Populus tremula) across Europe by means of whole-genome resequencing of a collection of 411 individual trees. We dissected admixture patterns between aspen lineages and observed a strong genomic mosaicism in Scandinavian trees, evidencing different colonization trajectories into the peninsula from Russia, Central and Western Europe. As a consequence of the secondary contacts between populations after the last glacial maximum, we detected an adaptive introgression event in a genome region of ∼500 kb in chromosome 10, harboring a large-effect locus that has previously been shown to contribute to adaptation to the short growing seasons characteristic of Northern Scandinavia. Demographic simulations and ancestry inference suggest an Eastern origin—probably Russian—of the adaptive Nordic allele which nowadays is present in a homozygous state at the north of Scandinavia. The strength of introgression and positive selection signatures in this region is a unique feature in the genome. Furthermore, we detected signals of balancing selection, shared across regional populations, that highlight the importance of standing variation as a primary source of alleles that facilitate local adaptation. Our results, therefore, emphasize the importance of migration–selection balance underlying the genetic architecture of key adaptive quantitative traits.Understanding local adaptation has become a key research area given the ongoing climate challenge and the concomitant requirement to conserve genetic resources. Perennial plants, such as forest trees, are good models to study local adaptation given their wide geographic distribution, largely outcrossing mating systems, and demographic histories. We evaluated signatures of local adaptation in European aspen ( FST and absolute divergence DXY  after several filtering steps . A deeper analysis of the composition of the species structure with NGSAdmix revealed five different ancestral populations and different levels of hybridization in particular geographic regions. Chinese and Russian samples, represented by solid colors in Due to the broad geographic coverage of Given these observations, we took a closer look at the genomic mosaicism of the Scandinavian population. We used EIGMIX, taking Russian, Scottish and Latvian populations as proxies of the ancestral Siberian, Western and Central European populations, respectively . These pFST estimations , or Latvia and Southern Scandinavia , whereas comparisons between populations without signs of admixture, such as China and Russia, reached values as high as 0.35, which approach FST values observed between closely related aspen species.These observations were also supported by imations , as we oP. tremula for potential introgression events that could have occurred given the admixed background we observed in Scandinavia. We followed a four-taxon approach for these scans, keeping the Chinese population as an outgroup in all our comparisons. We calculated the fdM introgression statistic , which combines both fd and distance estimations and that is less sensitive to the timing of gene flow. Both estimators give positive values when introgression occurs between P3 (donor population: Russia or Latvia) and P2 (Northern Scandinavia), and negative values if it occurred between P3 and P1 (Southern Scandinavia), on a scale from −1 to 1. When evaluating gene flow from Russia into Scandinavia, we obtained fdM and df mean genomic values of −0.008 and 0.014, respectively (fdM = 0.85 (FDR = 3.6e−3) and df = 0.89 (FDR = 1.33e−50). As expected, there were differences in DXY and FST patterns between subpopulations at this region: FST is remarkably high between Nordic subpopulations and any other geographic location, whereas DXY decreases when northern Norwegian, Swedish, and Russian subpopulations are contrasted  genes, are encoded in this introgressed genomic block: Potra2n10c20839  and Potra2n10c20842  and the region surrounding these two genes is known to have been subject to a selective sweep in the northern Swedish population  did not reach the accepted significance threshold to be confidently classified as regions targeted by positive selection or 2) were not geographically limited, that is, they are present in at least two distant subpopulations  online. FST values did not differ between BS-affected regions and genome-wide estimations, however , with an average value of 0.17 on the BS bins versus 0.11 at the genome-wide level B; averag however  online.  however C. The in[FDR] < 0.01 and ADD ≥ 0.5 (dosage form donor A(Latvia) = 0.76; dosage form donor B(Russia) = 0.24), where the predicted up/downstream gene models correspond to Potra2n15c28701/02, annotated as presenilin-like and exocyst subunit Exo70 family protein-coding genes, respectively. Similarly, in the Nordic populations of Scandinavia, we identified regions of Russian ancestry with SNPs displaying high β-scores on chromosome 4  = 0.29; dosage from donor B(Russia) = 0.71) only 3 kb downstream of the gene model Potra2n4c9007 annotated as a gibberellin 3-beta-dioxygenase coding gene, and on chromosome 19  = 0.29; dosage form donor B(Russia) = 0.71), upstream of gene model Potra2n19c34341, annotated as and autophagy-related protein 13 coding gene.We took a deeper look at gene models in close proximity to SNPs with high β-scores and high ADD in hybrid populations, as the bias in allele dosage from any of the donor population could give insights on the beneficial impact of those alleles  online. P. tremula across the Eurasian continent provides a good scenario to test for changes in population sizes across time. We evaluated population size changes through time using the stairway plot method, which is based on the composite likelihood of the site frequency spectrum (SFS) of the species and/or selected subpopulations. We used 50 random samples from our collection to generate the overall behavior of the species, as well as for specific collection sites (Russia and Northern Scandinavia). We observed the strongest reduction in Ne ∼700–800 ka and this sharp decrease in Ne was observed across all subpopulations, which means it was a bottleneck that affected the entire species and that predated its dispersal in the Eurasian continent. A second bottleneck occurred ∼150–170 ka in the populations from Central Europe and Scandinavia, and a third, mild bottleneck spanned from ∼10–35 ka, a period representing the last glacial era in the Northern hemisphere. These two bottlenecks were not present in the Russian subpopulation and instead, we only observed a second decrease of Ne around 60–80 ka in this population (The expansion of pulation .4), whereas the divergence of both lineages occurred 760 ka, consistent with the first bottleneck seen at the species level in the stairway plot analyses.Given the admixed nature of the Scandinavian individuals, we also evaluated 19 different demographic models including simple models of divergence with and without migration, models with instantaneous size changes, ancient migration or secondary contact, ancient migration plus instantaneous size change, and island models of vicariance and founder events. We used two levels of resolution for this analysis: We ran the 19 models at the chromosome level  online, P. tremula is not a novel idea, in fact, it has previously been documented between divergent lineages in Europe using data from microsatellite loci , Russia, Scandinavia (Sweden and Norway), Eastern European (Latvia) and what we suggest may correspond to Central and Western Europe (Scotland and Southern Norway + Iceland). Although Chinese and Russian individuals appear to be partly isolated from Western populations , the genomic mosaicism is evident particularly in Scandinavia, where the population can be divided into at least two different groups that follow the latitudinal gradient along the peninsula. Individuals from Northern Scandinavia have a strong Russian ancestral component, whereas those from Southern Scandinavia have strong influence from both Eastern and Central/Western European population. The observations of two independent routes of colonization of Scandinavia, one from the south and another one from the northeast along the ice-free Norwegian Atlantic coast, agree with findings in other organisms, such as brown bears , we observed that Russian and Northern Scandinavian populations have experienced symmetric migration following a secondary contact, making it possible for backcrossing to occur and thereby promoting introgression between the two populations. Second, using information only from the adaptively introgressed region on chromosome 10, we confirmed that, indeed, the secondary contact models are the most strongly supported (together with vicariance and late admixture) and that the migration in this region was stronger from Russia into Scandinavia than in the opposite direction  online. FT2 allele present in Northern Scandinavia displays partial dominance  in regions across multiple chromosomes shared by all the aspen lineages, indicated that BS has maintained ancestral polymorphisms in the species. Indeed, excess of diversity around selected loci may be due to the retention of ancestral polymorphisms or due to the accumulation of derived polymorphisms, which happens faster than expected under neutrality. The former is expected if the same allelic lineages have been evolving under BS for a long time, with drift and mutation determining polymorphism within each lineage. The latter would occur if new, more recent instances of BS appear, facilitating the establishment of new neutral polymorphisms ; given tArabidopsis thaliana  between individuals to minimize the risk of sampling identical genets. Leaf samples were stored in silica gel before shipping to Sweden. Geographical coordinates were recorded for the sampled trees. The individuals included in the study  and their geographic origins are listed . For the Icelandic samples, library preparation and sequencing were conducted by Génome Québec, Canada. Briefly, 1 µg of high-quality DNA was used for paired-end libraries construction. The 12 libraries were sequenced in three lanes of Illumina HiSeqv4 with read length of 125 bp. All other samples were subjected to paired-end sequencing using libraries with an average insert size of either 350 or 650 bp, and samples were sequenced by the National Genomics Infrastructure at Science for Life Laboratory, Stockholm, on an Illumina HiSeq 2000 or Illumina HiSeq X platform to a mean per-sample depth of approximately 17×.P. tremula v2.0  and found that they did not exceed 14% of the sequencing reads in individual libraries (ranging from 3% to 13.8%).Resequenced accessions were mapped against the reference genome of ula v2.0 , using Bula v2.0 ]—mem aliula v2.0 . Depth aula v2.0  online. P. tremula, available at ftp://plantgenie.org/Data/PopGenIE/Populus_tremula/v2.2/, using picard LiftoverVcf, which allowed for better resolution at the chromosome level. Further SNP pruning with vcf/bcftools removed positions with extreme depth values , absent in more than 30% of the samples, nonbiallelic or displaying an excess of heterozygosity .We used GATK v3.8 to call variants . First, We used plink .Next, we used vcftools to output the genotype likelihood information contained in the pruned VCF file; the resulting beagle-formatted file was input into NGSAdmix to estimate individual admixture proportions across a varying number of ancestral populations . We treated each chromosome independently; the corresponding vcf file was converted to .geno format using parseVCF.py. From these files, we calculated diversity and divergence values for each subpopulation  in nonoverlapping 10-kb windows, using the script genomics.py. Finally, we used the script ABBABABAwindows.py to compute the four-taxon D statistic and f estimators in nonoverlapping windows of 10 kb as well. For these calculations, we tested gene flow from the Russian  or Latvian  subpopulations into Northern Scandinavia  or Southern Scandinavian , setting the Chinese  samples as the outgroup. Since we were interested in estimating shared variation between Russian and Northern Scandinavian individuals, we focused on the fdM introgression statistic, described by fdM gives positive values when introgression occurred between P3 and P2, and negative values if it occurred between P3 and P1. In order to avoid stochastic errors that could produce meaningless values, we only considered windows with at least 100 biallelic SNPs.We combined several statistics developed to identify introgressed genomic regions. These included the classic ABBA–BABA test in its developed df  was 15, which we chose as the lower threshold to keep chromosome fragments considered to have BS signals. We then revised the gene content in such windows, allowing for sites to be within and up/downstream of the gene models. We used topGO v.2.36.0 (In addition, we ran betascan , to detev.2.36.0  to look A, supplementary fig. 11A, Finally, we combined iSAFE and β-scores with ELAI calculated ADD to assess the effect of hybridization in each scan of selection C and 5C.supplementary figure 11We chose StairwayPlot  to inferPopulus (In addition, we aimed to confirm the directionality of the gene flow events we detected on chromosome 10 between the Russian and Scandinavian individuals. For this, we explored alternative demographic models using the diffusion approximation method of dadi  to analyPopulus . We ran Populus  online fPopulus  online.In order to generate confidence intervals for the parameters estimated from the demographic models with the highest likelihood, we created 100 bootstrap replicates of the spectra and calculated the standard deviations of the best-fit parameters using the Godambe methods .Molecular Biology and Evolution online.msab229_Supplementary_DataClick here for additional data file.


The Joanne Knight Breast Health Cohort was established to link breast cancer risk factors, mammographic breast density, benign breast biopsies and associated tissue markers, and blood markers in a diverse population of women undergoing routine mammographic screening to study risk factors and validate models for breast cancer risk prediction.Women were recruited from November 2008 to April 2012 through the mammography service at the Joanne Knight Breast Health Center at Washington University in St. Louis, Missouri. Baseline questionnaire risk factors, blood, and screening mammograms were collected from 12,153 women. Of these, 1,672 were excluded for prior history of any cancer (except non-melanoma skin) or diagnosis of breast cancer within 6 months of blood draw/registration for the study, for a total of 10,481 women. Follow-up is through linking to electronic health records, tumor registry, and death register. Routine screening mammograms are collected every 1–2 years and incident benign breast biopsies and cancers are identified through record linkage to pathology and tumor registries. Formal fixed tissue samples are retrieved and stored for analysis. County-level measures of structural inequality were derived from publicly available resources.Cohort Composition: median age at entry was 54.8 years and 26.7% are African American. Through 2020, 74% of participants have had a medical center visit within the past year and 80% within the past 2 years representing an average of 9.7 person-years of follow-up from date of blood draw per participant. 9,997 women are continuing in follow-up. Data collected at baseline include breast cancer risk factors, plasma and white blood cells, and mammograms prior to baseline, at baseline, and during follow-up.This cohort assembled and followed in a routine mammography screening and care setting that serves a diverse population of women in the St. Louis region now provides opportunities to integrate study of questionnaire measures, plasma and DNA markers, benign and malignant tissue markers, and repeated breast image features into prospective evaluation for breast cancer etiology and outcomes. Many of the current cohorts of women for cancer study in the USA are insufficient for examining factors from the biology to the environment that are associated with breast cancer risk among a diverse group of women. Most of these ongoing cohorts of women for the study of cancer have extensive questionnaire risk factor data collected for all participants, yet they are made up predominantly of White women. Smaller subcohorts have blood samples for prospective analysis of hormones, metabolic markers, and DNA . These cDr. Colditz and colleagues established the Joanne Knight Breast Health Cohort at Siteman Cancer Center to collect, store, and ultimately share comprehensive data sets and tissue specimens for future research. The screening mammography service at Siteman and Washington University School of Medicine offered us the potential to recruit a diverse population of women  and to bThe Joanne Knight Breast Health Center at Siteman Cancer Center at Washington University School of Medicine, St. Louis, Missouri provides mammography services for women from varying socioeconomic and racial backgrounds in the St. Louis region, including those with coverage through the Missouri breast and cervical cancer screening programs (Centers for Disease Control and Prevention and state funded), the Komen Fund and Barnard Fund coverage for the uninsured, and regularly insured women with private insurance or Medicare coverage. All women are screened with the same technology (Hologic). The mammography service stores all images and as of 2019 all screening used tomosynthesis (Hologic).Posters describing the study were placed in waiting areas and women attending mammographic breast screening or diagnostic procedures at the Joanne Knight Breast Health Center were approached to participate, all of them completing extended data collection for breast cancer risk estimation. The Joanne Knight Breast Health Center screens approximately 25,000 women and does high risk and diagnostic screening for another 15,000 women per year [The variables needed for the simplified Rosner–Colditz breast cancer risk prediction model  (these iCohort participants consented to (1) retrospective and prospective review of medical records ; (2) one-time 20 ml blood draw; (3) access to tissue not required for clinical care ; and (4) optional future contact for the purposes of long-term follow-up and/or to recruit for other related research projects. Record linkage identifies new mammograms, biopsies, and other visits to BJC Health Care facilities. BJC is a non-profit health care organization serving metro St. Louis, mid-Missouri, and Southern Illinois.Enrollment from November 2008 to April 2012 included 12,153 women who provided blood and risk factor data. A survey of 158 women who opted not to enroll over a two-week period in October 2009 showed most women who did not participate cited a lack of time to give the blood sample (30.4%). The next largest group (19.0%) wanted more time to think about participation. The remaining reasons for not participating included not wanting to give a blood sample (8.9%), not wanting researchers to have access to their medical records (8.9%), and (13.9%) provided no answer. The majority of the women came in for screening mammogram and a subset for diagnostic follow-up . Of these enrolled women, 1,672 had a history of cancer at enrollment leaving 10,481 women free from cancer at baseline.Follow-up of cohort participants as determined by mammography and other clinic visits through December 2020 was 78% seen in 2019 or 2020; a further 4.4% seen most recently in 2018 and a further 2.4% in 2017. All women remain under surveillance for return to follow-up mammography. Follow-up is passive through medical record linkages every 6 months, annual tumor registry searches, and annual mortality searches. This results in over 80% active follow-up for women seen within the last 36 months. The average person-years of follow-up through most recent contact is 9.2 person-years.2 freezers.At enrollment a baseline questionnaire, blood draw, and mammogram were obtained along with address for follow-up and for geocoding for measures of structural inequality. Baseline blood samples were taken and stored in multiple aliquots; DNA extraction  and plasma aliquots of 1 ml (6 per participant) and placed into cryovials and stored at − 80 °C in LNWomen self-reported breast cancer risk factors on entry to the cohort. These are drawn from established and validated measures . The basa = almost entirely fat, b = scattered areas of fibroglandular density, c = heterogeneously dense, d = extremely dense). Routine screening mammograms were obtained using Hologic machines.Mammograms: a screening mammogram 12–24 months prior to baseline, at baseline, and subsequent follow-up screening have been identified and stored. These images are stored along with BI-RADS density report recorded  in SAS 9.4  and theory-driven choices. The data were publicly available and previously compiled by the Health Inequality Project . We alsoThe cohort free from cancer at baseline includes 10,481 women. The distribution by race/ethnicity reflects the racial distribution in our catchment area and is summarized in Table 2 and of note it was higher for Black women (32.8 kg/m2) vs White women (28.0 kg/m2). During follow-up linkages to cancer registry and pathology records have identified 272 incident invasive breast cancers and 116 in situ lesions through October 2020. A total of 623 benign biopsy samples from 6/28/2010 through 12/31/2020 have been identified and are stored for centralized pathology review and classification. Through January 2021, we have confirmed 329 deaths within this cohort.Breast cancer risk factors at baseline are summarized in Table Socioeconomic status varied among participants. 45.6% were living in counties with debt of any kind at or above 30% of the population (Table Plasma samples from the cohort have been evaluated for carotenoid concentrations and risk of proliferative benign breast disease diagnosed from baseline through April 2016 . Among wThis new cohort brings breast images and pathology from routine care in a clinical setting that serves a diverse population into prospective epidemiologic investigations for breast cancer. The integration of blood markers in addition to questionnaire-based risk factors and tissue samples for all breast biopsies, in addition to repeated mammograms on participants, brings unique strengths to this cohort. Furthermore, the diversity of this population that is approximately one-quarter African American fills gaps in both breast cancer etiologies, risk prediction development, and validation of breast cancer risk models in diverse populations.Although repeated visits to the breast health center for screening mammography could facilitate updated or repeated blood measures, the epidemiologic evidence and resources to justify this have not been assembled to date. However, because breast images are the product of the mammography visit, improving approaches to maximize use of the information in these repeated images appears to be the most efficient approach to improve risk stratification as part of routine breast health services.Through IRB approval of deidentified data, plasma or tissue samples can be shared with investigators. Applications submitted to Dr. Colditz are reviewed by an internal Siteman committee, including breast pathology, mammography, and Tissue Procurement Core leadership. Material Transfer agreements are developed once access is approved and data, tissue samples, or blood samples are shipped as agreed. The overall study is approved by the institutional review board at Washington University in St. Louis.


Background: Few preclinical studies have shown that Knee osteoarthritis (KOA) is linked to gut microbiome dysbiosis and chronic inflammation. This pilot study was designed to look at the gut microbiome composition in KOA patients and normal individuals with or without vitamin D deficiency . Methods: This pilot study was conducted prospectively in 24 participants. The faecal samples of all the participants were taken for DNA extraction. The V3-V4 region of 16s rRNA was amplified, and the library was prepared and sequenced on the Illumina Miseq platform. Results: The mean (±SD) age was 45.5 (±10.2) years with no defined comorbidities. Of 447 total Operational Taxonomic Units (OTUs), a differential abundance of 16 nominally significant OTUs between the groups was observed. Linear discriminate analysis (LEfSe) revealed a significant difference in bacteria among the study groups. Pseudobutyrivibrio and Odoribacter were specific for VDD, while Parabacteroides, Butyricimonas and Gordonibacter were abundant in the KOA_VDD group, and Peptococcus, Intestimonas, Delftia and Oribacterium were abundant in the KOA group. About 80% of bacterial species were common among different groups and hence labelled as core bacterial species. However, the core microbiome of KOA and VDD groups were not seen in the KOA_VDD group, suggesting that these bacterial groups were affected by the interaction of the KOA and VDD factors. Conclusion: Parabacteroides, Butyricimonas, Pseudobutyrivibrio, Odoribacter and Gordonibacter are the predominant bacteria in vitamin D deficient patients with or without KOA. Together these results indicate an association between the gut microbiome, vitamin D and knee osteoarthritis. Osteoarthritis (OA) of the knee is a degenerative joint condition that leads to progressive deterioration of the articular cartilage, subchondral bone sclerosis and synovial inflammation, resulting in joint pain and disability . There iBesides, various studies show that the gastrointestinal microbiome contributes to a triggering of this low-grade systemic inflammation in obesity ,15, throOn the other hand, observational studies found that low levels of vitamin D were associated with a higher prevalence of KOA, along with an increased risk of disease progression ,23,24. I2, history of hospitalization in last eight weeks, on antibiotics for last 12 weeks, gastrointestinal comorbidities  or on pharmacologic doses  of vitamins or minerals were excluded. The Institutional Ethics Committee clearance was obtained (IRB Min No: 9432-29/04/2015), and the patients were recruited after getting their written informed consent. The KOA patients were categorized into one of the following two groups; Group 1 with vitamin D levels <30 ng/mL were classified as vitamin D deficient , and Group 2 with vitamin D levels > 30 ng/mL were classified as normal vitamin D status . The asymptomatic individuals  were categorized into Group 3  and Group 4 . There were no differences in age, geography and gender between the four groups.This observational study was conducted in a tertiary care teaching hospital between June 2015 and December 2016 to investigate the association of gut microbiome and serum vitamin D status in knee osteoarthritis (KOA) patients and in normal healthy individuals. The diagnosis of KOA was based on the American College of Rheumatology Criteria. All patients of primary KOA between 30 to 60 years of age and willing to participate in the study with a follow up of six months were included. Patients excluded were those with varus deformity > 5 degrees, valgus deformity, flexion deformity > 5 degrees, on anticoagulants, BMI > 30 kg/mThe demographic profile, socioeconomic status and medical history were entered into a predesigned proforma. The dietary intakes of food groups and macronutrients were calculated from 24 h dietary recall and food frequency. The daily ingestion of macro and micronutrients were matched with recommended dietary allowances for Indians using the DIGEST programme by ICMR . The KOAFor KOA_VDD and KOA groups, plain radiograph of both Knees  in standing position were taken. The severity of KOA was classified as per Kellgren-Lawrence grade. Blood samples were collected in all four groups, and the following parameters were checked: serum haemoglobin level, erythrocyte sedimentation rate, C-reactive protein, serum creatinine, serum uric acid, serum calcium/phosphate, serum alkaline phosphatase, parathyroid hormone and thyroid function test and vitamin D levels. The faecal samples were collected from all participants in a plastic container on ice and then aliquoted in the lab, within 2 h, into a 2 mL Eppendorf tube for storage at −80 °C.2 and 1U of High-Fidelity DNA polymerase . The PCR amplification involved initial denaturation at 95 °C for 5 min followed by 1 min each 95 °C, 50 °C and 72 °C for 25 cycles and with a final extension at 72 °C for 7 min. The PCR amplicons were purified using Ampure XP beads  by following the manufacturer’s instructions. The amplicon Library Quality control was checked based on the size of the amplicon using Agilent Bioanalyzer DNA 7500 chip. All the libraries showed an expected size range (~600), indicating V3–V4 region amplification. The QC passed libraries was pooled in an equimolar concentration. The pooled libraries were sequenced using Illumina Miseq v2 Paired-End Reagents and Miseq instrument to generate 250 bp paired reads. FastQC and Rqc were used to check the quality parameters of sequence data like base call quality distribution, % bases above Q30, GC% sequencing adapter contamination etc. [A 200 mg of faecal sample from the Eppendorf tube was taken for DNA isolation with a modified DNeasy PowerLyzer Power Soil kit . The eluted DNA was quantitated using Nanodrop and checked using 1% agarose gel electrophoresis. The extracted faecal DNA was used to amplify the V3-V4 region of the 16s rRNA gene using region-specific primers  with Illion etc. .For downstream analysis of the metagenome data, “Mothur” software bundle was used. The quality-filtered sequence reads were imported into Mothur, and the read pairs were aligned with each other with a minimum of 30 bp overlap to form contigs. Reads with ambiguous base calls were rejected. The high-quality contigs were checked for identical sequences, and duplicates were merged. The quality-filtered contigs were classified into taxonomical outlines based on the Silva NR v123 database. The classified contigs were filtered for any undesirable lineage like Mitochondria, Chloroplast, Fungi or Archaea. The contigs were then clustered into Operational Taxonomic Unit (OTUs) based on the phylotype. The abundance of each OTU in the samples was estimated. The alpha and beta diversity of OTUs between the groups was tested by the Kruskal–Wallis test after normalizing the OTU abundance. The relative abundance of bacterial groups was analyzed using Linear discriminant analysis effect size (LEfSe) . Canonicp < 0.05) from all the three-study group; however, BMI among the three-study groups was not different. Their haemoglobin levels were 13 ± 1.7 (mean ± SD) gm/dl, and none of them was on any treatment. KOA and KOA_VDD groups had a chronic history of pain ranging from four weeks to three years, and they performed only mild to moderate physical activity and had a WOMAC score of 57.6 ± 15.1 and 54.3 ± 10.7 (mean ± SD), respectively. All participants in study groups KOA_VDD, KOA and VDD had an occupation that kept them indoors.A total of 24 participants, aged 45.5 ± 10.2 (mean ± SD) years, were recruited into four groups, with seven, four, seven and six participants in KOA_VDD, KOA, VDD and Normal (NVD) groups respectively . These pp = 0.12).We determined the gut microbiome composition through the analysis of the faecal microbiome. To identify bacterial taxa, we sequenced the V3–V4 hypervariable regions of the bacterial 16S rRNA gene using an Illumina MiSeq system. After quality control and filtering, ~3,401,904 high-quality sequences with an average length of 600 bp were recovered for further analysis, with an average of ~141,746 reads per sample  . The rarp = 0.13) statistically, the two groups appear to cluster differently.To assess the effect of Knee osteoarthritis disease on the gut microbiome, we compared the gut microbiome composition of individuals with normal vitamin D status, diagnosed with Knee osteoarthritis disease (KOA) and those considered as “healthy” (NVD). We first evaluated the overall differences in beta diversity between faecal microbiome samples of both groups using canonical correspondence analysis (CCA), based on Bray–Curtis distances A. AlthouWe estimated α-diversity using the observed OTU and Shannon index (using the Shannon index measuring how evenly OTUs are distributed in a sample) in both groups B. KOA saTo investigate differentially abundant taxa between both groups, we then performed LEfSe analysis to compare the abundance of bacterial taxa in KOA patients and healthy subjects. A histogram of the Linear Discriminate Analysis scores was computed for features that showed differential abundance between healthy (NVD) subjects and KOA patients C. The Lip = 0.19) statistically, the two groups appear to cluster differently.To assess the effect of vitamin D deficiency on Knee osteoarthritis disease on the gut microbiome, we compared the gut microbiome composition of individuals with Knee osteoarthritis patient with vitamin D deficiency (KOA_VDD) and those with vitamin D deficient (VDD). We first evaluated the overall differences in beta diversity between faecal microbiome samples of both groups using canonical correspondence analysis (CCA), based on Bray–Curtis distances A. Althoup = 0.7; and Shannon index, p = 0.42).We estimated α-diversity using the observed OTU and Shannon index (using the Shannon index measuring how evenly OTUs are distributed in a sample) in both groups B, and thHowever, when LEfSe analysis was performed between KOA_VDD and VDD, the gut microbiome of KOA_VDD was found enriched with Phascolarctobacterium, Gordonibacter, Delftia, Parabacteroides, Candidatus-saccharimoanas and Butyricimonas. In contrast, the gut microbiome in VDD was formed predominantly by Alloprevotella, Odoribacter and Oribacterium C.p = 0.055) (We assessed the effect of the vitamin D deficiency on the gut microbiome of KOA patients by comparing the gut microbiome of KOA patients with and without vitamin D deficiency (KOA vs KOA_VDD). Although the CCA analysis presents two distinct clusters representing the KOA and KOA_VDD groups, this difference is not significantly different  A. We als= 0.055) B. Howeve= 0.055) C.p = 0.43). We observed that the vitamin D deficiency tended to increase the alpha diversity  and normal . Globally, as shown, the CCA analyzed A the VDDiversity B  was negatively associated with OA and rheumatoid arthritis [Faecalibacterium prauznitzii) in the gut with ageing. Besides, our study showed that Faecalibacterium is enriched in the NVD group compared to the KOA_VDD group, suggesting that the KOA-associated microbiome could participate in the inflammation process and the degeneration process in the joint.Previously, Jackson et al. 2018 reported that the abundance of specific gut microbes , Pseudobutyrivibrio and Odoribacter are retrieved specifically in VDD patients. Interestingly we also observed an effect of the vitamin status on the KOA-associated dysbiosis . The gutIn support of this observation, the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis of our study revealed that the gut microbiome involved in lipopolysaccharide biosynthesis and lipopolysaccharide biosynthesis protein is seen in the KOA_VDD group . Along wPropionibacterium acnes into the shoulder joint developed erosive arthritis, showing microorganisms and bacterial components in the inflammatory process of the joint. The common bacterial pathogens detected in joint inflammation correspond to bacteria retrieved in the gut microbiome, such as Pseudomonas sp., Shigella sp. and Escherichia coli species.Vitamin D is known to induce E-cadherin expression and improve the epithelial cell junction in the gut. In contrast, vitamin D deficiency causes the breakdown of the junction resulting in a leaky gut and favouring the passage of bacterial components and product from the bacterial metabolism. In this way, KOA patients with a deficiency in D vitamin could have increased permeability of the gut barrier. Bacterial peptidoglycans were detected in the synovial fluid of patients with reactive arthritis and OA, suggesting that these antigens result in inflammation of the joints. In a study, rats injected with Lactobacillus casei  with type II collagen (250 mg/ kg body weight) and glucosamine (250 mg/kg body weight) reduced pain, cartilage destruction and pannus formation compared to Glucosamine or L. Casei alone in rat RA model_ENREF_15. L casei is a probiotic that suppresses the proinflammatory cytokines  and increases the anti-inflammatory cytokines (IL-10 and IL-4) in the rat OA model. To date, there are no human studies that have investigated the role of probiotics in osteoarthritis.Osteoarthritis requires lifelong treatment either for a cure or for arresting the progression of the disease. Because of its plasticity, the gut microbiome is an exciting target for preventive or therapeutic interventions. The data generated by our study show promising leads for effectively relieving indications of KOA. The microbes observed in VDD without KOA  may offer protection from KOA, despite a deficiency of vitamin D as these patients did not develop KOA. Specifically, identifying bacterial species belonging to these taxa could lead to identifying newer probiotics with potential benefits for the KOA. As shown in our study’s PICRUSt analysis , the modThese findings are based on the analysis of a few individuals, and they need to be confirmed on a larger cohort. However, the patient groups included in our study were homogenous in terms of dietary intake, smoking, alcohol intake, oral medication usage, physical activity, sun exposure and BMI, which confirms that the observed interaction between KOA and vitamin D status is not produced by other confounding factors and strengthens our data.To conclude, the gut microbiome in knee osteoarthritis patients with or without vitamin D deficiency presents different bacterial abundances. Form these data; we suggest that the impact of vitamin D deficiency in the KOA disease could be mediated through the gut microbiome. These observations recommend a larger vitamin D interventional study in KOA patients to confirm the beneficial effects of resetting the observed dysbiosis on the progression of KOA.


The objective of this study is to use DNA methylation analysis to detect DNA methylation loci linked to obesity phenotypes, adj = 0.048). Cg16170243 corresponds to a 50 bp-length human methylation oligoprobe located within the AC090241.2 gene that overlaps ST8SIA5 gene. No significant association was observed with waist-to-hip ratio adjusted for BMI (Padj > 0.05).Two-hundred and ten healthy European participants from the STANISLAS Family Study (SFS), comprising 73 nuclear families, were comprehensively assessed for methylation status using Illumina Infinium HumanMethylation450 BeadChip. An epigenome-wide association study was performed, which identified a CpG site cg16170243 located on chromosome 18q21.2 significantly associated with waist circumference, after adjusting for BMI (β = 2.32, SE = 0.41, PA novel association between DNA methylation and WC was identified, which is demonstrating that epigenetic mechanisms may have a significant impact on waist circumference ratio in healthy individuals. Further studies are warranted to address the causal effects of this association.The online version contains supplementary material available at 10.1186/s12920-021-01077-9. Epigenetic changes are covalent modifications of cytosine bases, histones and changes in nucleosome positioning that can modulate the effect of a genotype on a particular phenotype and thus affect physiological mechanisms as well as the pathophysiology of many diseases . The mos2. However, clinical evidence suggests that diseases, such as diabetes, are more associated with central obesity, where visceral adipose tissue is stored [Obesity is a systemic disease and a growing health problem that contributes to the increased risk of many common medical conditions . The excs stored . Therefos stored .Increasing prevalence of obesity worldwide is mostly due to changes in the environment, whereas a person’s genetic profile is considered one of the main causes of individual difference in predisposition to weight gain. A high heritability of this phenotype has been confirmed in several epidemiological studies  and examin uterus development and in early childhood was associated with an increased risk of a range of chronic diseases in adulthood, showing that early environmental influences can cause permanent effects [i.e. HIF3A [ABCG1 and CPT1A [Furthermore, obesogenic environment during  effects . This in effects –14. Epig effects . Severale. HIF3A  ABCG1 annd CPT1A , 16.In order to make further contribution to the comprehension of obesity trait, in this study, we are examining the methylation levels associated with central obesity, measured by WC and waist-to-hip ratio adjusted for BMI, in a healthy population. Using healthy individuals can help to avoid the discrepancies caused by extremes of obesity and comorbidity in population-specific cohorts. Our findings provide new insights into genetic regulation of visceral fat accumulation and are presenting a new variant, which may increase the susceptibility to chronic diseases..). Descriptive characteristics are presented in Table 210 healthy related individuals from 73 families of the STANISLAS Family Study (SFS) have been enrolled in this study, including 115 adults and 95 children. The SFS is a 10-year longitudinal cohort that includes three visits at 5-year intervals. It involved 1,006 French families from Vandoeuvre-lès-Nancy, France, first that were first recruited between 1993–1995 . All subThe study protocols were approved by the institutional ethics committees and all subjects gave written informed consent for their participation in the study. For the participants of less than 18 years old, consent was also given for their participation by their parents. Since SFS is a cohort of families, the parents and their children participated in the study and after being informed by the researchers, they all gave written consent for their participation and the participation of their children.2 (m)2. Data collection has been previously described [2 for adults and for children BMI ≥ 97th percentile for age and sex calculated on French BMI curves [Data and blood samples were collected during the baseline visit of the SFS. All measurements of the clinical indicators were performed in the laboratory of the Centre for Preventive Medicine (CMP) in Vandoeuvre lès Nancy, France. Blood count was performed using standard methodology in the laboratory of the CMP. WC was taken at the midpoint between the lower margin of the last palpable rib and the top of the iliac crest (hip bone). Trained professionals recorded measurements to the nearest 0.1 cm. BMI was calculated by the Quetelet index formula as weight (kg) divided by heightescribed , 18. ObeI curves .p-value was generated for every CpG in all samples, indicating the quality of the signal. Poor quality probes were excluded from the analysis using a detection p-value cutoff (˃0.05). Probes, missing in more than 5% of samples were excluded from all samples. Background correction and normalization were performed with Illumina background correction and SWAN [Blood samples were taken in the morning between 8 and 9 a.m. following an overnight fast. Whole blood DNA was extracted by the Miller technique  and was and SWAN  to all iWe further excluded probes already annotated in HumanMethlyation450 annotation files  sites). Finally, the probes containing cross-reactive and target polymorphic CpGs were excluded. All downstream analyses were carried out with software R.Genotyping was performed by the Infinium CoreExome Illumina assay. Significant WC-associated SNPs, located in the same chromosome as the significant methylation sites were selected from the NHGRI-EBI GWAS catalog . These Sρ2 from constant (random model) of G*Power software was used in order to calculate statistical power [ρ2 and H0 ρ2), α error probability, total sample size, and number of predictors.Waist-to-hip ratio and WC were not normally distributed and were therefore transformed to the e-log scale. Individual analyses have been performed for the adult population, the children population and the combined population (children and adults together). A linear mixed-effects model was used to analyze the association between methylation levels at each probe and log-transformed phenotypes. The model used included sex, age, BMI, family structure, and individual white cell counts  as covariates and chip array as random effect. Bonferroni correction and false discovery rate (FDR) methods (< 0.05) were used for the correction of the results for multiple testing. The association analyses of methylation values and the assessed phenotypes were performed by using the package CpGassoc in R . HumanMeal power , specifiIn order to investigate whether the observed association between WC and methylation level was related to genetic variants associated with WC, a linear mixed-effects model with age, sex, BMI, family structure, methylation batch array and white cell counts as fixed effects, and methylation chip as a random effect was carried out for association analysis using the R statistical package nlme .The significant CpGs were localized on the Human genome (GRCh38.p12) using Ensembl browser. In silico gene expression was obtained using the BLUEPRINT Data Analysis portal  and GTExPoor quality probes (n = 764) were excluded from the sample. In total, 77% of probes passed quality control, excluding probes containing SNPs, sex chromosomes, SBE sites, cross-reactive and target polymorphic CpGs, thus leaving 373 626 probes for association analyses Fig. . The posOne individual was excluded after quality control checks of the methylation array data (outlier of the plotted median of the methylated against unmethylated samples), thus 210 participants were included in the analyses.Padj = 0.048) in the combined population. QQ plot of the EWAS is presented in Fig. The results of the analysis showed one novel significant positive association of cg16170243 probe with WC adjusted for sex, age, BMI, family structure, and individual blood cell counts , whereas in the opposite strand the probe is located 245 base pairs upstream of the ST8SIA5 gene  and contains transcription factor binding sites (TFBS).The cg16170243 probe maps on chromosome 18q21.1 and is located in the forward strand of the ST8SIA5 gene is expressed in several cell-types of the bloodline: neutrophils, peripheral blood mononuclear cells, eosinophils and monocytes  between WC, adjusted for BMI, and methylation at the probe cg16170243. There was no association of cg16170243 with three candidate SNPs previously associated with WC. Therefore, the underlying mechanism of the CpG’s effect on WC is probably differentiated from the mechanisms linked to these genetic variants.We have identified a novel significant positive association  gene, coding for a sialyltransferase enzyme, involved in the synthesis of gangliosides GD1c, GT1a and GQ1b from GM1b, GD1a and GT1b, respectively [ST8SIA5 which would lead to an affected synthesis of gangliosides.The pression . The oveectively . Ganglioectively . Cg16170Studies showed that an inadequate ganglioside expression in mediobasal hypothalamic neurons deregulates neuronal leptin  and insuST8SIA5 gene in blood cells reflects a process that also occurs on a larger scale in neuronal cells when methylation at cg16170243 site is present, causing a disruption of the insulin signaling pathway and contributing to the accumulation of visceral fat (Fig. Therefore, it is plausible that the effect of cg16170243 on . has shown that variation in DNA methylation is most often a consequence of adiposity [Despite the above-proposed mechanisms of the methylation impact on the WC via insulin deregulation, we cannot exclude the possibility that methylation could be a consequence of modified WC, rather than a cause. Indeed, a meta-analysis from S. Wahl et aldiposity . Thus, pEpigenetics studies of obese phenotypes on healthy individuals have been previously done , howeverA limitation of our study is the small sample size. Although we have identified a significant result with high statistical power, we did not have the possibility to replicate it in a population-specific study. As epigenetic changes can be tissue-specific, the limitation of our study is also the use of blood samples without tissue-specific replications. Even though the within-subject correlation of CpG specific sites from blood and adipose tissue was previously confirmed, the use of methylation markers in blood to mirror the corresponding profile in the target tissue should be taken with caution .ST8SIA5 transcription, resulting in a perturbed synthesis of gangliosides.We identified a novel association between DNA methylation and WC. This association could be due to the modification of the regulatory region of Additional file 1. Peer review reports.


Pancreatic cancer is one of the deadliest types of cancer, with a five-year survival rate of only 10%. Nanotechnology offers a novel perspective to treat such deadly cancers through their incorporation into radiotherapy and chemotherapy. However, the interaction of nanoparticles (NPs) with cancer cells and with other major cell types within the pancreatic tumor microenvironment (TME) is yet to be understood. Therefore, our goal is to shed light on the dynamics of NPs within a TME of pancreatic origin. In addition to cancer cells, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were examined in this study due to their important yet opposite roles of suppressing tumor growth and promoting tumor growth, respectively. Gold nanoparticles were used as the model NP system due to their biocompatibility and physical and chemical proprieties, and their dynamics were studied both quantitatively and qualitatively in vitro and in vivo. The in vitro studies revealed that both cancer cells and CAFs take up 50% more NPs compared to NFs. Most importantly, they all managed to retain 70–80% of NPs over a 24-h time period. Uptake and retention of NPs within an in vivo environment was also consistent with in vitro results. This study shows the paradigm-changing potential of NPs to combat the disease. It is thought that if humans live long enough everyone will eventually develop cancer. The current human lifetime risk of developing cancer is about 40%, and the risk of dying from it is about 21% ,4,5. DesIs there a difference between in vitro uptake of NPs in tumor cells, NFs, and CAFs of pancreatic origin?What is the potential of retaining of these NPs within those cells in vitro for efficient delivery of therapeutics?Do we see retention of NPs within tumor tissue in vivo for an extended period of time as being successful for the delivery of therapeutics?As illustrated in 2COONa)2 2H2O) to 10.53 mL of double-distilled water to create a 1% solution. The gold solution was prepared by adding 0.12 g of Tetrachloroauric (III) acid trihydrate (AuCl4H·3H2O) to 10.35 mL of double-distilled water to create a 1% solution. A total of 300 µL of the 1% gold solution was then added to 30 mL of double-distilled water in an Erlenmeyer flask and was stirred and heated until boiling. Once boiling, 600 µL of the 1% reducing agent was added quickly to the flask and was stirred while boiling for 10 min. The solution’s color gradually changed from clear to a crimson red during this time, indicating the creation of GNPs. The heat was then turned off and the solution was stirred at room temperature for 10 more minutes.Spherical gold nanoparticles (GNPs) of 17.73 nm ± 0.17 nm diameter were produced using a citrate reduction method due to its simplicity, its relatively short preparation time, and its low chemical, physical, and environmental dangers. The reducing agent was prepared by adding 0.12 g of sodium citrate tribasic dihydrate (HOC(COONa).The negatively charged GNPs surfaces are coated with polyethylene glycol (PEG) at a density of 1 PEG per nmPEG, and GNPPEG−RGD shape, size, and concentration were characterized using Perkin Elmer λ 365 ultraviolet visible (UV-VIS) spectrophotometers, while Anton Paar LiteSizer 500 dynamic light scattering (DLS) and ζ potential were used to measure the hydrodynamic radius and surface charge, respectively. UV-VIS shows a measure of the amount of light being absorbed at a particular wavelength by the sample and is dependent on the size and shape of the nanoparticle. Transmission Electron Microscopy (TEM)  was used to verify the shape and size of GNPs used in this experiment.GNPs, GNP® CRL-1469™) were obtained from the American Type Culture Collection. Human cancer-associated fibroblasts (CAF-98) and matched normal pancreas fibroblasts (NPF-98) were derived from resected PDAC tumor tissue from a consenting patient through the Gastrointestinal (GI) Biobank at the Vancouver General Hospital. The study was approved by the University of British Columbia research ethics board (protocol #H20-00948). A small piece of tumor tissue was placed in FGM-2 media (Lonza cc-3132) to support the spontaneous outgrowth of NPs and CAFs. When confluent, the cells were passaged and further propagated in FGM-2 media. During the experiment, all cells were cultured in high glucose Dulbecco’s modified Eagle medium  enhanced with 10% fetal bovine serum , 1% penicillin/streptomycin (Gibco), and 4 mM of GlutaMax (Gibco). Trypsin–EDTA(Gibco) was used for all cell detachment from flasks and all cell fixations were performed using paraformaldehyde . Cell cultures were washed with Phosphate-Buffered Saline (PBS) and all cell line incubations happened at 37 °C with 5% CO2.Human pancreas cancer cell lines MIA-PaCa-2 (ATCC#: CRL-1420™) and PANC-1 . The media of the cells for the retention study was replaced with fresh media and cells were placed in the incubator for an additional period of 24 h. After the incubation period, cells were washed three times with PBS, fixed, and cover slips were mounted on microscope glass slides for imaging as described previously. For live cell confocal imaging, 1 × 105 cells were grown on 35 mm coverslip-bottom dishes  with 2 mL of media and were left in the incubator for 24 h. Cells were then dosed with 20 µg/mL of GNPPEG-CY5-RGD  and were placed in the incubator for another 24 h. Prior to imaging, the cultured media of the cells for the uptake study was substituted with the same amount of colorless media  and 4 drops of NucBlue® Live reagent  (DAPI) in each dish to stain the nucleus of each cell. Cells were incubated for another 20 min before imaging. For the retention study, the media was replaced with fresh media after an incubation time period of 24 h with NPs and cells were placed in the incubator for an additional period of 24 h. Prior to imaging, the cultured media was substituted with colorless media  and 4 drops of NucBlue™ Fixed Cell ReadyProbes™ Reagent (DAPI) in each dish to stain the nucleus. Cells in these dishes were then incubated for 20 min before imaging. Acquisition settings were kept the same for both studies.Darkfield (DF) coupled with hyperspectral imaging (HSI) CytoViva microscope  and Zeiss LSM 980confocal imaging microscopy  were used to determine GNP distribution. Fixed cells were imaged for DF and HSI under a 60× objective, while live-cell confocal imaging was done using 60× oil-immersion lens. For DF and HSI, 1 × 105 cells were seeded in 6-well dishes with 3 mL of media for a period of 24 h. All cells were dosed with 20 µg/mL of GNPPEG−RGD and were placed in the incubator for 24 h. For the NP uptake study, cells were washed three times with PBS after 24 h NP incubation time period to remove any GNPs that were not within the cells. In order to remove cells from well for quantification, 1 mL of trypsin was added to each well and placed in an incubator for 5 min. Once cells were detached from tissue culture wells, an additional 5 mL of media was added and mixed well before counting using a hemocytometer. Once the cells were counted, 1 × 105 cells from each well were transferred to glass tubes for quantification.To measure the gold content in the cell lines, 1 × 105 cells from each well were transferred to glass tubes for quantification.For the NP retention study, the media containing GNPs was replaced with fresh media following a 24 h NP incubation time period and cells were incubated for an additional period of 24 h to determine their potential of retaining NPs. After the incubation period, cells were also washed 3 times, trypsinized, diluted, and counted for quantification. Similar to the method used in the uptake study, 1 × 103(VWR)) to each glass tube and placed them in a mineral oil bath at 90 °C for ~35–45 min until solutions were free of fragments. This was followed by adding 100 μL of hydrogen peroxide (VWR) to each tube before they were returned to the mineral oil bath for ~30 min to confirm full consumption of cell contents. Lastly, the samples were diluted to 2.5% v/v acid content with deionized water, and the amount of gold in each sample was measured by Inductively Coupled Plasma Mass Spectrometry  in parts per billion (ppb) or ng/mL. Standard gold chloride solutions were used to produce calibration curves.To process the collected cells for quantification, we added 50 µl of aqua regia  mice of 6–8 weeks were used. To derive subcutaneous xenografts, 1.5 × 106 tumor cells in a volume of 100 μL were injected using a 28-gauge needle in the lower left dorsal flank. Tumor measurements were converted into tumor volume using (L × W2/2), where L and W are the larger and smaller diameters, respectively; tumors were measured every 2 days with calipers. When xenografts reached a volume of ~250 mm3, they were randomly divided into groups of 6 for all studies. Each mouse was administered a gold dose of 1 mg/kg in a volume of 100 μL via tail vein using a 28-gauge needle. The concentration of GNPs used for injections was 200 µg/mL and they were suspended in PBS. Animals were monitored for any signs of physical toxicity over the duration of each study. Experiments were conducted in accordance with the Animal Care Committee guidelines of the University Health Network and the approved protocol number is 2979.Human pancreas cancer cell line  was cultured in Dulbecco DMEM-Dulbecco’s Modified Eagle Medium  together with 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin G, and 100 μg/mL streptomycin . Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 being excluded.All serial sections (50 µm separation between sections) were cut from frozen block tumor tissue. Sections were stained for CA9  and CD31 . Secondary antibodies were used alone to control for nonspecific backgrounds. Sections were counterstained with 1 μg/mL DAPI to outline the nuclear area. Images were scanned on the TS4000 (Huron Technologies) at 0.5 μm/pixel. Regions of tumor, necrosis, stroma, and folds were specified, creating a training ruleset for tissue recognition using Tissue Studio . Cellular analyses included nucleus identification and separation, with objects < 10 µmFor acute toxicity, blood, liver, kidney, and overall toxicities were measured through proteins, enzymes, metabolites, and plasma electrolytes. Blood samples were collected through terminal cardiac puncture prior to sacrifice. Samples were analyzed using a vetscan2 Autoanalyzer . Mice were observed for any physical toxicity over a 50-day period. These physical indicators included body weight changes, dull sunken eyes, rapid/shallow breathing, hunched back, and lethargy.PEG [PEG to avoid the immune system as a nonforeign object and extend its blood circulation [PEG-RGD . The RME process of NPs occurs through interactions between the ligands on the surface of the NP and receptors on the cell membrane. In this study, we used the RGD peptide as a targeting ligand . This waMost NP uptake studies have mainly focused on tumor cells. However, TME consists of many other cell types which could significantly influence the outcome of NP-based therapeutics. Among the cells present in TME, our focus was on NFs and CAFs in addition to tumor cells. This is mainly due to the anti-tumorigenic properties of NFs and the tumorigenic properties of CAFs. CAFs promote the proliferation of a tumor through release of cytokines and chemokines, while NFs suppress it ,19,20,37In order to map the GNP uptake cross section among these four cell lines, we incubated cells with ications ,39,40,41(ICP-MS) B. GNP upBased on our quantification data, we believe that we can deliver therapeutics that target tumor cells and CAFs very effectively, not only to kill tumor cells but also to silence CAFs which promote tumor growth. This type of novel therapeutic is very much needed in treating pancreatic cancer where survival is very low. A recent study has demonstrated the feasibility of silencing CAFs of melanoma origin using both GNPs and radiation . GNPs usGNPs are taken up by cells via the endocytosis process where they are trapped in endosomal compartments upon entering, get processed through fusing with lysosomes, and are excreted to the extracellular medium, as explained in A clear reduction in the number of GNPs was apparent in all four cancer cell lines compared to normal media. This could be attributed to two reasons, the first being the redistribution of GNPs from the parent cell to the daughter cells following cell division, and the second reason being the nonequilibrium excretion of GNPs from cells via exocytosis without having enough GNPs getting into the cell due to lack of GNPs in the surrounding media . Even thOur in vitro studies discussed earlier  demonstrAs illustrated in schematic Modification of size and surface properties of NPs allowed us to have a significant number of GNPs present even 48-h post injection, which would facilitate better delivery of nanomedicines in future cancer treatments. In one of the pioneering studies using GNPs as radiosensitizers, radiation treatment had to be performed minutes after the injection of NPs since NPs were excreted much faster . Our appTo generate our in vivo tumor model for this study, we used Mia PaCa-2, which is a standard tumor cell line. However, this type of tumor model may lack the complex nature of TME where CAFs play a significant role. Therefore, our future studies will include generating tumors from patient-derived xenograft models of pancreatic origin or co-injection of both tumor cells and CAFs to create real tumor-like environments for testing nanomedicines in a more meaningful manner. This type of model will allow us a better comparison between our in vitro and in vivo studies for advancing cancer medicine for future clinics.Despite the latest advancements in radiotherapy, chemotherapy, immunotherapy, and surgical techniques for the management of pancreatic cancer, the majority of pancreatic cancer patients will die from the disease. Therefore, new treatment strategies are undoubtedly needed. One approach is to integrate cancer nanotechnology into current curative modalities, such as in radiotherapy to enhance the local radiation dose, and in chemotherapy to facilitate drug delivery. Most studies have so far focused on tumor cells. However, cancer-associated fibroblasts (CAFs) are key players in the tumor microenvironment (TME). They are responsible for promoting growth and metastasis of the tumor. As such, CAFs are associated with poor prognosis and have emerged as a focus of anticancer research. Our in vitro study shows the potential of targeting GNPs not only to tumor cells but also to CAFs. Furthermore, NP uptake by CAFs was over 10% higher than that of tumor cells. This will play a significant role in reducing the activity of CAFs when GNPs are used as radiosensitizing agents or as drug carriers. Most importantly, the percent retention of NPs 24-h post injection in CAFs and tumor cells was over 70%. The ability of CAFs and tumor cells to retain most NPs over a 24-h period allows for a better flexibility in the delivery of NP-based therapeutics. It was also noticeable that uptake of NPs by NFs was two-fold less compared to CAFs. This could shed light on concerns regarding normal tissue toxicity due to NPs. Results of our in vivo study were also very promising as we noticed 10% of the injected NP dose within the tumor 24-h post injection. After 48 h, over 70% of the NPs still remained within the tumor. We noticed a similar behavior of GNPs retention in our in vitro study as well. Our study concludes that NP-based targeting of both tumor cells and CAFs could be used as a smart novel strategy towards improving current anti-cancer therapies which include radiotherapy and chemotherapy. For an even better representation of a patient TME, our future in vivo studies will involve generating tumors from patient-derived xenograft models. We believe that a collective effort among biomedical researchers, radiation oncologists, medical oncologists, and pathologists could bring these novel therapeutics to the forefront for developing an effective strategy to increase the cure-rate in patients with pancreatic cancer.


It also showed a conductive bridge effect where the PEDOT:PSS acted as an anchor or support to Ag NW during mechanical strain and PEDOT:PSS also enhanced the electrical conductivity of the Ag NW. Therefore, to prove the real time performance of the electrode as a wearable device, we fabricated transparent electroluminescence devices and thin film heater devices which are highly flexible and demonstrated excellent performance proving that the AP electrode is more suitable candidate for future wearable transparent devices.In this study, we demonstrated poly: poly(styrene sulfonate) (PEDOT:PSS) as a composite with Ag nanowire (Ag NW) to enhance the stretchability of the Ag NW network electrode. The composite Ag NW/PEDOT:PSS hybrid ink (AP ink) was prepared at a ratio of 1 : 10, 1 : 20, and 1 : 30, respectively and bar coated on polyurethane substrate. The different ink ratios were studied and optimized with a sheet resistance of 14.93 Ω sq PEDOT:PSS, the highly stretchable and conductive polymer, can enhances the stretchability of Ag NW electrodes when mixed with Ag NW. Because PEDOT:PSS is still connected to the disconnected Ag NWs, the electrode has enhanced stretchability. Metal nanowires (Ag or Cu) are promising candidates, with Ag NW having the highest electrical conductivity of 6.3 × 107 S m−1 among all metal electrodes, including copper (Cu). It is noted that Ag NW has better corrosion resistance than Cu. Moreover, carbon nanotubes (CNTs) have high mechanical stability, flexibility, and transparency though the resistivity is extremely high and also flexible graphene is difficult to manufacture in a large scale with restacking and agglomeration problems.15 Although Ag NW has better electrical and optical properties, it has inherent limitations in that the polyvinylpyrrolidone used in Ag NW production introduces an insulating barrier around the Ag NW, increasing the overall sheet resistance of the electrode.16 Moreover, common coating strategies of the Ag NW electrode using various solution-based processes such as bar coating,17 spray coating,18 spin coating19 and vacuum filtration techniques20 are used to make flexible transparent conducting electrodes (TCEs). For example, Dong et al. demonstrated a high performance flexible Ag NW–polyimide (PI) electrode spin coated for non-fullerene solar cell with a power conversion efficiency of 11.6%.21 Zhang et al. studied Ag NW–PEN  spin coated as TCE showed a similar comparable result in transparency and conductivity as commercial indium tin oxide (ITO) electrode.22 However, Ag NW has significant disadvantages, including high junction resistance at the contacts of the nanowire, non-uniform morphology, poor mechanical–chemical stability and poor adhesion to the polymer substrate. Meanwhile, although PEDOT:PSS conductive polymers are highly flexible and cheap with a high work function of approximately −5.2 eV, where PEDOT:PSS has been used as a hole conductor, they have a low conductivity and suffer from humidity issues.23 However, the majority of the research was focused on conductivity, with flexibility and base conductive change receiving less attention.24 Therefore, for studying next-generation stretchable devices, research on how to improve stretchability and increase their resistance to water is needed.With the advancement of transparent electrode technology, innovations based on wearable portable gadgets have gained popularity for their human convenience and light weight. Therefore, the necessity and urgency for light weight stretchable electrodes is continuously increasing.via bar coating. We suggested a stretchability enhancing mechanism called ‘conductive bridge effect’ by mixing PEDOT:PSS and Ag NW. The Ag NW provided high conductivity while PEDOT:PSS provided high stretchability for the electrode. The network of bare Ag NWs is usually disconnected from stretching. On the other hand, the highly elastic PEDOT:PSS which strongly bonded Ag NWs in AP electrode, interlocked the fractured Ag NWs during heavy stretching without significant interrupting the flow of the current. As a result, the electrode has high electrical property and stretchability. We previously demonstrated that PEDOT:PSS composite with Ag NW can enhance the stretchability of Ag NW electrode with brush painting method.25 Though the brush painting method is both simple and cost effective, it is hard to control both the thickness and uniformity control for electrode. Thus, we used bar coating method to control the thickness and uniformity of the electrode by adjusting various parameters such as coating speed, mixing ratio of AP ink, and diameter of coating bar which is called Mayer bar. Moreover, for subjugation of the humidity issue, we added a protective layer called polydimethylsiloxane (PDMS) on top of the AP electrode by spin coating.23,26 The PDMS is cost-effectiveness,27 high transmittance,28 low elastic modulus,29 low surface energy,30 good insulating property,31 and high chemical resistance.32 Thus, applying PDMS as a protective layer would seal the electrode as well as reduce the degradation of the electrode. The highly stretchable transparent AP electrode was demonstrated via comparing the stretchability of AP electrode and bare Ag NW electrode. The AP electrode endured 10% higher strain and more than twice the repeated stretching test compared to the Ag NW electrode. Moreover, the AP electrode was proved to be highly flexible through 10 000 times of various repetitive mechanical tests.In this study, we developed an Ag NW/PEDOT:PSS composite ink (AP ink) at various mixing ratios and fabricated the electrode To verify the applicability of the electrode as wearable devices, the electroluminescence (EL) devices and thin film heater (TFH) devices were manufactured using AP electrode. The EL devices worked well in stretched and after stretching state. The performance of the TFH devices was not degraded in the bent or twisted states, however it was slightly degraded in the stretched state. These results showed the high potential of the cost-effective and easily manufactured AP electrode.2.2.1.−1 during bar coating was used to adjust the thickness of the electrode.The hybrid ink was prepared by adding 10 ml of water-based Ag NW solution  with 1 ml of PEDOT:PSS solution  and stirring them for 1 hour using a magnetic stirrer. It was then coated using bar coating technique on to a polyurethane (PU) substrate. The samples were then dried at 70 °C for 10 min. The electrode was optimized by changing the hybrid ink ratios of PEDOT:PSS and Ag NW ink to around 1 : 10, 1 : 20, and 1 : 30, respectively. The velocity movement of the bar at 17.5, 35, and 70 mm s2.2.n-hexane  at a ratio of 1 : 2 and the solution was spun coated at 2000 rpm for 30 s on top of the Ag NW/PEDOT:PSS electrode. The PDMS-coated Ag NW/PEDOT:PSS electrode (AP electrode) was then dried in a convection oven at 80 °C for 2 hours.The fabrication of the hybrid Ag NW/PEDOT:PSS electrode and the protective PDMS (base elastomer: curing agent = 10 : 1)  was mixed into 2.3.Electrical and optical properties of AP hybrid electrode were measured using Hall effect measurement  and UV/vis spectrometer  at a light of wavelength 400 nm to 800 nm, respectively. Surface energy data was collected by measuring the contact angle of droplets of DI water and diiodomethane on samples (Phoenix-MT(A), SEO Co.). The surface morphology of hybrid electrode before and after stretching test were analyzed by field emission-scanning electron microscope (FE-SEM) . The mechanical flexibility and stretchability of electrodes were measured by laboratory-designed stretching, bending, folding, rolling, and twisting tester. For measuring the stretchability, resistance change of electrodes was noted with increasing strain rate and stretching repetition test at 20% strain. For determining inner/outer critical bending radius of electrodes, resistance change of electrodes was measured by decreasing bending radius until 1 mm. Furthermore, dynamic fatigue tests were used to measure the endurance of AP hybrid electrode. Inner/outer bending fatigue tests were performed 10 000 times at a bending radius of 2 mm. Inner/outer folding tests and rolling tests were done 10 000 times at a bending radius of 3 mm and twisting test was done 10 000 times at a twisting angle of 35°.2.4.33 The mixed phosphor ink was brush painted on bottom AP electrode then the top AP electrode was attached and dried at 80 °C for 2 hours in convection oven. The alternating current (AC) potential was introduced to phosphor layer of EL devices using AC power supply.For fabricating stretchable EL devices, ZnS:Cu phosphor powder (Shanghai KPT company) and PDMS were mixed at a weight ratio of 2 : 1.2.5.AP electrode (5 × 2.5 cm) was coated with Ag paste to create contact points on the edges of the samples and dried at 80 °C for 10 min. The Ag contact points were then covered with Cu tape to ensure that they made contact with the power source. The PDMS layer was spin coated to protect the electrode. Direct current (DC) voltage was applied to TFH devices using DC power supply. The temperature was monitored using a thermocouple temperature measuring system that was placed on the surface of the TFH devices. The infrared (IR) thermal images were taken using IR camera tools .3.T10/Rsheet).34 where, T is transmittance and Rsheet is sheet resistance.The Ag NW/PEDOT:PSS ink was tested with various concentration ratios to optimize the electrode. 23,35 Also, moisture is the primary cause of silver corrosion. Therefore, water or moisture in the air can have an effect on the reliability of a silver electrode, such as a silver nano wire.26,36 In order to protect the electrode from the adverse effects of water, the PDMS was coated on the AP electrode. Because of the Cassie–Baxter state, the PDMS is hydrophobic due to the weak intermolecular interactions known as the London dispersion forces of the methyl group (–CH3) in PDMS and the helical molecular structure of PDMS that prohibits methyl groups from being on the same plane.37–39The hydroscopic property of PEDOT:PSS allows humidity to be absorbed in the polymer. Then the PEDOT:PSS expanded, increasing the distance between PEDOTs. As a result, the average hopping distance increased while interactions decreased. The dipole moments of absorbed water can also influence the conductivity of PEDOT:PSS. As such, due to the influence of water, the electrical property of PEDOT:PSS may deteriorate.40–42 The surface energy of film (γs) was calculated with Owens–Wendt method.43 The γs can be separated into the dispersion part (γds) and the polar part (γps) so that each part can be calculated from the contact angle of diiodomethane (DM) for disperse part and deionized water (DI) for polar part using below equations which well-known as Young equation  against their vapor, sl is the interface between liquid and solid, and θ is contact angle. The superscripts d and p refer to the dispersion part and the polar part. By plugging In these equations, γdl, γpl, and γlv of DM and DI, the free energy of film can be calculated with contact angles. The results in 45Using the already known R = (R − R0)/R0 was calculated where, R is the measured resistance and R0 is the initial resistance. The resistance was tested in situ under stretched conditions. The AP electrode was subjected to stretching as illustrated in 46 The extremely flexible PEDOT:PSS in the AP electrode, on the other hand, still interlocked the Ag NW despite repeated fatigue tests, and the electrode demonstrated improved mechanical durability. The mechanical fatigue test was carried out to further demonstrate the flexibility of the AP electrode in Electroluminescence (EL) devices were fabricated from the Ag NW/PEDOT:PSS electrode as shown in 47V is voltage, hc is convective heat transfer constant and Ti is the initial temperature of TFHs. They are constant with increasing strain rate. R is resistance of electrode and A is area of TFH. The resistance increased when the strain rate increased, and this increased value made the saturation temperature Ts decrease. However, the performance of stretched TFH was still enough to function as a heater. In 4.n-hexane mixture on the electrode surface. The optical, electrical, and mechanical properties of an electrode were investigated. The optimized AP electrode has 14.93 Ω sq−1. of sheet resistance and 88.6% of transmittance at 550 nm wavelength. The PDMS protective layer has a low surface energy and a hydrophobic property, which can protect the electrode from humidity-induced washing and corrosion. In the dynamic stretching test, the AP electrode endured over 40% stretch strain rate and 20% stretching repetition test for more than 350 times. These results showed enhanced endurance for stretching when compared with bare Ag NW electrodes. Other mechanical tests such as bending, folding, twisting, and rolling showed remarkable performances. For applications of electrode, the AP electrode was fabricated as EL devices and TFH devices. Various designed EL devices were created using brush paintable PDMS/ZnS:Cu phosphor ink. After the stretching cycle test, these devices performed well when stretched and worn 300 times. Moreover, TFH performed effectively in the twisted and bending states. These results demonstrated the potential of AP electrodes in a variety of stretchable or wearable applications.In this study, we demonstrated that mixing PEDOT:PSS into Ag NW enhanced the stretchability of Ag NW electrode by conductive bridge effect where the added PEDOT:PSS interlinked with the disconnected Ag NW network. The electrode was fabricated using bar coater with optimized AP ink on PU substrate and spin coater with PDMS/There are no conflicts to declare.


Global freshwater biodiversity has been decreasing rapidly, requiring the restoration and maintenance of environmental flows (EFs) in streams and rivers. EFs provide many ecosystem services that benefit humans. Reserving such EFs for aquatic ecosystems, implies less renewable water availability for direct human water use such as agriculture, industry, cities and energy. Here we show that, depending on the level of EF protection, global annual renewable water availability for humans decreases between 41 and 80% compared to when not reserving EFs. With low EF protection, currently 53 countries experience different levels of water shortage, which increases to 101 countries for high EF protection. Countries will carefully have to balance the amount of water allocated to humans and the environment. Freshwater species have been disproportionally impacted and decreased by 84%. Almost one in three freshwater species is threatened with extinction1, which is consistently higher than for their terrestrial counterparts2. The violation of environmental flows (EFs) due to the water footprint of humanity5 is a major reason for this rapid decline in aquatic biodiversity6. Such EFs are required to maintain ecosystem integrity in streams, rivers, wetlands, riparian zones and estuaries. EFs also provide many additional ecosystem services, with direct links to specific Sustainable Development Goals (SDGs)8. As an example, EFs sustain fish stocks and other aquatic life, which contribute as nutrition biomass directly to SDG 2 “zero hunger”. In the Mekong river basin, the second most aquatic biodiverse river basin in the world9, freshwater fish biomass contributes the bulk of animal protein intake. A dish with fish and rice therefore requires EFs as well as enough water for agriculture. Restoring and maintaining EFs is thus essential for humanity10.Globally, monitored population sizes of mammals, fish, birds, reptiles and amphibians have declined by 68% on average between 1970 and 201611. With over 200 existing EFs methods10, it is a challenge to quantify how much water should be reserved to sustain ecosystems and how much water is available for direct human use such as agriculture, industry, cities and energy.Although locally, empirical quantitative relationships between various degrees of flow alteration and ecological responses have been derived, EFs are still unknown for the vast majority of freshwater and estuarine ecosystems11, which attributes 80% of natural monthly river flows as EF (EFPROT). As a measure representative for minimum flow recommendations (EFMIN), we use the monthly Q95, that is, the flow exceeded for 95 per cent of each month. We then substract EFs from national renewable available water and identify the corresponding levels of human water shortage for each country. Both EFPROT19 and EFMIN25 are widely used in different water management studies.Here we assess national per capita renewable water availability worldwide, accounting for high respectively low aquatic ecosystem protection by means of two well established EF methods. As a measure for high ecosystem protection, we use the presumptive standard for EFs by Richter et al.26. Natural or pristine water availability is water availability without human interventions, such as water use or water infrastructure . The model works at a spatial resolution of 0.1 degrees (11.1 km at equator), with a daily time step for the period 1980–2018, and generates natural water availability as the sum of renewable surface and groundwater.We compute renewable water availability in high spatial and temporal detail, with the established hydrological open-access model LisfloodMIN = WA_noEF − EFMIN) and human renewable water availability with high EF protection (WA_EFPROT = WA_noEF − EFPROT).We aggregate daily simulated water resources to monthly values in order to account for the intra-annual variability in water availability and EFs, and compute the resulting per capita water availability and shortage on an annual level. We distinguish between natural or pristine water availability, referred to as renewable water availability without EF protection  to 5 (absolute water shortage). Above that threshold, two classes without water shortage are defined.Water shortage estimations are derived by combining average annual country renewable water availability estimates with population statistics. Based on the corresponding per capita water availability, different water shortage classes are defined for each country Table . Followi28.National per capita water availability and potential water shortage are quantified for the year 2020, both as internally produced water availability and total water availability when water enters a country through inflow from upstream river basins. We use the transboundary river basins database to address the contribution of this externally produced water availability8. We do not account for economic water scarcity8.27 is a water shortage indicator.Water shortage measures water availability per person. Given a certain water endowment and per capita water requirement, water shortage can therefore be seen as population-driven scarcity. The Falkenmark indicatorWater stress measures water use relative to water availability. Water stress can be seen as demand-driven scarcity, potentially occurring even when population is low, for instance because of large water-use for producing agricultural or industrial products for populations elsewhere. SDG indicator 6.4.2 is a water stress indicatorWe use water shortage as indicator. There are two main physical water scarcity indicators: water shortage and water stress3 (or 7669 m3 per person per year), with high temporal and spatial variation in availability spread over the globe  for direct human use, so 41% less as compared to WA_noEF. Ensuring high monthly EFs adds up to a global annual EFPROT amount of 48,107 km3, leaving 12,025 km3 (WA_EFPROT) for direct human use. This is 80% less as compared to WA_noEF.Estimated global annual natural renewable water availability (WA_noEF) amounts to 60,132 kmPROT and 20% for WA_EFPROT related to WA_noEF are spatially homogeneous, there are wide spatial variations in the proportions of EFMIN and WA_EFMIN related to WA_noEF , results in 130 countries (with a population of 4879 million) without water shortage and 75 countries (with a population of 2912 million) with water shortage. Ensuring high EFs (EFPROT), results in 73 countries (with a population of 1731 million) without water shortage and 132 countries (with a population of 6060 million) with water shortage. There is a substantial increase in the amount of countries and population under water shortage when ensuring EFPROT instead of EFMIN, with populous countries such as China, Mexico or Iran moving to water shortage.For the population status in the year 2020, internal and total water shortage levels differ substantially among the 205 countries and between the different levels of EF protection Fig. . When onMIN to 74 (with a population of 2881 million) for WA_EFPROT  as well as island states . Many island states in the Caribbean as well as Cyprus, which do not have inflow from abroad, shift to class 3 for WA_EFPROT.The amount of countries with very high to absolute internal water shortage (classes 3 to 5) increases from 18 (with a population of 224 million) for WA_noEF to 21 (with a population of 235 million) for WA_EFMIN) results in 152 countries (with a population of 5687 million) without water shortage and 53 countries (with a population of 2104 million) with water shortage. Ensuring high EFs (EFPROT) results in 104 countries (with a population of 2498 million) without water shortage and 101 countries (with a population of 5293 million) with water shortage.When accounting for water inflows from upstream countries, renewable water availability in different countries increases and the amount of countries as well as number of people under water shortage decrease Fig. . Not accPROT = 2099 m3 per person per year), as 99% of total water availability comes from abroad  include Bangladesh (87% from abroad), Thailand (52–57% from abroad), Romania (74–77% from abroad), Ukraine (66–67% from abroad), Sudan (82–84% from abroad), Zimbabwe (67–71% from abroad) and Mozambique (69% from abroad).Inflow from abroad changes the water shortage situation of some countries dramatically. Most notably, the inflow from the Nile shifts Egypt from class 5 water shortage to no water shortage  for WA_EFPROT , the Middle East , North and Southern Africa , Europe  as well as many island states spread over the world.When accounting for total renewable water availability Fig. , the amoPROT), results in 101 countries (with a population of 5293 million) experiencing different levels of water shortage, of which 49 (with a population of 2016 million) with very high to absolute internal water shortage (classes 3 to 5). But even low EF protection (EFMIN) results in 53 countries (with a population of 2104 million) with water shortage, of which 16 (with a population of 92 million) with extreme to absolute internal water shortage.We compute a first-time detailed national population-driven water shortage analysis under no, low and high EF protection, by means of a state-of-the-art global hydrological model. We base our analysis on estimated monthly water availability and EF amounts, data we provide for each country in Database 27, a well-established and widely used indicator31. Reasons for the wide acceptance of this indicator are multiple: it is simple and intuitive, and data on human population are readily available.The population-driven water scarcity metric we use, is strongly based on the Falkenmark indicatorBy 2030, implement integrated water resources management at all levels, including through transboundary cooperation as appropriate”.Also, although water should be properly managed within river basins, even when they cross international borders, most countries are primarily concerned about water availability within national borders. Our assessment provides such water availability estimations and has shown that several countries are highly dependent on inflow from abroad. This clearly shows the necessity for bringing together different sectors and stakeholders at all scales from local to transboundary in river basin management. SDG 6 therefore has a dedicated target 6.5 which aims at: “3 per person a proxy for the lowest need in a modern semi-arid country27 (the cut-off for water shortage class 3), has in recent times further reduced its total per capita water abstraction to less than 300 m3 per year32, by implementing measures such as highly efficient irrigation, water recycling and desalination. It has thereby surpassed its peak water demand, decoupling GDP from water abstraction, in the same way many developed nations including the USA have34. According to Gleick33, such a decoupling of water withdrawals from population and economic growth has occurred in the USA due to three key reasons. First, substantial technological improvements reduced the amount of water required to meet urban, industrial, and agricultural needs. Second, changes in the overall structure of the US economy have also played a role in this transition, including a shift in water-intensive manufacturing to overseas locations. Third, a change away from water-intensive once-through cooling systems for thermal power plants has reduced the amount of water required to produce a unit of energy.Our indicator does not grasp efforts of countries to reduce their actual water abstraction. Israel, representing with 500 mThe threshold value for class 5 (minimum domestic water demand) is a universal amount. The threshold value for class 4  is a realistic target value which can be achieved in a developed country. The threshold values for water shortage classes 1 to 3 are dependent on climatological conditions and are to be regarded as indicative, not absolute.35 in water availability. Another indicator on water scarcity, i.e. water stress, the ratio between water use and water availability, can grasp such information30, but is not in the scope of our study. Nor does our study give any indication on the status of national water supply infrastructure . Our study should therefore be seen a first-pass assessment that gives indications on national water shortage, to be examined in detail on country level. In addition, we do not account for the impact of climate change on future water shortage.Our assessment does not address differences in water demands between countries or intranational differencesPROT and EFMIN we have determined in our analysis.Countries will carefully have to balance how much water they attribute as EFs and how much for direct human use. Which EFs are eventually optimal in specific locations requires further investigation of environmental needs through sophisticated EF assessment methodologies, such as habitat simulation methods and holistic methods, supported by local field measurements campaigns. We expect such local EF flows to be situated within the range between EF36, surpassing peak per capita demands, by increasing water efficiency and productivity41, water recycling, addressing the groundwater component of EFs42, holistic river basin management and end-of-supply-chain interventions such as food waste reduction, dietary shifts and choice of energy sources47. In addition, river fragmentation by infrastructure such as dams and weirs will have to be managed and river connectivity ensured in order to rehabilitate and maintain EFs50, to bend the curve on global aquatic biodiversity loss6 and to ensure the multiple ecosystem services provided to humans by EFs. The EU, e.g., has in its biodiversity strategy for 203051, targeted that at least 25,000 km of rivers will be restored into free-flowing rivers by 2030, through the removal of primarily obsolete barriers and the restoration of floodplains and wetlands. To bend the curve on global aquatic biodiversity loss, in addition, good water quality and habitats need to be restored54, global warming limited55 and overexploitation56 and invasive species57 tackled. Overall, many different actions need to be undertaken to speed progress towards SDG 636, addressing win–win situations and trade-offs within the water-energy-food-ecosystem (WEFE) nexus59. This requires the inclusion of many different actors at different scales60.Human water use will have to be managed and optimized26, a well established model used in different studies from local to global level63. Lisflood accounts for rainfall-runoff-routing in the river network, as well as several surface and sub-surface hydrological processes, including plant interception, evapotranspiration, soil freezing, snow accumulation and melting, surface runoff, lakes and reservoirs, water abstraction, infiltration, preferential flow, redistribution of soil moisture within the soil profile, drainage to the groundwater system, groundwater storage, and base flow. Surface runoff is produced at every grid cell and routed through the river network using a kinematic wave approach64.To compute natural water availability, we used the open-source distributed semi-physically based hydrological model Lisflood65 for elevation and the global river network database66 for river network and flow directiontopography maps . We use the Shuttle Radar Topography Mission (SRTM)67 for soil informationsoil . We use SoilGrids1km68 for land use and the SPOT-VGT data69 for monthly maps of Leaf Area Index.land use . We use GlobCover 200970 for river widthschannel geometry . We use the Global Width Database of Large RiversHigh-quality spatial datasets in hydrological modeling are crucial to avoid over‐parameterization and reduce the dimensionality of the model calibration. Spatial datasets used in Lisflood include:62, a state-of-the-art global streamflow reanalysis with median scores at 1226 calibration stations within 66 world countries of Kling-Gupta Efficiency KGE = 0.67 and correlation r = 0.8. All atmospheric variables to run the model were extracted from the ERA571 reanalysis and regridded from the original resolution of 31 km to the model resolution (~ 11 km) using nearest neighbour interpolation. Lisflood requires, as input, near surface air temperature, precipitation and potential evapotranspiration. The latter was estimated with the Penman–Monteith equation as described in Supit et al.72, using daily average temperature, wind speed, relative humidity and solar radiation as input.We used the Lisflood setup and parameterization of the GloFAS-Reanalysis v3.0We run a 40 year simulation for 1980–2018, excluding all the human influence, i.e., without the effect of reservoirs and of all water abstractions. Resulting daily discharges of natural or pristine flows were aggregated at monthly resolution. The simulated discharges incorporate surface water flows as well as base flows (renewable groundwater), resulting in total renewable water availability.3 as average for the period 1980–2018. Past assessments found a range of WA_noEF amounts, depending on modelling methodology, time period, inclusion of anthropogenic water use as well as inclusion of Greenland or not73. LISFLOOD is on the higher end of the multi-model range of water availability computations73. A recent assessment for the period 1970–200515 quantified for three models an average WA_noEF of 54,100 km3, a value close to our own estimate here.As a result, we calculate a global annual renewable WA_noEF of 60,132 kmPROT: the presumptive standard for EFs by Richter et al.11, which defines 80% of the (monthly) natural flow as EF; the remaining 20% is considered as water available for human use.EFMIN: the monthly Q95 (the flow exceeded for 95 per cent of the month). The Q95 is often used as low flow indicesEFWe use two well-established hydrological EF methodologies to account for minimum and maximum EF protection:These EFs are calculated with a daily time step. Both selected methods are hydrological methods. EF methods can be classified into four types: hydrological methods; hydraulic rating methods; habitat simulation methods; and holistic methods. Latter methods require large amounts of data which are not available at the global level. As such, we choose only hydrological methods.74. We distinguish between:renewable water availability without EF consideration (WA_noEF)MIN = WA_noEF − EFMIN)human renewable water availability water with low EF protection (WA_EFPROT = WA_noEF − EFPROT).human renewable water availability water with high EF protection  adds to the internal water availability  of a country. When a river forms the border between two countries, we assume that this flow is fully available to both countries . As such, we compute a maximal total renewable water availability. These values are of course theoretical and represent an overestimation. In reality, upstream countries alter (monthly) water availability for downstream countries by means of water storage infrastructure such as dams as well a consumptive water use8.To compute annual national total renewable water availability, we add inflow to internal amounts. The inflow is determined by means of the inflow from upstream river basins, for which we use the transboundary river basins database8 Figure . As ruleSupplementary Information 1.Supplementary Information 2.