proximal to expansive ice sheets in both hemispheres has been proposed to explain limited ocean ventilation and a habitat requirement for marine and higher-trophic terrestrial fauna. Nonetheless, their existence remains equivocal, not least due to the hitherto paucity of sufficiently characteristic proxy data. Here we demonstrate polynya formation in front of the NW Eurasian ice sheets during the Last Glacial Maximum (LGM), which resulted from katabatic winds blowing seaward of the ice shelves and upwelling of warm, sub-surface Atlantic water. These polynyas sustained ice-sheet build-up, ocean ventilation, and marine productivity in an otherwise glacial Arctic desert. Following the catastrophic meltwater discharge from the collapsing ice sheets at ~17.5 ka BP, polynya formation ceased, marine productivity declined dramatically, and sea ice expanded rapidly to cover the entire Nordic Seas. Polynyas potentially played a role in sustaining marine life during the last glacial, yet their presence and importance remains equivocal. This multi-proxy study reconstructs a corridor of polynyas in the Nordic Seas during the last glacial maximum, and reveals a strong association with biological productivity. A major source of bottom waters is found along the East Antarctic coastline in a myriad of coastal polynyas that form in response to strong katabatic winds originating from the adjacent ice masses4. High rates of sea ice growth and consequential ejection of salt in these polynyas contribute to the production of dense shelf waters (brines), and thus deep water renewal in the global ocean. Although this model was proposed initially to explain persistent ocean convection during past cold phases  in the glacial Nordic Seas5, it was subsequently questioned, since brine-enriched shelf waters are isotopically indistinguishable from deep water generated by ocean convection7. More recently, however, Keigwin and Swift8 found evidence for glacial deep water in the western North Atlantic that may have been formed through enhanced sea ice growth rates and brine rejection in the Labrador Sea, potentially associated with polynya formation. In the Nordic Seas, Thornalley, et al.9 also discussed the possibility of limited deep-water formation as a consequence of brine rejection from coastal polynyas. The existence and location of these polynyas, however, remain unresolved.Coastal polynyas on Arctic and Antarctic shelves today are widely recognized to be sites of deep water formation12. The presence of the grounded ice sheet at the shelf edge maintained the formation of coastal polynyas between ca. 25 and 19 ka13, which then became inactive when the ice sheet retreated. Apart from their importance for deep water formation, coastal polynyas adjacent to expansive ice sheets are also regional moisture sources16. Further, they are recognized as key sites for enhanced primary and secondary productivity associated with sea-ice controlled seasonal nutrient supply during advance and retreat, and thus represent oases for higher-trophic life in an otherwise glacial desert17.In Antarctica, coastal polynyas are inferred to have occurred beyond the glacial ice-sheet margin, with their formation probably enhanced by offshore katabatic winds and amplified further by diurnal tides during the last glacial period and the Holocene20, the margins were preconditioned for the formation of coastal polynyas. Previously, the dominance of planktic and benthic foraminifera and the occurrence of the seasonal sea ice biomarker IP25 in LGM sediments from the northern Barents Sea margin have been used to propose the katabatic wind-driven formation of coastal polynyas supported by upwelling of Atlantic-derived water masses16. Meanwhile, at the western margin, advection of Atlantic water (AW) and seasonally sea ice free conditions prevailed during the LGM22. Although no direct inferences of polynya formation have been made so far in this region, Bauch, et al.14 postulated that sub-surface Atlantic water advection into the Nordic Seas was possibly facilitated by polynyas.Corroboration of coastal polynyas as suppliers of moisture to the build-up of adjacent ice sheets, and as potential sea-ice factories that facilitate regional ventilation and primary productivity in the glacial Nordic Seas, requires confirmation of their existence through multi-proxy evidence preserved in the sedimentary archive. Since the Barents Sea shelf was fully glaciated during the Last Glacial Maximum (LGM) between 26.5 and 19 kaIn the present study, we combine multi-biological proxy data with sedimentary physico-chemical characteristics to show that coastal polynyas indeed existed along the entire Svalbard-Barents Sea margin during the LGM, based on the evidence of a highly dynamic sea ice cover coupled with enhanced plankton productivity adjacent to a grounded ice sheet at the continental shelf. In contrast to previous studies, we propose the existence of an open water corridor in front of the NW Eurasian ice sheet that was controlled, primarily, by a combination of strong katabatic winds blowing seaward off the ice shelves and upwelling of relatively warm intermediate Atlantic water masses. This polynya activity along the entire continental margin of the Barents Sea was the ultimate reason for weak (but constant) ocean convection and persistence of higher-trophic life in otherwise heavily sea-ice covered Nordic Seas during the LGM. The duration of this polynya scenario is closely tied to the stability of the marine-based Svalbard-Barents Sea ice sheet (SBIS). Thus, with the onset of the SBIS deglaciation at ~19.5 ka, biological activity in the polynyas ceased, and finally stopped when the ice sheet collapsed at ~17.5 ka, triggering the formation of perennial sea ice cover over the entire Nordic Seas as a consequence of extreme freshwater modulation of the surface ocean and significant weakening of the AMOC.www.mareano.no)23. During two cruises with RV G.O. Sars in 2012 and 2014, a gravity core  and a giant piston core  were retrieved from the same location on the upper continental slope in ~949 m water depth , which transports water of Atlantic origin northward through the Norwegian Sea29. North Atlantic Water is characterized by high salinity (>35‰) and temperature (>6 °C) at the mid-Norwegian margin, and is typically found on the upper slope shallower than ~700 m water depth30.The study area Fig.  was mapp3, water content between 40 and 60%, and low magnetic susceptibility (MS ~50 10−5 SI). Sequences enriched in IRD are slightly denser (~2.1 g/cm3) and/or characterized by elevated MS values. Facies (2) This facies is more heterogenous with higher amounts of coarse-sand, higher density (2.2 g/cm3) and lower water content (~25–30%). The two short intervals around 442 and 540 cm with Facies 2 characteristics are interpreted as mass flow deposits. The core top (ca. 20–30 core depth cm) is sandy-rich and most likely a distal deposit of nearby reported sandwaves23  and mixed benthic and planktic foraminiferal assemblages from parallel core GC08  by applying the Calib7.1 program and the Marine13 calibration curve31. No local reservoir age (ΔR = 0) was applied. We are aware of large uncertainties in reservoir ages in the Nordic Seas during the LGM and early deglaciation32 and the consequence of younger ages when applying local reservoir ages between ΔR = 0 to 400–500. Here, we elected to use ΔR = 0 to enable us to make a direct comparison to other key records from the North Atlantic and Nordic Seas. To generate a common depth model for both cores, we used the XRF Ca records to identify several tie-points (0–5) 34  of GS14-190 is constrained by 6 accelerator mass spectrometry (AMS) radiocarbon datings obtained from tests of planktic or mixed benthic and planktic foraminifera . The LGM is characterized by the heaviest δ18O values (ranging from 4.9‰ to 5.2‰) and rapid δ18O depletion during the deglacial phase at ~17.5 ka BP  reliably reflects the overlying sea ice conditions. Thus, absent IP25 has been reported for sites of year-round ice free conditions (including those close to the GS14-190 core site). Relatively low concentrations are found close to the winter sea ice margin, with highest abundances in regions of more extensive seasonal sea ice cover, which are mainly >76°N38  from glacial inception at ~30 ka BP to the LGM (~21 ka BP), superimposed by short events of maximum rates during first shelf edge glaciation (24–23 ka BP) and initial ice sheet collapse (~20–19 ka BP) indicate a slow, but continuous movement of the ice sheet towards the shelf break  occur between ~20–19 ka BP and suggest enhanced iceberg calving and debris release from the retreating grounding line , slightly reduced IP25 and dinosterol concentration, as well as moderately abundant planktic foraminifera do not support proximal water mass perturbations 56.The commencement of ice sheet disintegration in the SW Barents Sea is not, however, followed by large-scale meltwater injections into the Norwegian Sea. Indeed, heavy δ18O ~ ‰, sligh BP Fig.  may be c BP Fig. 52, changait Fig.  and sub-19, the ice sheet disintegrated rapidly after ~17.6 ka, with a modeled rate of ~670 gigatonnes per year (Gt a−1)45. This enhanced rate is reflected in a large δ18O planktic anomaly in the study area57 and indicates a massive meltwater injection into the eastern Nordic Seas5 and Arctic Ocean22 during the onset of HS1  where marine and terrestrial faunal changes were found to be highly correlated with sea ice dynamics in coastal polynyas12. It has also been postulated that the existence of polynyas during glacial periods was essential for the survival of marine and terrestrial life17. Indeed, previous reports of unusually high abundances of the benthic foraminifer Cassidulina neoteretis and smaller-sized planktonic specimens of Turborotalita quinqueloba in LGM sediments off western Svalbard51 indicate the existence of a productive benthic oasis in an otherwise glacial desert at this time, with nutrient supply and organic matter production in a marginal ice zone supported by upwelling of nutrient-rich Atlantic-derived water masses.From a broader perspective, the finding of large scale polynyas in front of LGM ice sheets in this study support recent inferences by Keigwin and Swift51 in an environment that is characterized by a marginal sea ice zone in close proximity to the Svalbard-Barents Sea continental margin and upwelling of nutrient-rich, sub-surface flowing Atlantic waters during this period. Polynya activity likely collapsed when ice sheets disintegrated and freshwater injections to the eastern Nordic Seas caused rapid sea ice expansion in the entire Nordic Seas and AMOC weakening during Heinrich Stadial 1, reflected by a significant δ18O anomaly and near complete cessation of sympagic and pelagic productivity. Overall, our findings confirm the crucial importance of coastal polynyas for survival of planktic and benthic species during otherwise harsh glacial conditions in the Arctic.Hence, we propose the presence of large-scale coastal polynyas in front of the extensive NW Eurasian ice sheets during the Last Glacial Maximum in an otherwise Arctic desert, characterized by a perennial sea-ice cover. This conclusion is built on the co-existence of high amounts of sea-ice algae, primary and secondary plankton producers, and a high diversity of benthic and planktic faunaWhole core measurements, i.e. wet bulk density (WBD) and magnetic susceptibility (MS) were conducted on the gravity and piston cores using the Standard MSCL-S core logger  at 1 cm resolution with 5 s measurement time. MS measurements for whole cores were done with a Bartington MS2C loop sensor with 130 mm coil diameter. For control of the MS2C sensor, a certified sample piece with known magnetic susceptibility was measured. After lengthwise splitting and surface cleaning core surface images were taken with the GeoScan IV color line-scan camera. The camera was equipped with a Nikon AF Nikkor 50 mm f/1.8D lens and three detectors using three 2048 pixel charge-coupled device CCD arrays for red, green and blue light. The core surface was continuously imaged with 100 µm down and cross core resolution. Each core section was measured with an X-rite ColorChecker as reference for basic color control.X-ray images (XRI) of split cores were taken with the Geotek MSCL-XCT . The device is equipped with a Thermo Kevex PSX10-65W X-ray source  and a Varian PAXScan 2520 V X-ray detector . Voltages of 87 and 120 kV and electric currents of 125 and 140 µA were used for imaging of both cores.4P4O7×10H2O, MerckPA) and sonicated. To compensate for elevated silt contents compared to traditional methods (Pipette and Sedigraph), we applied the method published by Rise and Brendryen64. Grain sizes >250 µm were determined by dry sieving on a duplicate of each sample (relative error ± 10%) and identified as ice-rafted debris (IRD).The grain size distribution (0.4 µm – 250 µm) was determined with a Coulter LS 200. To prevent charging and agglomeration of particles, de-carbonated samples were treated with 5% sodium pyrophosphate , aluminum (Al), and calcium (Ca) concentrations (ppm) were used for this study.X-Ray fluorescence (XRF) core logging was carried out with the Standard MSCL (MSCL-S) core logger  and an attached DELTA Handheld XRF sensor. The XRF sensor is equipped with a 4-W Rh Tube anode and Si drift detector. Prior to core measurements the XRF sensor was standardized and SRM 2710a Montana soil I standard sampleorg) were performed with a LECO SC-632. For TC determination subsamples of 300–400 mg were combusted at 1350 °C and the release of CO2 determined. For Corg analysis, sub-samples of 400–450 mg were placed in carbon-free pervious ceramic combustion boats. These were placed on a heating plate at 50 °C ( ± 5 °C) and treated with 10 vol.% hydrochloric acid (HCl) to remove inorganic carbon (carbonate) and subsequently rinsed with distilled water and dried in the drying oven prior to analysis. Results are given in weight percentage (wt. %) and the standard deviation of the TC and Corg measurements based on the repeated measurement of a standard was ± 0.026 w. t%  and ± 0.028 wt. % , respectively.Analyses of total (TC) and organic carbon  to freeze-dried sediments (ca. 1–3 g), extraction  and purification of extracts using silica column chromatography . Further purification to remove saturated hydrocarbons was achieved using Ag-ion chromatography , followed by separate elution of unsaturated hydrocarbons including IP25 using acetone (2 mL). Sterol fractions were obtained following internal standard addition  to freeze-dried sediments, saponification  and back extraction into hexane (3 × 2 mL). Silica column chromatography was used to remove impurities, including hydrocarbons and long-chain ketones , and for sterol elution . Partially purified IP25- and sterol-containing fractions were analyzed using gas chromatography-mass spectrometry (GC-MS) according to established methods67. IP25 and sterols were identified by comparison of total ion current (TIC) mass spectra and retention indices to those of authentic standards or those presented previously68. Biomarker quantification was carried out in Selective Ion Monitoring (SIM) mode by comparing the peak areas of molecular ions of individual biomarkers to those of the internal standards, followed by correction for sediment mass and ion fragmentation efficiency. In the current study, we confine the sterol analysis to that of dinosterol.The sea ice biomarker IP18O), monospecific planktic foraminifera Neogloboquadrina pachyderma ) were hand-picked from the >100 µm size fraction. For each interval, 5–10 specimens were analyzed. δ18O values were determined using a Thermo Scientific MAT253 mass spectrometer coupled to a Finnigan Gasbench II at UiT, The Arctic University of Norway in Tromsø. Results are reported relative to the Vienna Standard Mean Ocean Water (VSMOW) standards in per mil (‰) notation. External precision was ± 0.07‰ based on analysis of NBS-19.For planktic foraminiferal concentration/abundance the sediment core was sampled in ca. 20 cm intervals. Approximately 300 specimens from >125 µm fraction were picked and identified per sample, using a micro-splitter if the number of foraminifera was higher. Only one sample contains less than 200 planktic foraminifera (depth 368 cm); one sample (depth 345 cm) is barren of foraminifera. For oxygen stable isotope analyses (δSupplementary InformationPeer Review File


Childhood diarrheal disease causes significant morbidity and mortality in low and middle-income countries, yet our ability to accurately predict diarrhea incidence remains limited. El Niño-Southern Oscillation (ENSO) has been shown to affect diarrhea dynamics in South America and Asia. However, understanding of its effects in sub-Saharan Africa, where the burden of under-5 diarrhea is high, remains inadequate. Here we investigate the connections between ENSO, local environmental conditions, and childhood diarrheal disease in Chobe District, Botswana. Our results demonstrate that La Niña conditions are associated with cooler temperatures, increased rainfall, and higher flooding in the Chobe region during the rainy season. In turn, La Niña conditions lagged 0–5 months are associated with higher than average incidence of under-5 diarrhea in the early rainy season. These findings demonstrate the potential use of ENSO as a long-lead prediction tool for childhood diarrhea in southern Africa. Here, Heaney et al. show that La Niña conditions are associated with higher than average incidence of childhood diarrheal disease in Botswana in the early rainy season. This finding could help to predict childhood diarrhea outbreaks in southern Africa. Rates of under-5 diarrhea in Africa are particularly high, with an estimated incidence of 3.3 episodes of diarrhea per child each year and 25% of childhood mortality caused by diarrhea3. Despite the high burden in this region, our ability to predict and prepare for diarrheal case surges remains limited.Diarrheal disease is a controllable disease but continues to cause significant morbidity and mortality in low and middle-income countries. In these regions, diarrhea is the second leading cause of death in children younger than five years of age, with 72% of deaths occurring in the first two years of life8, and drought conditions can concentrate animal activity increasing the movement of diarrhea-causing pathogens into surface water resources12. High ambient temperatures can further promote diarrhea transmission by enhancing pathogen replication rates or by changing water usage behaviors and hygiene and sanitation practices13. Identifying these potential couplings between meteorological drivers and disease outbreaks in a particular location can contribute to an enhanced ability to improve disease forecasting and outbreak preparedness.Infectious diarrhea is caused by many different pathogens  and meteorological conditions can have critical influence on pathogen exposures, in particular those associated with waterborne transmission. For example, extreme rainfall events may contaminate drinking water by flushing diarrhea-causing pathogens from pastures and dwellings into drinking water supplies14, limited attention has been directed at understanding how El Niño-Southern Oscillation (ENSO) moderates climate–diarrhea relationships. ENSO is a coupled ocean-atmosphere system spanning the equatorial Pacific Ocean. ENSO oscillates between two end-member extremes, El Niño and La Niña, which manifest in part as periodic departures from average sea surface temperatures (SSTs) in the central equatorial Pacific Ocean. ENSO has a 3–7 year periodicity between El Niño events , and La Niña events, . These SST deviations are associated with changes in convection over the Pacific that produce large-scale waves in the atmosphere that affect local weather patterns around the world, including temperatures, winds, and precipitation.While the impacts of meteorological variability on diarrhea incidence have been well documented17, Bangladesh19, China20, and Japan21, but studies of the effects of ENSO on diarrheal disease in Africa have been limited to cholera22. Increases in cholera incidence have been linked to El Niño driven rainfall increases in Uganda23 and the Great Lakes Region24. Moore et al.25 demonstrated an El Niño driven geographic shift in cholera incidence from Madagascar and West Africa to East Africa. In this study, higher cholera incidence was observed in regions with both increased rainfall and decreased rainfall, suggesting a complex relationship between ENSO and cholera transmission25. Cholera is indeed an important agent of childhood diarrhea, but recent etiological analyses estimate that it causes less than 1% of childhood diarrhea cases in Africa27. Additionally, the health impacts of cholera are mostly ephemeral while all-cause diarrhea has been linked to long term health effects like stunting and impaired cognitive development29.El Niño events have been linked to diarrhea outbreaks in Peru32, and decreased vegetation across southern Africa33. These effects are specifically apparent in Botswana, where El Niño events have been consistently associated with below normal rainfall during the summer rainy season35. The large meteorological shifts associated with ENSO provide a useful natural experiment to understand the relationships between climatic change and infectious diseases. Indeed, previous studies have shown that ENSO variability is strongly linked to anomalous rainfall patterns in Botswana from December–February and can be used to predict malaria dynamics37.Here, we explore the link between ENSO and under-5 diarrhea incidence in Botswana. A strong ENSO teleconnection has previously been documented across southern Africa. Warmer than average SSTs in the Pacific Ocean have been linked to lower summer (December–March) rainfallShigella, Salmonella, Cryptosporidium, Giardia lamblia, rotavirus, and norovirus41. While rotavirus infections have been shown to peak in the cool dry winter season in Botswana41, the seasonal patterns of other diarrhea-causing pathogens in this region remain uncharacterized. A monovalent rotavirus vaccine (RV1) was introduced across Botswana in July, 2012 with high uptake across the country42.Botswana is a middle-income country in southern Africa whose government provides heavily subsidized healthcare and has a well-developed water treatment and reticulation infrastructure, yet still experiences significant under-5 diarrhea burden annually. Etiological studies show that under-5 diarrhea in Botswana is caused by a multitude of pathogens, including 44, with attack rates highest for children younger than one year of age46. In this system, variability in rainfall, Chobe River height, and temperature are significantly associated with under-5 diarrhea case reports44.Chobe District is a hydrologically dynamic region in northeastern Botswana that experiences annual wet (November–March) and dry (April–October) seasons. The Chobe River floodplain system, which is the primary source of water for the district, floods annually with a peak flood height occurring in the late wet season/early dry season (March) Fig. . Our preGiven the established links between ENSO and rainfall in Botswana, we hypothesize that ENSO variability is an important driver of diarrhea dynamics in this region. Specifically, we explore the following questions: (1) Does ENSO influence local environmental conditions in Chobe district? (2) Does a relationship exist between ENSO and under-5 diarrhea incidence in Chobe district? and (3) Does the introduction of a rotavirus vaccine in 2012 affect any identified ENSO-diarrhea association?n = 127 months), there were 10,946 cases of under-5 diarrhea reported across the ten health facilities in Chobe district, Botswana. On average, diarrhea case reports peaked in January in the wet season and August in the dry season  and positively correlated with minimum temperature  at lags of 0–3 months.ENSO was significantly associated with local environmental conditions in Chobe District. Across all seasons and years, monthly La Niña (El Niño) conditions were associated with cooler (warmer) conditions and higher (lower) Chobe River heights Table . SpecifiAnalyzes at seasonal time scales revealed that Niño 3.4 anomalies had significant negative associations with total rainfall in December, January, February (DJF)  and average DJF Chobe River height (0–8 months lags), and a significant positive association with average DJF minimum temperatures (0–9 months lags) Fig. . At an 8Examination of the broader regional association of rainfall with ENSO revealed the source of the significant teleconnection with Chobe River height. The Chobe River is part of the Zambezi River Basin and is fed by the Cuando River system in the Angolan highlands. Satellite estimated rainfall upstream of Chobe River was negatively associated with Niño 3.4 during DJF Fig. . This fi44, we hypothesized that higher than average Chobe River flooding would lead to increased concentrations of organic material (including diarrhea-causing pathogens) in Chobe River. Indeed, we observed that Niño 3.4 was negatively associated with DJF concentrations of E. coli in the Chobe River at 0–8 months lags .Based on our previous workp < 0.001, Pearson’s correlation)  Table . At a 5-n) Table .Fig. 4AsThese results were robust to sensitivity analyzes accounting for the monovalent rotavirus vaccine (RV1) introduction in July 2012. A strong El Niño event  during 2014/2015 co-occurred with much lower than average under-5 diarrhea cases Fig. . Before Our findings confirm the existence of a teleconnection between ENSO and environmental conditions in the northern region of Botswana and demonstrate an association between ENSO variability and local childhood diarrhea incidence. Specifically, we show that cooler than average Niño 3.4 SSTs were associated with cooler and wetter conditions in the Chobe District as well as the upper reaches of the Chobe River basin, and were also associated with increased under-5 diarrhea cases. The effects were strongest in the DJF season, when the Chobe River height increases annually due to local and upstream rainfall. Due to the long lead-lag associations with ENSO in the Chobe region, under-5 diarrhea cases during DJF could be predicted up to 5 months in advance.37. Thomson et al.37 found a negative association between Niño 3.4 and DJF rainfall across Botswana, and linked Niño 3.4 and DJF rainfall to annual malaria incidence. These ENSO-malaria relationships were then used in conjunction with multi-model ensemble climate predictions to forecast malaria incidence up to 5 months in advance37. These studies in combination with our results underscore the potential use of publicly available ENSO information for long-lead infectious disease prediction systems in Botswana and other regions with strong ENSO teleconnections.Existing studies that linked ENSO to malaria incidence in Botswana reported similar findings to those presented here47, famine48, and infectious diseases22. Climate change projections suggest that Botswana will become progressively hotter and drier49. Between 1926 and 2011, rainfall decreased in Botswana by an average of 0.861 mm/year50, which has led to a significant decrease in river flow throughout the country. Here, we show that El Niño conditions, i.e., drier than normal conditions, are associated with fewer cases of under-5 diarrhea in the wet season. These results may presage changes in the burden of under-5 diarrhea in Botswana as a result of anthropogenic climate change.ENSO can represent a natural experiment that is useful for inferring the impacts of climate change on various phenomena, such as global conflict51. Niño 3.4 was significantly associated with DJF rainfall at 0–4 months lags, DJF river height at 0–8 months lags, DJF minimum temperature at 0–9 months lags, and DJF under-5 diarrhea incidence 0–5 and 8–9 months lags. However, Niño 3.4 did not significantly predict DJF hydrometeorological conditions or under-5 diarrhea when lagged 10–12 months. Existing work has shown that ENSO forecasts initialized in February–May have much lower accuracy than forecasts initialized during other months of the year. Similarly, ENSO indices such as Niño 3.4 have strong temporal autocorrelation, which allows for predictions to be made at long-lead times, but the temporal autocorrelation becomes inconsistent during February–May51. As a result, it is difficult to predict DJF ENSO conditions in the previous February–May. Here, we show that Niño 3.4 accurately predicts local DJF meteorological conditions and diarrhea incidence when lagged 0–8 months (corresponding February–previous June), but has poorer predictive ability when lagged 9–12 months (previous May–previous February), as would be expected, given the spring predictability barrier.Our results also confirm the well-documented ENSO spring predictability barrier52, here we find that La Niña conditions were associated with increased under-5 diarrhea incidence. This divergence emphasizes the importance of considering local teleconnections and climate–disease relationships when making ENSO-based predictions. ENSO teleconnections are geographically diverse;53 while El Niño events produce colder and drier conditions in Botswana, they drive increases in temperature and rainfall in Peru, eastern Africa, and Bangladesh (sites of previous studies). Furthermore, any associations between ENSO and diarrhea cases will be mediated by local transmission pathways, water treatment infrastructure (discussed more fully below), and health behavior. In short, while ENSO can be a powerful prediction tool for diarrhea incidence, results from ENSO-diarrhea studies should not be generalized to other regions.While existing studies have linked El Niño events to diarrhea outbreaks54, and we found that ENSO mostly influences under-5 diarrhea cases during DJF, when pathogens other than rotavirus are the principle drivers of diarrhea. Second, ENSO has a similar association with DJF under-5 diarrhea incidence when we stratified the analysis before and after the RV1 introduction. Childhood diarrhea in Botswana is caused by a multeity of pathogens other than rotavirus, including Shigella, Salmonella, and Cryptosporidium55. Therefore, while rotavirus prevalence has declined, other diarrhea-causing pathogens are still circulating and susceptible to environmental perturbation. These results also emphasize the importance of accounting for environmental changes such as ENSO events when evaluating the effects of interventions such as vaccines. Two analyses have determined that the rotavirus vaccine was successful in lowering under-5 diarrhea incidence in Botswana56; however, these studies may have overestimated the effectiveness of RV1 because they did not account for the possible effects of the 2014/2015 El Niño event on the reduction of overall diarrhea cases.Our findings indicate that ENSO remained an important driver of under-5 diarrhea dynamics in northern Botswana after the introduction of a rotavirus vaccine (RV1). While the RV1 introduction may have modified the observed ENSO under-5 diarrhea relationships, we believe it did not significantly affect these associations for two reasons. First, rotavirus transmission almost exclusively occurs from June to October44. Here, we provide further evidence that higher than average Chobe River flooding and rainfall during the wet season, driven by La Niña conditions, is associated with increased E. coli concentrations in the river and higher childhood diarrhea incidence. While the associations between Niño 3.4 and E. coli were not statistically significant  is associated with about a 30% increase in diarrhea incidence during DJF. There are 305 diarrhea cases on average during DJF across all years, so a 30% increase would roughly translate to an increase of 92 cases. Advanced stockpiling of necessary medical supplies, preparation of hospital beds and organization of healthcare workers could dramatically improve the ability of health facilities to manage higher than expected diarrheal disease incidence, potentially reducing morbidity and mortality impacts in children under-5. Long-lead prediction systems may also allow the development of other interventions including public health messaging campaigns and distribution of household water disinfection supplies. Utilizing climate information to forecast diarrheal disease could be particularly useful in low resource settings where healthcare infrastructure and surveillance systems may be less developed.59. Chobe District is also home to the Chobe National Park, which provides important habitat for the largest elephant population in Africa, as well as an abundance of other wildlife species.Botswana is a semi-arid, landlocked country in southern Africa. The country has a subtropical climate with annual wet (November–March) and dry (April–October) seasons. Intra-annual and inter-annual precipitation variability is high, resulting in frequent droughts and flooding. This study focuses on Chobe District, located in the northeastern part of Botswana Fig. . The Chon = 127 months).As part of a program of syndromic disease surveillance, the Botswana Integrated Disease Surveillance and Response (IDSR) Program collates the weekly number of children under 5 presenting with diarrhea to health facilities. We extracted weekly reports from 10 of the 16 healthcare facilities in Chobe District. A diarrhea case was defined as the occurrence of at least three loose stools in a 24-h period within the four days preceding the health visit. Weekly incidence reports from the healthcare facilities were summed to obtain total monthly incidence estimates for Chobe District from January 2007 to June 2017 , were not included in the analysis. For weeks with greater than zero but fewer than ten clinics reporting cases, we used the total number of cases reported in a given week divided by the number of clinics/hospitals reporting that week. This method provides a weekly estimate of the total under-5 diarrhea incidence per clinic/hospital reporting. Weekly estimates of incidence per clinic/hospital were then multiplied by 10 to estimate the total weekly incidence from all l0 clinics/hospitals.  Monthly diarrhea anomalies were then calculated as follows:Daily measurements of minimum temperature, and rainfall were obtained from the Department of Meteorological Services under the Ministry of Environment, Wildlife and Tourism. Minimum temperature was measured daily in Kasane; rainfall was measured in both Kasane and Kavimba, and then averaged to produce daily estimates for Chobe District. The Water Affairs Department in Kasane provided daily measurements of Chobe River height. All environmental variables were aggregated to a monthly resolution and span 2007–2017.60. We aggregated TRMM daily rainfall across all of southern Africa to derive monthly estimates of total rainfall in each grid cell from 1998 to 2015.Regional rainfall data were obtained from the Tropical Rainfall Measuring Mission (TRMM) database across southern Africa. TRMM combines rainfall measurements from multiple satellites and available meteorological stations to produce a fine scale (0.25°) daily gridded rainfall dataset61. Niño 3.4 represents a three-month running mean of equatorial SSTs  and is presented as monthly anomalies with respect to 1971–2000 climatology. Monthly Niño 3.4 anomalies are interpreted as temperature in Kelvin above or below monthly mean SSTs. These anomalies represent variability in the ENSO cycle and can be used to indicate ENSO conditions. El Niño events are defined by the National Oceanic and Atmospheric Association (NOAA) as Niño 3.4 SST anomalies above 0.5 K for at least five consecutive months, and La Niña events are conversely defined as Niño 3.4 SST anomalies below −0.5 K for at least five consecutive months62. We also ran analyses with other ENSO indices, Niño 4, SOI, and MEI. Results of analyses using these ENSO indices were very similar to the results from analyses using Niño 3.4 and are shown in the Supplementary Tables To estimate the strength of ENSO, we used the Niño 3.4 index, which provides historical measures of sea surface temperatures (SSTs) in the equatorial Pacificn = 14 sampling points, July 2011–June 2014; June 2015–July 2017) located at 1 km intervals along the Chobe River above the two water intake stations for the community. Land use along this reach consists of protected area and urban land. Water quality assessments were conducted to estimate in river concentrations of Escherichia coli (E. coli) and total suspended solids (TSS). Water grab samples to measure E. coli concentrations were collected at transect points and vacuum-filtrated through sterile gridded nitrocellulose membrane filters . After filtration, the sides of the funnels were rinsed twice with 20–30 mL of sterile reagent-grade deionized water. Filters were then aseptically transferred to the surface of RAPID’E.coli2  agar plates, and incubated at 37 °C for 24 h prior to colony enumeration. TSS (mg/L) were evaluated using water samples filtered  and dried at 103–105 °C for 1 h before being weighed. The drying cycle was repeated until a constant TSS weight was obtained. The protocol used is explained in detail in Fox et al.63.Water quality samples were collected bimonthly from established transect sites , March–May (MAM), June–August (JJA), and September–November (SON). For each year and season, we calculated the average Chobe River height, average minimum temperature, total rainfall, and total number of diarrhea cases in Chobe District. We then used linear regression to predict seasonal environmental variables using monthly Niño 3.4, and negative binomial regression to predict seasonal under-5 diarrhea cases using monthly Niño 3.4. Separate regressions were run using Niño 3.4 lagged 0–12 months, i.e., seasonal outcomes in DJF were predicted by Niño 3.4 in the corresponding February (0 month lag), January (1 month lag), December (2 months lag), November (3 months lag), October (4 months lag), and September (5 months lag), etc. Although we had limited water quality data, we conducted a parallel exploratory analysis predicting seasonal In addition, we investigated seasonal teleconnections of ENSO with regional rainfall using correlations between monthly Niño 3.4 and monthly TRMM gridded rainfall estimates from 1998 to 2015 within each season. Correlations were calculated within each grid cell for each season and year.To explore whether the introduction of the monovalent rotavirus vaccine (RV1) modified the relationship between Niño 3.4 and under-5 diarrhea, we reran the negative binomial regressions using monthly Niño 3.4 to predict seasonal under-5 diarrhea incidence using data from January 2007 to July 2012 (the date of the RV1 introduction across Botswana) and August 2012 to June 2017. By grouping the data before and after RV1 introduction, we limited the sample sizes, but were able to explore whether the ENSO–diarrhea relationship was modified by the RV1 introduction.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary