We assessed soil organic carbon (SOC) stocks, sequestration rates and carbon origin of deep peat soils (1 to 6 m). We found massive amounts of SOC up to 2792 Mg C ha−1, the highest value reported in the literature so far. This SOC is primarily derived from highly preserved mangrove roots and has changed little since its deposition, which started over 3220 years ago (±30 BP). Most cenotes are owned by Mayan communities and are threatened by increased tourism and the resulting extraction and pollution of groundwater. These hot spots of carbon sequestration, albeit small in area, require adequate protection and could provide valuable financial opportunities through carbon-offsetting mechanisms and other payments for ecosystem services.Mangroves are among the most carbon-dense ecosystems on the planet. The capacity of mangroves to store and accumulate carbon has been assessed and reported at regional, national and global scales. However, small-scale sampling is still revealing ‘hot spots’ of carbon accumulation. This study reports one of these hotspots, with one of the largest-recorded carbon stocks in mangroves associated with sinkholes ( As a re2 of permeable limestone [cenotes, a word derived from the low-land Maya language ‘tz'onot’. There are over 2000 cenotes distributed throughout the Yucatan Peninsula. In the north, cenotes were formed by the impact of the Chicxulub asteroid creating a feature known as ‘Ring of Cenotes’ [cenotes are associated with the Holbox fault, which formed open water bodies parallel to the coastline. In these cenotes, seawater intrudes into the aquifer creating a habitat for dense mangrove forests nortes (north winds) from November to January with sporadic rains (mean monthly precipitation of 68.3 mm), a hot dry season from March to April (37.7 mm) and a hot wet season from May to October (133.9 mm), when tropical storms are common.Yucatan Peninsula is in the southeast of Mexico. The region has a tropical wet–dry climate (Köppen classification Aw) with a mean annual temperature of 25.7°C  and mean annual precipitation of 1122 mm  at the surface from marine water at the bottom . Cenote Yaxchen  is about 350 m from the sea, with an opening of 6500 m2, maximum depth of 7.5 m and a similar halocline profile as Casa Cenote. Finally, Cenote Jaguar  is located furthest from the sea, 2 km inland, with an opening of 500 m2, a maximum depth of 8 m and no halocline, with salinity values <3 ppt throughout the water column.We selected three cenotes associated with mangroves, where SCUBA diving was accessible . Casa Ce (b)cenotes . In Cenote Jaguar, the peat layer was 1 m deep; in Yaxchen, the peat reached 5 m and in Casa Cenote, it reached 6 m. The samples were refrigerated and transported to the laboratory, where they were oven-dried and weighed. Bulk density was determined from the dry mass per wet volume (expressed as g cm−3).Sampling was conducted in September 2019 with SCUBA equipment. We sampled two points at Cenote Jaguar and Yaxchen and four points at Casa Cenote. Within each point, underwater sampling was conducted with a 60 ml mini core, which resulted in minimum compaction. Sampling was done at each of the following depths: 0–15, 15–30, 30–50, 50–100 cm, and every 100 cm until the bottom of the peat [13C with an elemental analyser coupled with an isotope ratio mass spectrometer [13C values with depth. We expected that isotope values would remain constant with depth if carbon was stable through time and decomposition was low; on the contrary, isotopic values would increase if decomposition was high [13C would be close to −28‰, a value typical of plants with a C3 photosynthetic pathway and common for mangroves in the region [Soil samples were acid-washed with HCl to eliminate carbonates and analysed for soil organic carbon (SOC) and δtrometer   were radiocarbon dated at Beta Analytics . The results were corrected for isotopic fractionation and calibrated with the 2013 INTCAL program  and rounded to the nearest 10 years . Carbon . 3−3. The δ13C values were remarkably consistent with depth . Carbon stocks were very high at Casa Cenote and Cenote Yaxchen with a mean value of 1492 ± 549 and 1518 ± 561 Mg C ha−1, respectively, with one sampling point in Casa Cenote reaching 2792 Mg C ha−1. In comparison, SOC stocks at Cenote Jaguar were moderate with 429 ± 44 Mg C ha−1.The SOC for all sites was 38.5 ± 0.9% (mean ± s.e.), values close to vegetation, indicating that the samples were mostly peat, with little sediment . Bulk deR2 = 0.89; p = 0.024, y = 471.3x + 24.8; electronic supplementary material, figure S1). The radiocarbon age of the deep peat samples was 2490 ± 30 BP (75 cm deep), 3040 ± 30 BP and 3220 ± 30 BP (450 cm deep) in Cenote Jaguar, Cenote Yaxchen and Casa Cenote, respectively. Correspondingly, SOC sequestration rates were estimated at 0.12, 0.60 and 0.66 Mg C ha−1 yr−1  and deep soils, which have been preserved for millennia.We found massive SOC stocks below mangroves of the −1 yr−1 in coastal cenotes (Cenote Yaxchen and Casa Cenote) and 0.12 Mg C ha−1 yr−1 in inland cenotes (Cenote Jaguar). At present day, deep SOC continues to accumulate, probably at rates similar to those of current sea-level rise [We propose that the high SOC stocks of these mangroves are associated with the sea-level history of the region. Globally, after the last glacial minimum, sea-level rise accelerated and then decelerated towards the mid and late-Holocene . At the vel rise ,21  [cenotes [−1 yr−1) [δ13C values consistent with a mangrove origin .The SOC stocks in these n depth) . The rat[cenotes  and rate−1 yr−1) . While r−1 yr−1) . Thus, t (27–29‰ ). Althoucenotes in this region . The massive and stable SOC stocks from this study also provide a strong basis for the inclusion of these mangroves in carbon projects. For instance, a carbon project in a mangrove forest in this region with peat of 2 m would have approximately 1000 Mg C ha−1, from which more than 50% could be lost if the site is converted to another land use [cenotes complex) for 100 years could offset almost 13 341 Mg of CO2eq every year [2eq in an international market), a considerable amount of money for a rural community in Mexico. This economic incentive could also be complemented through other payments for ecosystem services given the high endemicity of species within these cenotes [The established relationship between SOC stock and depth provides a significant opportunity to reduce costs and effort when estimating SOC for mangroves associated with land use . If defoery year , which c cenotes  and impo−1 [cenotes within the Yucatan Peninsula.The exceptionally large SOC stocks stored in these unique ecosystems are threatened by poorly planned tourism, excessive water extraction and groundwater pollution. The latter is caused by the lack of water treating infrastructure in the region, which mostly relies on septic treatments that can leach nitrogen into the aquifer . Contemp−1 . Thus, t−1 . These a


Methylmercury is a neurotoxin and bioaccumulated with fish age, but other factors including selenium and other elements, diet and trophic level as assessed by stable isotopes of nitrogen (δ15N) and carbon (δ13C), as well as growth rate, condition, and geographic origin, also contributed depending on the species, even though all the fish shared a similar anadromous or sea-run life history. Although mean mercury concentrations for most of the species were ~0.09 µg·g−1 wet weight (ww), below the levels described in several jurisdictions for subsistence fisheries (0.2 µg·g−1 ww), 70% of lake trout were above this guideline (0.35 µg·g−1 ww), and 19% exceeded the 2.5-fold higher levels for commercial sale. We thus urge the development of consumption advisories for lake trout for the protection of pregnant women and young children and that additionally, periodic community-based monitoring be initiated. As mercury emissions continue and climate-mediated permafrost thaw increases the burden of this contaminant in northern waters, Inuit from a Northwest passage community in the Canadian Arctic Archipelago pressed for an assessment of their subsistence catches. Sea-run salmonids ( Ting melt . Hg leveing melt . The Arc2 with MeHg contamination, as well as with tissue accumulation due to rapid uptake and slow elimination [The most abundant form of Hg found in fish tissue is methylmercury (MeHg), and therefore total mercury (THg) levels including organic MeHg, a neurotoxin, are most consistently reported in food fish ,8. The mmination ,11,12,13mination ,15,16. Fmination . Moreovemination . THg levmination ,20. Common measures for normalizing levels of contaminants at size typically do not routinely consider environmentally malleable growth rates in fish, as well as variable growth between species, populations, and even individuals ,22,23,24Qikiqtaq, or King William Island, in the lower Northwest passage in the east Kitikmeot region of Nunavut, Canada. Salvelinus alpinus  has the most northerly distribution of any freshwater fish [Coregonus clupeaformis  as well as C. autumnalis  and C. sardinella , are also anadromous, overwintering in lakes but entering brackish waters on a seasonal basis, and according to Indigenous Knowledge (IK) or Inuit Qaujimajatuqangit, coming back to fresh water within days of the char migration peak. Although S. namaycush  have been reported to be the least salt-water-tolerant of the salmonids [Qikiqtaq and adjacent mainland sites during the autumn migration, with ice-fishing also occurring after “ice-up” and into the spring breakup. Here, otolith age assessments were used to investigate THg contamination in four popular subsistence salmonids at sites on or near ter fish , with thalmonids , they alalmonids . All theQikiqtaq community of Uqsuqtuuk, or Gjoa Haven, are regular consumers of these fish and requested that contaminants be assessed. Although THg has been intermittently assayed in select species in the Canadian north from the 1970s  and August–September (at open-water rivers or brackish waters prior to ice formation) were sampled over several years (2016–2019) using commercial (140 mm mesh) or multi-mesh subsistence (5 or 8 panels of 38–140 mm panels) fishing nets that were soaked for several hours. Occasionally, hand lines or spears were employed, ensuring that different populations of fish were sampled at dispersed fishing sites  using 400-, then 800-grit discs, leaving approximately 300–380 µm thin sections. These were microscopically viewed at 25, 40, and 100× magnification, and annuli, or growth discontinuities associated with the distal edge of translucent zones, were counted and tracked in three directions along the ventral, dorsal, and medial axes , with a log–log relationship developed for the entire sample. The antilog of the mean incremental value at each age provided the annual incremental growth standard curve  at age for each fish , and as previously noted in other regions . The pre3) were cut into small pieces using a sterile knife and dried at 70 °C for most elemental analyses, but at 21 °C for THg and MeHg. After drying and the calculation of mean moisture content, an aliquot of each sample was ground to a powder with a methanol-cleaned mortar and pestle, and after discarding the first grindate, the second grindate (500 mg) was removed to a glass vial. THg was quantified on the solid sample using cold vapor atomic absorption spectroscopy (CVAAS) at Queen’s University Analytical Services  following the United States Environmental Protection Agency (USEPA) Method 7473 [Frozen muscle samples  and carbon (δ13C) followed previously reported guidelines [Analysis of stable isotopes of nitrogen  to achieve normal distributions  prior to statistical analysis. Condition (K) was calculated based on Fulton’s equation (K = W·L3−1 [2 [2 used the F statistic by quantifying the ratio of two variances. Secondary explanatory variables (r2) were used to interpret interspecies differences in THg accumulation with age. Linear models for THg concentrations at a calculated standardized age were used to compare THg in fish from different sampling sites. Statistical differences in the THg means from grouped sites with unequal group sizes and heterogeneity of variances were performed using Welch’s tests with the Games–Howell post hoc test used to evaluate differences in means between grouped sites, with other statistical tests performed in IBM SPSS Statistics 25 and Statistix (version 9.0), except where noted otherwise. Biological data , in addition to THg and Se data, were log transformed (log= W·L3−1 ). Relati·L3−1 [2 ). The st13C, and δ15N, using XLSTAT 2020.4.1 , as well as age, length, weight, K value, δ.4.1 see . Concentn = 321, Spearman correlations) using XLSTAT 2020.4.1. Again, concentrations below detection limits were imputed with ROS in ProUCL 5.1. To better visualize comparisons between the THg from different fish, exploratory principal components analysis (PCA) was conducted with the same variables used for the correlation analysis  were consistent with previous reports showing that 95% of the THg was contributed by MeHg, which is known to be derived from dietary sources in addition to only a minor aqueous component from respiration [Mean THg concentrations for both lake whitefish and cisco at 0.09 µg∙g−1 ww  were conn Canada . Mean THectively ,47,48. Npiration . We considered that between-species THg variation could be due to differences in growth rate and trophic level. It has been reported that slow-growing fish bioaccumulate greater concentrations of THg than do fast-growing fish  had the greatest age range (8–62 years), with a mean age of 25.4, just higher than lake whitefish (n = 100), with a mean age of 21.3 (range of 4–47 years) (n = 197) had a younger mean age of 14.2 (5–29 years). Both species of cisco were pooled (n = 98), since as noted, the presence of hybrids made identification to species more challenging, and they had a mean age of 14.8 years (2–30-year range). Of the factors influencing THg, age as determined by otoliths was the only variable that was significant for all examined fish (p < 0.01), with age also explaining 30% of the variation in lake trout  = 57.36, p < 0.01). Although not as strong, age explained 14% of the Arctic char THg variation  = 32.5, p < 0.01) and 12% of the variation in cisco  = 12.72, p = 0.001). This association was further confirmed when the salmonids were pooled and analyzed together, showing a positive rank correlation between THg and age , as shown in Fork lengths are frequently used as a proxy for age, but as indicated, at high latitudes, fish grow slowly, and in our samples, growth leveled off after ages 5–10, underscoring the importance of using otolith year class for accuracy . Lake tr7 years) . Arctic ned fish , consistp < 0.000). Tying low THg levels with rapid growth is not adequate by itself, however, when one considers that lake trout had the second highest growth rate but accumulated the highest THg levels. The otolith age data, used to develop an incremental growth standard for each of the fish groups , showed p < 0.000), accounting for 14% of the variation (p = 0.016), with average Se concentrations in the latter fish being 1.4-fold higher than levels in the other three salmonids , but not so clearly in the Coregonus species (−1 ww), compared to a lake trout of the same age in good condition (K = 1.3) with low Hg levels (THg = 0.09 µg·g−1 ww). Another example is afforded by two 13/14-year-old Arctic char from the same freshwater site on Koka Lake, one with poor condition (K = 0.9) and a high THg level (0.35 µg·g−1 ww) vs. the other with good condition (K = 1.1) and a low THg level (0.02 µg·g−1 ww). Strikingly, both these comparisons align with the toxicity threshold of ~0.2 µg·g−1 ww THg, which is defined based on sub-lethal endpoints of growth, development, and behavior in fresh- and salt-water fish, with a lower threshold for reproductive effects [Differences in THg accumulation were also sought in secondary explanation variables. After age, condition factor (K) accounted for the greatest variance of THg in lake trout  = 22.80,  species . As fish effects ,60,61,6215N‰) and forage location (δ13C‰). Results for these isotopes in Arctic char and lake trout were very similar to those reported for anadromous populations in the west Kitikmeot region of the Arctic [15N values , but since lake trout and Arctic char had similar trophic positions but substantially different THg levels, this emphasizes our argument that trophic position by itself cannot explain THg accumulation.The possible role of seasonal diet in THg bioaccumulation led to the measurement of carbon and nitrogen isotopes to investigate the impact of proxy values for trophic position , or carbon sourcing (foraging habits). Fish that feed in the benthic zone tend to have higher δ13C than those that forage in the offshore, pelagic zones [13C is typically normalized to lipid content, and as previously reported, lipid assays of a subset of the samples (n = 101) showed that Arctic char was less fatty than the other three groups of salmonids that were all similar [13C values can be directly compared in lake trout, lake whitefish, and cisco. Efforts to increase the overall sample size by using the C/N ratio as a proxy for lipid assays, as used by other researchers [15N) and carbon sourcing (δ13C), was modeled for each of the fish groups (15N (‰) were significantly and positively related to changes in carbon sourcing for lake trout  = 8.22, p = 0.005), not significantly related in Arctic char and whitefish, and significantly negatively related for cisco  = 7.37, p = 0.008). It has been reported that at least for Arctic char, δ13C levels vary depending on the year, thus indicating broad prey choices [We considered that another explanatory variable for the low THg level in Arctic char could be assessed using δic zones . To unde similar , and theearchers ,70, coulearchers ). Similaearchers ,67,72. Rh groups . Changes choices . Overallp < 0.0001, unless stated otherwise) between THg and Rb (ρ = 0.530), Tl (ρ = 0.361), Ti , Mn (ρ = 0.235), Zn , Ca , Mg (ρ = 0.288), P (ρ = 0.283), K (ρ = 0.325), Na (ρ = 0.372), and S (ρ = 0.449), and negatively correlated with Ni (ρ = −0.237), Cr (ρ = −0.288), and Cu (ρ = −0.262), but not with As, Sr, Co, Fe, or Se. The association between these various factors in the sampled salmonids was further investigated with exploratory PCA. As might be expected based on the correlation analysis, there were associations between THg and other elements: the non-essential Tl and Rb, in addition to the essential nutrients K, S, Mg, and P, plotted in the same quadrant when the fish were analyzed together , and in contrast to the consistently high THg values in lake trout  higher mean THg levels than those obtained from mainland sites (groups 1–2 vs. 3–4) at 0.16 and 0.08 µg·g−1 ww respectively, such concentrations are still low , and 30t weight  and not  mg·kg−1 ,84,85,861 [−1 ww , and for1 [−1 ww . Concern1 [−1 ww . AdditioUqsuqtuuk community will continue to access Arctic char, lake whitefish, and cisco as healthy food choices and as a resource for economic benefits. Nevertheless, our data underscore a concern about the advisability of sourcing lake trout for community prenatal cooking classes, the “mums and tots” group, school food programs, as well as celebratory events and community feasts. Due to the special vulnerability of Arctic regions to atmospheric Hg emissions and thawing permafrost, we further recommend that monitoring of these food fish should continue on a periodic basis, both for the long-term health of subsistence consumers, and for the well-being and safety of any fishery operations.As indicated by a wealth of literature, in addition to this study that investigated multiple influences on the levels of THg, the bioaccumulation of this element depends on many biotic and abiotic factors, including age, growth rates, condition, trophic level, and concentrations of selenium and other elements , with th


Background: Psoriatic disease is a multifactorial inflammatory condition spanning from skin and nail psoriasis (Pso) to spine and joint involvement characterizing psoriatic arthritis (PsA). Monozygotic twins provide a model to investigate genetic, early life environmental exposure and stochastic influences to complex diseases, mainly mediated by epigenetics.Methods: We performed a genome-wide DNA methylation study on whole blood of monozygotic twins from 7 pairs discordant for Pso/PsA using the Infinium Methylation EPIC array (Illumina). MeDiP—qPCR was used to confirm specific signals. Data were replicated in an independent cohort of seven patients with Pso/PsA and 3 healthy controls. Transcriptomic profiling was performed by RNAsequence on the same 7 monozygotic twin pairs.Results: We identified 2,564 differentially methylated positions between psoriatic disease and controls, corresponding to 1,703 genes, 59% within gene bodies. There were 19 regions with at least two DMPs within 1 kb of distance and significant within-pair Δβ-values (p < 0.005), among them SNX25, BRG1 and SMAD3 genes, all involved in TGF-β signaling pathway, were identified. Co-expression analyses on transcriptome data identified IL-6/JAK/STAT3 and TNF-α pathways as important signaling axes involved in the disease, and they also suggested an altered glucose metabolism in patients’ immune cells, characteristic of pro-inflammatory T lymphocytes.Conclusion: The study suggests the presence of an epigenetic signature in affected individuals, pointing to genes involved in immunological and inflammatory responses. This result is also supported by transcriptome data, that altogether suggest a higher activation state of the immune system, that could promote the disease status. Psoriatic arthritis (PsA) and psoriasis (Pso) represent different manifestations of the same disease spectrum, i.e., psoriatic disease, a systemic chronic inflammatory condition, where genetic factors contribute to disease susceptibility and where biomarkers are virtually absent . Monozygloci to be further investigated as molecular biomarkers.We hypothesize that epigenetic changes, in specific DNA methylation, may contribute to the onset of Pso/PsA in discordant MZ twins. . We perfWe enrolled pairs of MZ twins previously identified through the collaboration with the Italian Twin Registry—Istituto Superiore di Sanità and residing in the area surrounding the Humanitas Research Hospital in Rozzano . Participants were evaluated jointly by a rheumatologist and a dermatologist to define the diagnosis of Pso according to the physical examination and/or PsA according to the CASPAR classification criteria . The stup-value was evaluated across all samples, and those probes with a p-value > 0.01 in one or more samples were discarded from the dataset. We also removed those probes where SNPs may affect the CpG, and underperforming probes, as suggested by Zhou and others , following manufacturer’s instructions. For each sample, 500 ng of genomic DNA was bisulfite converted using the EZ DNA Methylation Kit . Bisulfite-treated samples were then processed using the EPIC array , according to manufacturer’s instructions. The iScan system (Illumina) was used to scan the arrays and obtain the raw intensity data files. DNA methylation data were processed using the R package minfi , to obtad others . Finallyd others , the fund others .https://eforge.altiusinstitute.org/) (The eFORGE v2.0 (te.org/)  tool waste.org/) .elim algorithm  was used following manufacturer’s instructions. Briefly, genomic DNA was sheared using a BioRuptor sonicator  to produce 400 bp fragments, which were checked by gel electrophoresis. The obtained fragments were immunocaptured with a monoclonal antibody specific for 5-methyl cytosine (supplied by the kit). Methylated DNA was then washed and purified from beads with a DNA Isolation Buffer (DIB) and proteinase K provided by the kit. Following an incubation at 55°C and at 100°C (for 15 min each), the supernatant containing the DNA was used for qPCR analysis to evaluate enrichment.Whole blood was collected in a PAXgene Blood RNA Tube  and RNA extraction performed using the Maxwell simplyRNA Blood Kit , following the manufacturer’s instructions. RNA quality was assessed by LabChip GX Touch . Libraries were prepared starting from 500 ng of total RNA, using the TruSeq Stranded mRNA Library Prep Kit (Illumina) and following the manufacturer’s instructions. Samples underwent a paired-end 75 bp sequencing using a NextSeq 500 platform (Illumina).Sequencing reads were mapped to the human genome (hg19) using STAR (version 2.5.2). Transcript quantification from mapped reads was performed using HTSeq-count (version 0.6.1p1) and the human transcripts annotations from Ensembl database (GRCh37 version). Differential expression analysis was carried out using R and the DESeq2 package, considering an FDR<0.1 as threshold.The enrichment analysis was performed using the Enrichr online tool , the MSiThe estimation of the cellular composition of the samples was performed using the immunedeconv R package, and the quantiseq method .β-Actin used as housekeeping gene .Quantitative RT-PCR was performed following the protocol previously described . RT-PCR Forward and reverse (for and rev) primers are listed below:SMAD3 for 5′- CATCGAGCCCCAGAGCAATA_-3’; SMAD3 rev: 5′- GTGGTTCATCTGGTGGTCACT_3’; BRG1 for: 5′- AGTGCTGCTGTTCTGCCAAAT - 3’; BRG1 Rev: 5′-GGCTCGTTGAAGGTTTTCAG -3’.Proceedings of the National Academy of Sciences, 2012, 201120791; DOI: 10.1073/pnas.1120791109.Primers for SNX25 (for 5′- CCGTTGTTCTCGTGCGTTAA_3′) and (rev: 5′- CCCACCTCGTTTACCACTCG-_3′) were derived from Kato L, et al. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes, t-test to determine statistically significance in RT-PCR and MeDIP followed by qPCR experiments. Significance values were set to p < 0.05. In We performed one sample It was not appropriate or possible to involve patients or the public in the design, or conduct, or reporting, or dissemination plans of our research.The genome-wide DNA methylation profile was investigated using the Illumina Inflnium MethylationEPIC BeadChips (EPIC arrays). The general demographic and clinical characteristics of the MZ twins for which methylation data were available are illustrated in p < 0.005), 2,564 DMPs were observed  highlighted the high similarity between MZ twins .When evaluating whether the Pso/PsA-associated DMPs reside in genomic regulatory regions involved in transcriptional regulation  in different cell types and tissues we observed that the Pso/PsA-associated CpGs were enriched in H3K36Me3 specific histone modifications, especially in blood cells, such as monocytes, lymphocytes, NK cells and hematopoietic stem cells  as shown by the scatterplots in We found 19 regions with at least two DMPs within 1 kb, significant within-pair Δp-value (N = 662). Very interestingly, when considering deregulated genes, we observed an enrichment in the pathways related to “oxidative phosphorylation,” “inflammatory response,” and “MYC targets” , were associated to “Psoriasis vulgaris” and “Psoriasis” in the PheWeb database (p < 2.2e-16), confirmed the enrichment in genes involved in the “IL-6/JAK/STAT3” pathway, as well as in “inflammatory response” and “TNF-α signaling pathway” (p < 2.2e-16), was instead mainly enriched in “oxidative phosphorylation” pathway  is concordant with the methylation profile, with affected MZ twins showing higher methylation levels  (A Gene Ontology (GO) analysis of DMPs associated to the 19 regions showed an enrichment in transcription factor binding, transcription corepressor and transcription coactivator activity, SMAD binding and histone -lysine-N-methyltransferase activity (< 0.005) .www.string-db.org) (p < 0.005) localized in the SMAD3 and SMARCA4 genes, in affected and healthy twins separately, represented in pink and blue, respectively  and SMAD3 (mothers against decapentaplegic homolog 3), by performing protein-protein interaction analysis  . The BRG-db.org) . Based oectively  althoughEpigenetic changes have widely been proposed to be crucial in the pathogenesis of complex diseases  and, morp < 3.6 × 10−8 . A recent study evaluated the effect of PDK4-deficiency on the development of experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis, another autoimmune disorder, induced by pathogenic Th17 cells. Very interestingly, knock-out mice developed a less severe disease and showed a decrease in Th17 cells and an increased infiltration of Foxp3+ Tregs in the central nervous system (The enrichment analysis performed on transcriptome data suggested a significant role of “oxidative phosphorylation,” “inflammatory response” and “myc targets” pathways in disease susceptibility. Indeed, these pathways have been already associated to autoimmune disorders, and also to Pso/PsA. Regarding the “oxidative phosphorylation” pathway, it was observed that there is a direct link between dysregulated glucose metabolism in lymphocytes and autoimmunity . In facts system . All togAlso MYC is one of the key players that coordinate metabolic reprogramming and activity in immune cells , which pβ-value variation in all the probes distributed, either with increased or decreased methylation level between affected and healthy twins, is of primary importance in order to define a specific methylation signature associated with psoriatic disease. Among these seven genes, SNX25, a negative regulator of TGFβ pathway and intracellular trafficking, was previously found associated among the differentially methylated sites between psoriatic and normal skin (The finding that seven genomic regions are characterized by a concordant mal skin . Interesβ-induced transcriptional regulation at multiple genes, by means of chromatin remodeling and gene expression regulation. Although the methylation level of the CpG probes located in SMAD3 and SMARCA4/BRG1 was significant, this did not follow a consistent trend. Further, despite no significant changes in RNA expression were observed, a possible contribution of the differential methylation pattern in the definition of the alternative splicing profile of the gene cannot be ruled out.A small number of the 19 genes with at least two DMPs within 1 kb of distance and significant within-pair Δβ-values was found significantly associated with psoriatic disease. Among those, SMAD3, which is involved in the downstream signaling pathway of TGF-β, is associated with BRG1 to mediate TGF-A few studies evaluated the role of DNA methylation in the disease susceptibility specifically in psoriasis  at genomWe are aware of the strengths and limitations of our study. Among the former, the chosen model of MZ twins is a unique setting to investigate complex diseases, as hereditary factors are not the major determinants of immunological functions, as proven in healthy MZ twins . FurtherIn conclusion, our genome-wide study on the unique model of MZ twins demonstrates high similarity in whole blood-based methylomes of psoriatic disease-discordant samples. However, we identified DMPs specific for psoriatic disease and few candidate loci that warrant to be further evaluated. The evaluation of the causal relation between genetic variants, biomarker levels and DNA methylation is becoming of undisputed importance. Their interplay is relevant to define plausible biological pathways in psoriatic disease that are regulated by epigenetic mechanisms.


KIT mutation has the highest rate among all mutations in mucosal melanoma types. The KIT inhibitors (Tyrosine kinase inhibitors: TKIs) imatinib and nilotinib could be the treatment options. Moreover, immune checkpoint inhibitors combined with KIT inhibitors may potentially treat cases of resistance to immune checkpoint inhibitors. However, because of the different conditions and a small number of cases, it is difficult to evaluate the efficacy of immunotherapy and targeted therapy for gynecologic melanoma rigorously at this time. Further prospective cohort or randomized trials of gynecologic melanoma alone are needed to assess the treatment with solid evidence.Gynecologic melanomas are rare and have a poor prognosis. Although immunotherapy (immune checkpoint inhibitors) and targeted therapy has greatly improved the systemic treatment of cutaneous melanoma (CM) in recent years, its efficacy in gynecologic melanomas remains uncertain because of the rarity of this malignancy and its scarce literature. This review aimed to evaluate the literature of gynecologic melanomas treated with immunotherapy and targeted therapy through a PubMed search. We identified one study focusing on the overall survival of gynecologic melanomas separately and five case series and nine case reports concentrating on gynecologic melanomas treated with an immune checkpoint inhibitor and/or targeted therapy. Furthermore, the  Gynecologic melanomas are uncommon. They stand for 18% of mucosal melanoma , which accounts for 1% of all melanomas. The most common site is the vulva (70%), vagina, and the cervix of the uterus, in that order. Gynecologic melanomas also have a poor prognosis, with a 5-year overall survival (OS) of 37–50%, 13–32%, and approximately 10% for vulvar, vaginal, and cervical melanomas, respectively [+ T cell infiltration, and T cell exhaustion markers . When the MAPK pathway is reactivated, melanoma antigens are suppressed, and an immunosuppressive tumor microenvironment reemerges. If this pathway is inhibited subsequently by an MEK inhibitor, melanoma antigens are restored, and CD8+ T cell infiltration is promoted [Meanwhile, the systemic therapy for cutaneous melanoma (CM) has improved recently by immunotherapy (immune checkpoint inhibitors) and targeted therapy; this therapeutic approach inhibits the mitogen-activated protein kinase (MAPK) pathway including BRAF, NRAS, MEK, and KIT. In almost fifty percent of patients with metastatic melanomas, valine is replaced with glutamine in codon 600 of the serine/threonine kinase BRAF (BRAF V600 mutation) ,3,4. Howpromoted ,6,7.BRAF V600-activating mutation, BRAF inhibitor combined with MEK inhibitor is preferred . If no mutation is observed, anti-PD-1 monotherapy or combination therapy including pembrolizumab/nivolumab or nivolumab plus ipilimumab   is preferred. Other recommended options (category 2B) are pembrolizumab/low-dose ipilimumab, a combination of targeted therapy and immunotherapy . The maximum efficacy of these drugs in clinical trials with patients with CM is beyond the scope of this article.According to the National Comprehensive Cancer Network Guidelines version 2 (2021) , the folBRAF gene mutation in MM affects gene regions other than codon 600 or is a non-activating mutation, thereby not predicted to respond to targeted BRAF inhibitor [In terms of biological characteristics, MM and CM seem to be different. MMs have a considerably lower rate (3–15%) of BRAF gene mutations than CM without chronic sun damage ,10,11. Inhibitor .KIT mutated melanoma, including MMs.In this regard, KIT inhibitors (Tyrosine kinase inhibitors: TKIs) relevant to the treatment of MM include those listed in With regard to immunotherapy, randomized clinical trials of ipilimumab, pembrolizumab, and nivolumab for MM are currently unavailable, but these medicines are effective according to some studies. However, the efficacy of immunotherapy and targeted therapy in gynecologic melanomas is uncertain because this malignancy is rare, thereby obtaining scarce literature. Hence, this review aimed to evaluate the literature of gynecologic melanomas treated with immunotherapy and targeted therapy, and consider the possibility of such treatment in the future.We searched the literature in PubMed using the following search strings:“melanoma” AND “genital” AND “immunotherapy”; “MM” AND “genital” AND “ipilimumab”; “melanoma” AND “genital” AND “nivolumab”; “melanoma” AND “genital” AND “pembrolizumab”; “melanoma” AND “vulvovaginal” AND “ipilimumab”; “melanoma” AND “vulvovaginal” AND “nivolumab”; “melanoma” AND “vulvovaginal” AND “pembrolizumab”; “melanoma” AND “vulvovaginal” AND “immunotherapy”; “melanoma” AND “gynecologic” AND “immunotherapy”; “melanoma” AND “gynecological” AND “immunotherapy”; “melanoma” AND “vagina” AND “immunotherapy”; “melanoma” AND “uterine cervix” AND “immunotherapy”; “melanoma” AND “vulva” AND “immunotherapy”; “MM” AND “immune checkpoint inhibitor”; “melanoma” AND “genital” AND “targeted therapy”; “melanoma” AND “vulvovaginal” AND “targeted therapy”; “melanoma” AND “vulva” AND “targeted therapy”; “melanoma” AND “vagina” AND “targeted therapy”; “melanoma” AND “uterine cervix” AND “targeted therapy”; “melanoma” AND “gynecologic” AND “targeted therapy”; “melanoma” AND “gynecological” AND “targeted therapy”; “Mucosal melanoma” AND “tyrosine kinase inhibitor.”We included articles that focused on human studies exclusively, used the English language, and were published between 10 years ago and March 2021. We excluded articles concerning CMs, MMs of non-gynecologic organ origins, and gynecologic tumors other than melanoma. In the case of vulvovaginal melanomas, one question about the distinction between mucosal and cutaneous melanoma might have occurred. The location of the tumor from the vulva or vagina, specifically within X cm, was not mentioned in any literature. However, as it is assumed that gynecologic melanoma is included in mucosal melanoma in general, we treated all of them as mucosal melanoma.In total, we found 252 articles in PubMed. Among them, six studies and nine case reports were included.We identified five case series and nine case reports that focused exclusively on gynecologic melanomas treated with immune checkpoint inhibitors and/or targeted therapy . In addiNRAS and KIT mutations, respectively. However, KIT was most frequently mutated in four (13.8%) patients with vulvovaginal melanoma.A population-based study in the Netherlands evaluated the benefits of immune checkpoint inhibitors to MM in comparison with those to CM in patients with advanced melanoma between 2013 and 2017 . CM and We identified five retrospective case series. AJCC staging classification (eighth edition) was applied for vulvar melanomas, and vaginal or cervical melanomas were classified as local, regional, or distant . In one In a single-center study in Toronto over 15 years, 13 patients with advanced MMs were treated with immune checkpoint inhibitors . Their bp = 0.01, log-rank test and p = 0.15, log-rank test: The p-value of OS was not statistically significant). The response rate to immunotherapy was 28.5%.In another single-center study conducted in Milan between January 2011 and December 2016, seven patients who received immunotherapy for metastatic gynecologic melanomas were included . The PFSNRAS mutation (p.Q61R), and another melanoma carried a KIT mutation (exon 11). According to the OS achieved, 4 patients (66%) had a confirmed progressive disease (iCPD), 1 had an iSD (maintained for 11 months), and 1 had a good response in which the tumor burden was reduced by 89% and a long survival was achieved (31 months). The 1-year survival rate was 33%. Meanwhile, eight patients received nivolumab. One melanoma carried a BRAF V600E mutation (p.Q61R), and two melanomas carried a NRAS mutation (p.G12D). The best OS response was the iPR in four patients; the four other patients had an iCPD. The 1-year survival rate was 86%. One patient was treated with ipilimumab plus nivolumab as part of the clinical protocol CA209511. After 5 months from treatment initiation, an iPR (30% decrease in the target region) was obtained.In a skin cancer department in France between 2013 and 2018, a study including 15 patients with unresectable or metastatic vulvovaginal melanoma was conducted . Six patKIT (4/7) mutations, including 2 exons of 13 K642, 1 exon of 17 N822K, and 1 exon of 11 L576P mutations. However, no BRAF (0/5) or NRAS (0/2) mutations were detected. Ipilimumab, pembrolizumab, interferon pegintron , imatinib, and nilotinib were administered to 3, 1, 1, 3, and 1 patients, respectively. Imatinib and ipilimumab showed the best response, resulting in iPR in 2/3 (66%) of cases with imatinib and 1/3 (33%) with ipilimumab. However, only one patient who received pembrolizumab had an iSD after 3 months. Adjuvant treatment with pegintron at 100 µg/week subcutaneously for more than 5 years lead to a successful outcome in one patient of nodal relapse following lymph node dissection and remained disease-free.In another single-center study conducted between January 2006 and September 2013, 64 non-CM cases were collected, including eight vulvovaginal melanomas . Tumor mBRAF, NRAS, or KIT mutation did not occur in 3/4 patients, while genetic testing was not performed in the remaining one patient did. All four patients received at least three ipilimumab doses with concurrent external beam radiation. The OS was 16 months in one patient with recurrent vaginal melanoma, 20–38 months in patients with non-recurrent vaginal melanoma, and 19 months in one patient with recurrent cervical melanoma. Moreover, a study retrospectively evaluated four patients treated with combined immunotherapy and radiation for gynecologic melanomas between 2012 and 2015 . BRAF, NThis study evaluated one retrospective population-based study, five retrospective case series, and nine case reports with gynecologic melanomas treated with immunotherapy and/or targeted therapy. Because of the different conditions and a small number of cases, it would be inappropriate to statistically analyze them together. And it is difficult to evaluate the efficacy of immunotherapy and targeted therapy for gynecologic melanoma rigorously at this time. So, the aim of this article is to assess the findings currently available and consider the possibility of treatment in the future.KIT was the more frequently mutated gene in MM of the vulvovaginal region rather than in CM.In one study , patientKIT mutation was also frequently detected in gynecologic melanomas.Of the 57 patients included in the case series and case reports listed in Zeijl et al. reported that MM, including gynecologic melanoma, had a worse prognosis than CM , correlaKIT mutation rate than CM (39% vs. <3%), but the increased rate is mainly due to the increased frequency of vulvovaginal and anorectal melanomas, not head and neck melanoma [KIT mutation was most common in vulvovaginal melanomas (22%) in contrast to 3% in CM (p < 0.001). Meanwhile, NRAS mutations were rare in vulvovaginal melanomas in contrast to CM  [KIT mutations were found in 18% of vulvar melanomas, but none in vaginal tumors. In immunohistochemistry, moderate or strong KIT protein expression was present in 30 cases, including all tumors with KIT mutations and 6 of the 7 tumors with KIT amplification [KIT mutations, particularly in exon 11 [KIT mutation may also serve as a prognostic factor. Moreover, 95 patients with vulvar melanoma from 10 clinical institutions underwent molecular analysis by either targeted next-generation or direct sequencing. Detected mutations were in KIT (44%), BRAF (25%), NF1 (22%), TP53 (17%), NRAS (9%), and telomerase reverse transcriptase promotor (9%). In a univariate analysis, KIT mutations were notably related to a better PFS  [KIT mutations, vulvovaginal melanomas with wild-type KIT mutations tended to express molecular markers suggestive of platinum resistance (ERCC1), alkylating sensitivity (MGMT), and anthracycline sensitivity (TOP2A) [As shown in the literature being evaluated, MM has a higher melanoma . In one = 0.009) . In anotfication . In anot exon 11 . The KIT 0.0013) . Compare (TOP2A) .KIT mutations in gynecologic melanomas should be administered with KIT inhibitors such as imatinib, nilotinib, and dasatinib. The details of these drugs are shown in KIT mutations. In one report, six responses to KIT inhibition were detected in tumors with L576P or K642E mutation. In the same report, some patients had V654A and D820Y mutations, which are known to cause resistance to imatinib in GIST [KIT mutations reported in this previous study.These results suggest that patients with low TMB and frequent  in GIST ; hence, KIT mutation, that is, Del579 in exon 11, which is less frequent (2%). The efficacy may be attributed to the single effect of imatinib or sequential effect after PD-1 blockade. Compared with imatinib alone, the combination treatment of PD-1/PD-L1 blockade and imatinib leads to increased intertumoral CD8+ T cell proliferation and inflammatory cytokine production; hence, immune checkpoint therapy could increase the antitumor effect of imatinib by enhancing the function of T cell effectors [KIT mutation was p.N822K in exon 17.With regard to gynecologic melanomas and KIT inhibitors, we found six patients treated with TKI in one case series and two case reports, as presented in ffectors . In anotffectors . AvapritA phase I study demonstrated another possibility of the combination of an anti-PD-1 antibody  and an anti-vascular endothelial growth factor antibody (axinitib) . A totalAs mentioned a little before, we found no studies that solely compare MM and CM in terms of the evaluation index, such as PFS, OS, and 1-year survival. Currently, assessing the optimal treatment or its efficacy to gynecologic melanomas with solid evidence is difficult. The possible treatments are the extrapolations of MM and CM treatments. If conducting a prospective cohort or randomized trials is difficult because of the small number of cases, a system that can register individual cases  should be established. Gynecologic melanomas are likely to be included as part of MM, and the treatment is evaluated together with the other MM types. In particular, genitourinary MM is most common in non-Hispanic white people ; thus, aKIT is the most frequently mutated gene. A KIT inhibitor such as imatinib or nilotinib could be the treatment of choice. Moreover, a combination of an immune checkpoint inhibitor and a KIT inhibitor may potentially treat cases of resistance to immune checkpoint inhibitors. Further prospective cohort or randomized trials of gynecologic melanoma alone are needed to assess the treatment with solid evidence.Gynecologic melanomas are rare and have a poor prognosis. Because of the different conditions and the small number of cases, it is difficult to evaluate the efficacy of immunotherapy and targeted therapy for gynecologic melanoma rigorously at this time. And its possible treatments are the extrapolations of MM and CM treatments. Regarding genetic mutation,


Background and Objectives: Sudden cardiac death (SCD), particular among elite footballers, has attracted much attention in recent times. However, limited information exists on football-related SCD in Turkey. Autopsy-based studies of sports-related sudden deaths in Turkey are rare and often have small sample sizes. To address this, this study aimed to determine the population-based incidence and profile of football-related SCD nationally in Turkey. Materials and Methods: Due to a lack of national data on this issue, football-related SCD  between 1 January 2011, and 31 December 2019 were identified by dual, independent identification and screening of online media reports. Deaths were explored by sex, age group, season, and phase of exercise. Descriptive statistics were utilised. Age-specific mortality rates and proportional mortality rates were calculated. Results: In total, 118 football-related SCD were identified, a crude mortality rate of 0.41 per 100,000 population. All fatalities were males and the mean age was 35.5 years ± 10.4. Those aged 40–49 years recorded the highest mortality rate , three times the risk of those aged 50–59 years . Those aged 30–39 recorded the highest age-specific proportional mortality rate (0.86/1000 deaths). The highest risk occurred while playing football , with another 15% of deaths (n = 18) occurring within 1 h of play. Almost all fatalities  occurred during participation in recreational football. Conclusions: This study has identified football-related SCD most commonly occurs during recreational football among males aged 30–49 years. It is recommended males of this age participating in recreational football be encouraged to seek pre-participation heart health checks. Given the value of automated external defibrillators (AEDs) in responding to out-of-hospital cardiac arrest, future research should explore the feasibility and effectiveness of AEDs in preventing football-related SCD in Turkey including training of first responders in cardiopulmonary resuscitation and AED use. Football  is the most popular sport in the world with an estimated 260 million participants . As in aSudden cardiac death, although uncommon, can occur during sports and physical activity and includes unexpected deaths in people with symptoms onset within one hour of their death ,6. Some More than half of all sudden deaths in athletes are due to cardiovascular causes, the most common of which are hypertrophic cardiomyopathy and congenital coronary artery anomalies . A studyFootball is a sport commonly implicated in sudden deaths. Almost half (44.9%) of all sudden cardiac deaths in a study from Italy occurred during football , while 3Despite football being a popular sport in Turkey, there is scarce participation data and very little information on the incidence of sudden death among footballers . AutopsyAs autopsies are not performed for every death in Turkey , relyingThis study investigating football-related sudden cardiac deaths in Turkey using a retrospective and web-based design. Media reports were used to identify and determine the incidence of football-related sudden cardiac deaths in this study. This study was approved by The University of New South Wales Human Research Ethics Committee on the 29 September 2021 (HC210766) and conducted in accordance with Helsinki Declaration.The study involved deaths which occurred in Turkey during the study period and were reported in the media as being related to recreational or non-elite competitive football and were triggered by cardiac causes No lower or upper age limit was set for the study sample population. www.news.google.com). The following search terms in Turkish language were used: “sudden death” (“ani ölüm”) OR “sudden cardiac death” (“ani kardiyak ölüm”) OR “cardiac death” (“kardiyak ölüm”) OR “heart attack”  OR “cardiac arrest”  AND “football” (“futbol”) OR “football pitch” (“futbol sahası”) OR “astroturf pitch”.  In addition, we strengthened our search results by using the following phrases in Turkish language in a general Google search (www.google.com) to identify media reports not found via Google News searches: “sudden death in football”, (“futbolda ani ölüm”) “sudden death in football field”, (“futbol sahasında ani ölüm”) “cardiac death in football”(“futbolda kadiyak ölüm”), “cardiac death in football field” (“futbol sahasında kardiyak ölüm”), “heart attack in football” , “heart attack in football match”,  “cardiac arrest in football” , “cardiac arrest in football match” , and “death in football match” (“futbol maçında ölüm”).To identify cases of football-related sudden cardiac death which occurred between 1 January 2011 and 31 December 2019 retrospective screening was performed of online media reports via the search engine Google News (Searches were run and the data were collected and recorded independently by two researchers (A.I. and A.T.). The researchers then compared data to ensure completeness of data and accuracy of coding. Duplicate cases were removed. Cases with missing data, or an unidentified cause of death, were not included in the study. Because media reports were updated periodically, all data were last reviewed on 22 June 2020.As previously reported by Bohm et al. , we defiTM  and then into IBM SPSS V23 . For age group, the mean and standard deviation was calculated. Data on the resident population of Turkey  by age group were sourced from the Turkish Statistical Institute [All data were transferred from online Google News searches into Microsoft Excel 365 nstitute  and usedAge-cause-specific mortality rate: Data on all-cause mortality  by age group were also obtained from the Turkish Statistical Institute  and usedThe relative risk (RR) and 95% confidence intervals (95% CI) were calculated using population data for the age groups. The age group with the lowest number of cases (50–59 years) was considered as the reference point for calculating the RR and 95% CI. From 2011 to 2019, 118 football-related sudden cardiac deaths in Turkey were identified from media reports. The average number of football-related sudden cardiac death per year during this 9-year period was 13 (range 8–25) . This eqn = 41) of football-related sudden cardiac deaths occurred in individuals aged 30–39 years, followed by 40–49 year-olds . The highest age-specific mortality rate occurred among 40–49year-olds  and the lowest rate was in the age group 10–19 years . When considering all-cause mortality, the football-related sudden cardiac death rate was 0.30 per 1000 deaths . This rate was found to be lowest in the 50–59 age group (0.05 per 1000 deaths). The relative risk and confidence interval for football-related sudden cardiac death varied depending on the age of the individuals participating in football. The age group 40–49 years had the highest risk  compared to the reference age group 50–59 years , followed by after exercise . Just 2.5% (n = 3) occurred before exercise. Among 10–19 year-olds, no cases were found before or after exercise. Similarly, in the age group 40–49 years, no cases were noted before exercise. While 113 cases  occurred during recreational football, five cases (4.2%) were associated with non-elite competitive football , whereas Summer saw the largest proportion of deaths among 50–59 year-olds   .Although participation in football is a popular past time with many health and social benefits ,25, it iIn the present study, football-related age-specific mortality was found to be 0.41 per 100,000 population and proportional mortality was 0.30 per 1000 deaths. This compares favorably with previously published studies identifying mortality rates of 0.9 in physically active men aged 15–34 years in Norway , but is In Turkey, health examinations are mandatory for professional players, but there are no regulations for non-professional football players and recreational football players. Furthermore, professional football players undergo more in-depth health examinations compared to non-professional football players. A medical examination is required for amateur football players only when a football license is first issued . The degIn an autopsy-based study of football-related sudden cardiac deaths in Turkey between 2000 and 2005, 15 cases aged 10–48 years were reported. Thirteen of these cases occurred during recreational football . SimilarOur research found that nearly two-thirds of football-related deaths in Turkey occurred in males aged 30 to 49 years. This is a similar finding to the two previously published autopsy-based studies in Turkey which found people aged 30 years and older accounted for 47% and 80% of deaths respectively ,18. ThesOur finding that football-related sudden cardiac death in Turkey occurs most commonly during exercise 82%) is similar to that of other previously published studies ,36. As t2% is simRecreational sports activities have been reported as the activity with the highest incidence of sports-related sudden cardiac death . In our n = 6 and n = 15) [To the best of our knowledge, the present study is the most comprehensive epidemiological study on football-related sudden cardiac death in Turkey. Although autopsy-based studies have previously conducted in Turkey, they are limited to small sample sizes ( n = 15) ,18 as no n = 15) . For thi n = 15) ,19,20,21There are limitations associated with this research. Cases included in this study are sudden deaths deemed to be of cardiac origin from media reports only and may not be as accurate as case reports based on findings from an autopsy , or may Mortality rates are calculated using population data and do not consider exposure to the activity. Participation data for football in Turkey by age group would allow for more accurate rates to be calculated, however this data is currently unavailable. The use of media reports as the data source limits the risk factor data that can be identified, such as the use of illicit substances and tobacco use. It should also be noted that risk factors for SCD may differ significantly between different populations, potentially impacting age-specific mortality rates presented in this study. Although the inclusion of deaths <1 h within playing football is a reasonable approach, it must be noted that media reports may be less likely to report football as an association if the death occurs before or after a sporting event. Additionally, we were not able to analyze clinical and ECG data of the decedents. Football-related sudden cardiac death results in 13 deaths on average each year in Turkey, most commonly impacting males aged 30 to 49 years participating in recreational football. Although the age-specific mortality rate for football-related sudden cardiac death is lower than all-cause mortality for males in Turkey, prevention of such deaths is still an important public health issue. Promotion among both the general community and general practitioners of the importance of cardiac health checks prior to participation in recreational football should be conducted. Similarly, future research should explore the feasibility and acceptability in Turkey of AEDs at football fields, combined with training of community members in their use. This study has also identified the need for detailed data on out-of-hospital cardiac arrest in Turkey.


Here, we show that with rare exceptions, oceanic diamonds and the isotopically lighter cores of super-deep continental diamonds share a common organic δ13C composition reflecting carbon brought down to the transition zone by subduction, whereas the rims of such super-deep continental diamonds have the same δ13C as peridotitic diamonds from the lithospheric mantle. Like lithospheric continental diamonds, almost all the known occurrences of oceanic diamonds are linked to plume-induced large igneous provinces or ocean islands, suggesting a common connection to mantle plumes. We argue that mantle plumes bring the transition zone diamonds to shallower levels, where only those emplaced at the base of the continental lithosphere might grow rims with lithospheric mantle carbon isotope signatures.Rare oceanic diamonds are believed to have a mantle transition zone origin like super-deep continental diamonds. However, oceanic diamonds have a homogeneous and organic-like light carbon isotope signature (δ The positions of these deposits relative to plume magmatism and the large low sheared wave velocity province (LLSVP)2 suggest that they are related to mantle plume events5. A small fraction (1%) of the continental diamonds have mineral inclusions that suggest a deeper origin of between ∼ 300 and 1000 km depths6, and are known as super-deep or sub-lithospheric continental diamonds7. Super-deep continental diamonds have extremely variable carbon isotopic compositions (δ13C from − 25 to + 3.5‰)11, in contrast to a dominant mode of carbon isotope at around − 5‰ exhibited by lithospheric peridotitic and eclogitic diamonds12.The vast majority of diamonds have grown in the old continental lithospheric mantle between 150 and 300 km depths17 , presence of moissanite (SiC), and a large continuous range of δ15N isotopic composition (− 5.6‰ to + 28.7‰), not found in synthetic diamonds22. In situ occurrence of such natural diamonds have also been demonstrated by thin-section in situ petrographic observations24. Some studies proposed that diamonds found in ophiolites could have resulted from serpentinisation processes25. However, the presence of ultra-high-pressure and highly reduced mineral phases in such diamonds suggests their formation at > 300 km depths26, which cannot be explained by serpentinization27. Moreover, although diamond nano-particles can form under thermodynamic instability and low pressure–temperature conditions29, this mechanism is incompatible with the nano- to micro-meter sizes of the oceanic diamonds30, and cannot reproduce fluid inclusions with complex compositions  similar to kimberlitic and ultra-high pressure (UHP) metamorphic diamonds32.An even smaller fraction << 1%) of diamonds are found in the oceanic lithosphere sampled by mantle xenoliths or preserved in ophiolitic belts worldwide% of diam35, except for the exceptionally large CLIPPIR diamonds36) and are associated with a range of ultra-high-pressure and highly reduced mineral phases 16. It is thus believed that oceanic diamonds and super-deep continental diamonds formed in the volatile-rich regions of the mantle, most likely in the transition zone (410–660 km)16. It is widely accepted that such diamonds  formed from an oxidized, CO3-rich melt41 or reduced fluids produced when subducted slabs melted after entering the transition zone, where the pressure is 15–16 GPa, temperature ~ 1600 °C, and volatile contents 1–1.5 wt%43.On the other hand, these oceanic diamonds share similar characteristics to super-deep continental diamonds in that they, in general, are microdiamonds (< 1 mm in size13C from − 28 to − 20‰)44 11  mantle upwelling15. In the first model, gravity causes the subducting slab to roll back, which triggers return flows that brings the diamonds and ultra-high-pressure minerals to the shallower levels within the subduction channel. In the second model, mantle upwellings, possibly induced by the pondering of plume heads on the transition zone, brings the diamonds and ultra-high-pressure minerals to the lithospheric mantle, fragments of which are later accreted onto the continental margin during subduction.Two main mechanisms have been proposed to explain the transportation of oceanic diamonds from the transition zone to the upper mantle: (1) subduction return flow33, which includes geological and geochemical evidence for plume-related materials in ophiolites54. We show that most oceanic diamonds found in ophiolite belts are not associated with classic ophiolitic sequences that represent the normal oceanic lithosphere formed at mid-ocean ridges; instead, they are associated with plume-modified O-LIP or OIB lithospheric fragments preserved in ophiolite belts.In this contribution, we aim to achieve a coherent mechanism for the origin of both the oceanic diamonds and the super-deep continental diamonds, including processes that brought them to the surface. We test the various models by investigating the oceanic diamond record together with a compilation of oceanic large igneous provinces (O-LIPs) and ocean island basalts (OIBs) database, or O-LIPdb14 or ancient ophiolites16, and the oceanic mantle plume record33  ophiolite in Turkey is part of the eastern Tauride belt, and is characterised as an accreted OIB or oceanic plateau64 similar to the Mirdita ophiolite nappe65. Although the original authors interpreted the Ray-Iz and Myitkynia ophiolites to be of suprasubduction origin without plume involvement, we found that the mafic and ultramafic rocks from these two ophiolites share similar geochemical features to the plume-modified oceanic lithosphere  12 from one locality to another 8 continental super-deep diamonds exhibit carbon isotopic zonation, featuring very light C isotope fractions in their cores (δ13C from − 28 to − 20‰) and heavier, mantle-like C isotope compositions in the rims (δ13C − 15‰ to − 5‰)10  distinct carbon sources for the cores and rims (organic and inorganic)51, and (3) isotopic fractionation of carbon in the mantle78.Previously proposed models to explain the variability of carbon isotopic composition in super-deep continental diamonds include (1) primordial isotopic variability inherited from Earth's accretion13C − 24 ± 6‰), and the overall homogeneous negative values for the superdeep diamonds from Jagersfontein (δ13C up − 20 ± 4‰)11 and Monastery (δ13C up − 17 ± 1‰)11, are comparable to those of the relatively homogeneous carbon isotopic composition of oceanic diamonds (δ13C − 25 ± 4‰)  and nitrogen is not supported either due to the lack of correlation between δ13C and nitrogen in various growth zones8. This leads us to hypothesize that the very light cores of Juina-5 and Sao Luis super-deep diamonds, and superdeep diamonds from Jagersfontein and Monastery, share a common origin with the oceanic diamonds, as reflected by their carbon isotope signatures  very light isotopic composition of such diamonds  the highly negative carbon isotope values for the cores of the super-deep continental diamonds from Juina-5 and Sao Luis δ13C − 4 ± 6‰, a83, all above the large low-shear-velocity provinces (LLSVPs) in the lower mantle85. We envisage that mantle upwellings, caused by plumes87, entrain microdiamonds formed in the transition zone and transport them  to shallower levels93. In contrast, mantle convection around normal mid-ocean ridges, away from mantle plumes, do not contain such diamonds and ultra high-pressure minerals  oceanic lithospheric mantle59. Buoyancy caused by such depletion, along with plume-induced thermal buoyancy and the abnormal thickness of such plume-modified oceanic lithosphere94, makes it more resistant to subduction95, leading to components of it being accreted onto arcs and preserved in orogenic belts96. Subduction fluids modify the original chemical signature of the lithospheric mantle by melt-rock interactions at shallow depth during both accretion/obduction and exhumation in the spinel stability field (< 80 km), leading to the formation of large podiform chromite bodies, typical of subduction zones16. During the accumulation processes of podiform chromite, oceanic diamonds are incorporated in newly formed high-Cr chromite97. Such a process is not only consistent with the deep and highly reduced origin of oceanic diamonds and associated ultra-high-pressure minerals and chromite, but also the subduction origin of the high-Cr podiform chromite.Some mantle plumes are rooted from the lower mantle, whereas others could have a root near the transition zone, possibly as secondary plumes2, CH4), the pressure–temperature condition of the ambient environment (P > 130–150 km for T > 1000 °C), the redox condition (ΔlogfO2 (oxygen fugacity) <−2) which controls the speciation of carbon and its precipitation98, and the time available for the growth to occur. The P–T–fO2 of the lithospheric mantle beneath the oldest continents (aka cratons) is known to favor the growth of diamond99. Such lithosphere is typically thicker (up to 300 km), colder (< 900–1000 °C) and reduced (down to ΔlogfO2 <−4)100, contains sufficient amounts of carbon101, and is able to survive for a sufficiently long time. In such an environment, “purely” continental diamonds  , yet their cores, of super-deep origin, retain their original lighter carbon isotopic signature which is the same as that of the oceanic diamonds (δ13C − 25 to − 20‰) . The lithospheric mantle of oceanic plateaus and ocean islands is believed to be thinner (< 140 km)102, hotter (1000–1200 °C)59, more oxidised (ΔlogfO2 from − 3 to − 1 according to limited data on mid-ocean ridge peridotites)103, with less carbon available79, and recycled quickly through Wilson cycles. Oceanic microdiamonds, once incorporated in the thickened oceanic lithosphere by plumes, are thus suppressed from growth, or even totally frozen in size, shape  and low aggregation state (Type Ib), due to such unfavorable conditions 13  ophiolite (ca. 95 m.y) in the eastern Tauride belt, Turkey; the Tibetan ophiolites ; the Mirdita ophiolite (c.a. 160 m.y.) in Albania; the Myitkynia ophiolite (ca. 170 m.y.) in Myanmar; the Hegenshan ophiolite (ca. 295 m.y.) in inner Mongolia, China; the Sartohay ophiolite (ca. 395 m.y.), part of the West Jungar belt, Xianjiang, China; and the Ray-Iz ophiolite (ca. 420 m.y.), in polar Ural, Russia.We compiled the known occurrences of both present-day oceanic diamonds and those found in ophiolite belts Table 13,14,16.108, with the exceptions of the Mirdita and Pozanti-Karsanti ophiolites which are often described as Penrose-type ophiolites109. We compiled the geological and geochemical information for the mafic and ultramafic rocks (when available) of these ophiolites at] > 0.91), low Al2O3 (1.5–0.2 wt%) and very minor SiO2 enrichments  their inclusions115 that defines their paragenesis3 and ultimately their lithospheric or sub-lithospheric provenances117.In this study we classify diamonds based on (1) nitrogen and boron contents as well as their configuration in the diamond lattice to define the “type” classification systemLithospheric diamonds are diamonds formed in the continental lithospheric mantle and have mineral inclusions of eclogite and peridotite typical of continental lithosphere mantle, including forsterite, pyrope, omphacite, diopside, enstatite, and sulfides. Lithospheric diamonds are commonly subdivided into “eclogitic” and “peridotitic”, depending on the association of inclusions. For example, diamonds with almandine and omphacite inclusions are classified as eclogitic while diamonds with forsterite and pyrope are classified as peridotitic.3-walstromite, jeffbenite, majoritic garnet and retrogressed bridgmanite. Superdeep diamond inclusions also indicate the depth of the diamonds, for example, diamonds with majoritic inclusions are believed to have come from the transition zone while diamonds with retro-morphosed bridgmanite are interpreted to have come from the lower mantle (a much rarer occurrence).Superdeep (or sub-lithospheric) diamonds have mineral inclusions typical of the upper mantle, including ferro-periclase, CaSiOSupplementary Table S1.Supplementary Informations.


Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) is a critical signaling molecule activated downstream from a variety of cell surface receptors that contain an intracellular immunoreceptor tyrosine-based activation motif. These receptors recruit kinases such as Syk, BTK, and BLNK to phosphorylate and activate PLCγ2, which then generates 1D-myo-inositol 1,4,5-trisphosphate and diacylglycerol. These well-known second messengers are required for diverse membrane functionality including cellular proliferation, endocytosis, and calcium flux. As a result, PLCγ2 dysfunction is associated with a variety of diseases including cancer, neurodegeneration, and immune disorders. The diverse pathologies associated with PLCγ2 are exemplified by distinct genetic variants. Inherited mutations at this locus cause PLCγ2-associated antibody deficiency and immune dysregulation, in some cases with autoinflammation. Acquired mutations at this locus, which often arise as a result of BTK inhibition to treat chronic lymphocytic leukemia, result in constitutive downstream signaling and lymphocyte proliferation. Finally, a third group of PLCγ2 variants actually has a protective effect in a variety of neurodegenerative disorders, presumably by increased uptake and degradation of deleterious neurological aggregates. Therefore, manipulating PLCγ2 activity either up or down could have therapeutic benefit; however, we require a better understanding of the signaling pathways propagated by these variants before such clinical utility can be realized. Here, we review the signaling roles of PLCγ2 in hematopoietic cells to help understand the effect of mutations driving immune disorders and cancer and extrapolate from this to roles which may relate to protection against neurodegeneration. IP3 and DAG then facilitate secondary signaling for membrane-bound immunological and growth factor receptors (Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) belongs to a group of intracellular enzymes that cleave the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIPte IP3) A. IP3 an A. IP3 aB). Interaction between the cSH2 domain and residues surrounding the catalytic active site results in autoinhibition of PLCγ2. Upon Y759 phosphorylation, the cSH2 domain is removed from the catalytic active site and interacts with phosphorylated residue, allowing the substrate PIP2 access to the active site (B) is essential and sufficient for activation by Rac2, with the crucial amino acids at the PLCγ2–Rac2 interface determined by NMR spectroscopy  domain, and SH3 domain B. Interaive site . Biochemive site B is essetroscopy . Rac2-mePLCγ2 is also a known risk factor and an important driver in a multitude of diverse disease circumstances including those with an immunological basis such as inflammation, autoimmunity, immunodeficiency, and allergy, as well as in hematological malignancies . Moreove3, DAG, and calcium flux, PLCγ2 activation results in activation of phospholipase D and cell spreading  is stimulated by its cognate antigen. Early studies of PLCγ2 function, typically conducted in the chicken B cell line DT40, revealed that BCR signaling through PLCγ2 required its association with other signal transduction molecules including Syk and Btk in a complex located in a lipid raft, or signalosome. PLCγ2 would then be phosphorylated at four tyrosines . Mopreading , 16, 17,preading . Studiespreading . Upon BCmembrane .in vitro for PLCγ2 phosphorylation and resultant calcium flux after BCR stimulation  with a neomycin cassette . Unlike ult mice . Indeed,antigens , 27, 28.−/−PLCγ2−/− mice where floxed enzymatic PIP2 domains of PLCγ2 were inducibly deleted  associating with the BCR and via binding its known ligand, herpes virus entry mediator and activating SHP-1 -mediated proliferation were normal in the absence of PLCγ2 , 26, witIn summary, PLCγ2 plays a fundamental role in B cell development affecting the survival of mature B cells and antibody production, as well as being an absolutely necessary and immediately proximal component of the BCR signaling cascade. While PLCγ1 plays a greater role in T cells, PLCγ2 is still important in terms of TCR signaling and the calpain-calpastatin system and when combined with PLCγ1 deletion. Considering these vital roles for PLCγ2 in the adaptive immune system, it is of little surprise its dysregulation or malfunction is also then of crucial importance in a number of disease settings of an immunological and hematological nature.PLCG2 gene  and common variable immunodeficiency (CVID). PLAID is specifically driven by a gain-of-function amino acid mutation in the PLCγ2 gene , 64, wheCG2 gene . PatientCG2 gene , 66. TheCG2 gene . Chemotain vitro . Patientin vitro .In vitro studies revealed that after BCR signaling, the cSH2 domain of PLCγ2, coupled with poor phosphorylation of Syk, Btk, and BLNK, stabilized the early signaling complex , and mast cells often because of its utility in facilitating downstream signaling after FcR engagement. Moreover, PLCγ2 is reported to be involved in essential signaling processes associated with integrin activation and signaling downstream of critical myeloid receptors including cytokine receptors and TLRs. This signaling drives subsequent antibacterial functions including degranulation and production of reactive oxygen species (ROS) and inflammatory responses.3 production, degranulation, and inflammatory cytokine release  receptor, essential for macrophage development, also associates with PLCγ2, leading to its activation, resulting in PI3K activation, thus showing a potential role in myeloid cell differentiation , 77. Bloin vivo ) in the macrophage cell lines  is a fundamental cytokine governing bone formation and destruction nd NF-κB . In contnd NF-κB . They alnd NF-κB . Moreovend NF-κB , 100. Thnd NF-κB . Anothernd NF-κB . Ephrin nd NF-κB . These nvia engagement of their antigen-specific IgE receptors that in turn release their proinflammatory payload. PLCγ2 is implicated in allergic responses, specifically with regard to the function of mast cells and their crosslinking of FcεRI (IgE-binding receptor) and FcγR and subsequent degranulation  in PLCγ2 resulting in spontaneous inflammation  and innate system  as well as various forms of inflammation , as evident by an upregulation of the PLCγ2 gene signature of patient peripheral blood mononuclear cells . Indeed,oduction . Pathogeoduction .PLCG2 also link the enzyme to type 1 diabetes (Lyn tyrosine kinase autoactivation (gain-of-function) mice, after BCR stimulation, showed constitutive phosphorylation of Syk/PLCγ2, with a resultant increased calcium flux, observed in their resting B cells . These cdiabetes  and venodiabetes , which cOverall, PLCγ2 clearly exhibits an important role in the manifestation of autoinflammatory disease with hypermorphic PLCγ2 signaling resulting in spontaneous autoinflammatory disease. These PLCγ2 signaling-induced autoimmune diseases produce a range of inflammatory phenotypes in both patients and disease models, typically driven by autoantibodies (with the exception of APLAID) and broad autoimmune cell expansion. These disease situations highlight the potential to develop PLCγ2-modulating therapies that treat immune disease but must be tailored so as to avoid effects on other disease relevant processes such as cancer and neurodegeneration. Next, we explore recent insights and advances in these fields.The crucial nature of PLCγ2 in both the innate and adaptive immune system is further underscored by its vital roles in hematological malignancy. This is perhaps most evident in chronic lymphocytic leukemia (CLL), but PLCγ2 is also observed to play a role in diffuse large B cell lymphoma (DLBCL), the most common form of non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myelodysplastic syndrome, endemic Burkitt lymphoma (EBL), MALT-associated gastric lymphoma and multiple myeloma. Finally, PLCγ2 has also been shown to be a relevant part of Epstein–Barr virus (EBV) transformation of B cells.A powerful tool for validating the roles of oncogenes and tumor suppressors is the Eμ-Myc mice lymphoma model where all mice will succumb to lymphoma. Using this Eμ-Myc lymphomagenesis mouse model in the absence of PLCγ2 demonstrated that PLCγ2 was indeed vital to early B cell development, resulting in an accumulation of large pre-B cells . PLCγ2 dWhile the absence of PLCγ2 can further enable lymphogenesis in certain murine lymphomas, in humans, PLCγ2 signaling can drive leukemogenesis in some cases. Ibrutinib, which inhibits Btk and thus blocks PLCγ2 signaling, has become an important and effective treatment for CLL. However, a subset of patients become refractory to this treatment over time and relapse, with PLCγ2 as a primary driver of the recurrent disease , 123. MuPLCG2 were noted to be correlated with CLL resistance to Ibrutinib  signaling, a common relapse patient PLCγ2 mutation, suggesting SYK and LYN as alternative targets in Ibrutinib resistance  of brutinib . It was sistance . SYK/PLCsistance , 134. Fu disease .in vitro suppressed their proliferation and induced apoptosis and cell cycle arrest  . Treatmee arrest . Howevere arrest . Downrege arrest . The useCγ2 mice  in a modlymphoma . These m T cells . Mutatio T cells . These m T cells .via the virally encoded integral latent membrane protein 2A in combination with BLNK , osteosarcoma, esophageal squamous cell carcinoma, esophageal adenocarcinoma, cervical adenocarcinoma, Birt-Hogg-Dube tumors, non–small-cell lung cancer, and breast cancer. PLCγ2 was implicated in the Wnt signaling pathway necessary for kidney development and development of Wilms' tumor . In addiof PLCG2 . PLCγ2 wof PLCG2 . This isof PLCG2 . PLCγ2 wisplatin . Howeverisplatin . Populatisplatin , in non–isplatin , and as isplatin . Indeed,isplatin , 153.2, also resulted in PLCγ2 upregulation in human osteosarcoma cell lines  has been associated with decreased risk of AD by genome-wide association study  and neurofibrillary tangles. A rare variant in the on study . The inion study , 162. Reon study  Fig. 4)PLCG2 genon study . In conton study . On a fuon study .Figure 4PLCG2 gene expression in the cortex of AppNL-G-F/NL-G-F mice Fig. 4). Taken together, these results provide further evidence that PLCγ2 may play an important role in AD pathophysiology. Future studies investigating how these variants in PLCγ2 modulate its function and microglia phenotypes in AD could lead to the identification of novel therapeutic strategies.In summary, PLCγ2 is a fascinating enzyme regulating diverse biological functions through critical immunological molecules used by a variety of extracellular receptor signaling pathways. Conclusive genetic evidence demonstrates how important normal PLCγ2 function is to immune health, the overaction of which can lead to immunodeficiency, autoimmunity, or autoinflammation. Some of these same mutations can also drive hematopoietic cell proliferation, for example, in Ibrutinib-refractory CLL, and so inhibition of PLCγ2 could be considered as a therapeutic modality in some malignancies and immune disorders. However, such intervention would need to be approached with caution, as the constitutive roles of PLCγ2 are important, as are other gain-of-function variants that provide protection against neurodegenerative disease. Therefore, improved understanding of PLCγ2 functions could inform cellular and temporal targeting of this pathway for therapeutic benefit in the future.S. L. M. is a scientific advisor for IFM Therapeutics. J. T. J., E. M., and S. L. N. declare no conflicts of interest.


Reports have suggested that TiO2 is a relatively inert material that has low toxicity to animals. However, as various types of NPs increasingly accumulate in the ocean, their effects on aquatic life-forms remain unclear. In this study, a zebrafish model was used to investigate TiO2NP-induced injury and mortality. We found that the treatment dosages of TiO2NP are positively associated with increased motility of zebrafish and the bacterial counts in the water. Notably, gill but not dorsal fin and caudal fin of the zebrafish displayed considerably increased bacterial load. Metagenomic analysis further revealed that gut microflora, such as phyla Proteobacteria, Bacteroidetes, and Actinobacteria, involving more than 95% of total bacteria counts in the NP-injured zebrafish gill samples. These results collectively suggest that opportunistic bacterial infections are associated with TiO2NP-induced mortality in zebrafish. Infections secondary to TiO2NP-induced injury could be a neglected factor determining the detrimental effects of TiO2NPs on wild fish.The large amounts of engineered titanium dioxide nanoparticles (TiO Industrial production of TiO2 occurs at a large scale, and an estimated 165,050,000 metric tons of TiO2 were produced worldwide between 1916 and 2011 [2 nanoparticles (TiO2NPs), such as sunscreen, cosmetics, paints, and semiconductors, are widely manufactured in various industries [2 in the form of anatase enable it to catalyze H2O to release reactive oxygen species [2 has led to its use in skin-protecting sunscreens and cosmetics [2 is a vital component of these everyday products, its use means that human-made TiO2NPs will inevitably be released into the ecosystem. Experimental data–based safety guidelines for the release of TiO2NPs into fresh or salt water are not yet available. For example, neither the United States Environmental Protection Agency Aquatic Life Criteria nor the United Kingdom Environmental Quality Standards clearly indicate specific standards for the release of TiO2NPs [2NPs has been proven to negatively affect aquatic life-forms, primarily through direct NP-induced toxicity [Titanium dioxide TiO forms nadustries . For exa species –6, which species , 5, 7–12osmetics –15. Althtoxicity , althougDanio rerio) are widely used for vertebrate models in the study of diseases and is increasingly being used in preclinical and toxicological studies [Zebrafish  [2NP solutions were prepared immediately before use through dilution of the stock solutions with distilled deionized water and sonication  for 20 min. In our studies, the particle size distributions and ζ-potential were estimated using the dynamic light scattering method (DLS) with a Zetasizer Nano ZS  [2NPs in solution was 22.08 ± 0.32 mV, with pH value around 5.35–5.45. The TiO2NPs obtained (from Sigma Aldrich) has an averaged particle size about 20 nm according to company specification, but in medium the particles aggregated and measured to be 100 nm.The chemicals used in this study were purchased from Sigma-Aldrich . To prepare the stock solutions of 1 mg/mL Degussa P25  TiO ± 5 nm) , 8, 35, ern, UK) , 37. The2NPs in well water for 21 days, and the mortality was recorded each day. The Kaplan Meier curves are plotted using the Online Application for the Survival Analysis of Lifespan Assay (http://sbi.postech.ac.kr/oasis) [http://www.kinovea.org/) in accordance with methods used in previous studies [Adult wild zebrafish were used in the experiment and were kept in a semistatic system with charcoal-filtered tap water (pH 7.0–7.4) at 28 ± 0.5°C as recommended in a previous study , with a r/oasis) –44. Fish studies , 46. Aft studies , 47. The studies . The zebAll methods on the collection and analyses of zebrafish samples were performed in accordance with Animal Protection Act, Taiwan, and were approved by the Institutional Animal Care and Use Committee of Tzu-Chi University, Hualien, Taiwan .The bacterial genomic DNA was extracted from the 200-mg frozen gill samples with a QIAamp Fast DNA Stool Mini Kit . After isolation, the DNA yield was approximately 1–2 μg. The DNA sample was stored at −20°C before polymerase chain reaction (PCR) amplification.5’-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3’; reverse, 5’-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3’. The PCR products were then purified with a GenepHlow Gel/PCR purification kit .The DNA samples were adjusted to 25 μg/mL. Forward and reverse primers that were complementary upstream and downstream of the V3-V4 region of 16S were designed with Illumina overhang adapters, and used to amplify templates from bacterial genomic DNA. The following 16S amplicon PCR primer sequences were shown: forward, The Illumina sequencing adapters and dual indices were attached to the PCR products using the Nextera XT Index Kit . Next, AMPure XP beads were used to clean up the final libraries, and the expected size on the Bioanalyzer trace of the final libraries was approximately 630 bp.Libraries were normalized and pooled and then sequenced on the MiSeq System using v3.0 reagents .Because microbiota profiling using specific hypervariable regions of 16S ribosomal RNA cannot reach taxonomic levels lower than the family or genus level , we obtahttp://sbi.postech.ac.kr/oasis) [t test was used to assess the statistical significance of differences in antimicrobial effects. A P -value of less than 0.05 (P < 0.05) was considered significant. The statistical tests were performed and output to graphs using Microsoft Excel  and SigmaPlot .The mortality of zebrafish was calculated using Online Application for the Survival Analysis of Lifespan Assay (r/oasis) –44. A t 2NPs were used in the investigation of TiO2NP-induced mortality in adult zebrafish  of TiOebrafish . We founectively .2NPs). For this reason, we hypothesized that the zebrafish in the groups treated with 5 mg/L TiO2NPs may have been subject to TiO2NP-induced injury prior to mortality, and the resulting release of blood or tissue fluids would subsequently cause optical-density changes in the water. Consequently, to assess the physical condition of the fish, the motility . We found that when compared with the untreated groups, the groups treated with 5 mg/L TiO2NPs exhibited remarkably reduced motility, as indicated by decreases in both the average swimming speed and spot swimming speed of the zebrafish . This result indicates that TiO2NP-induced zebrafish injury involves gill infection.Pure TiOre of UV , 40, 49.ondition , 11. To Proteobacteria, Bacteroidetes, and Actinobacteria, all of which are bacteria found in normal zebrafish gut microbiomes [2NP-induced zebrafish gill injury is associated with opportunistic infection of the gut normal flora.Because the plating method can only reveal culturable bacteria , 51, metrobiomes –55, acco2NPs are toxic and induce mortality in zebrafish embryos but are less toxic and cause less mortality in adult zebrafishes; this finding is likely attributable to the fact that these studies have tended to focus only on observations of acute stimulation [Zebrafish have been used to study engineered nanomaterials and NPs in various fields, such as biomedical research , 56–58 amulation , 61–64. mulation , 61–64, 2NPs led to reduced motility, reduced body weight, and increased mortality. Additionally, we also found that the adverse effects of TiO2NPs were associated with gill infection. Accordingly, we postulate a hypothetical model, in which TiO2NPs-induced gill injury the fish at the first stage, while opportunistic gill infection may further exacerbate the injury and then lead to mortality  of TiOortality . This suProteobacteria, Bacteroidetes, and Actinobacteria, accounted for more than 95% of the total bacteria count; these types of bacteria are all found in normal zebrafish gut microflora [2NPs may theoretically be more toxic to wild fish and lead to higher mortality. As an increasing amount of NPs are released into rivers, lakes, and oceans [Through metagenomic analysis, we discovered that bacteria found in the gill samples of zebrafish with NP-induced injury, such as the phyla croflora –55. Thisd oceans , 66, infk__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Sphaerotilus.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Sphingobacteriales;f__env.OPS_17;g__Ambiguous_taxa;s__Ambiguous_taxak__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Aeromonadales;f__Aeromonadaceae;g__Aeromonas.k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Corynebacteriales;f__Mycobacteriaceae;g__Mycobacterium.k__Bacteria;p__Fusobacteria;c__Fusobacteriia;o__Fusobacteriales;f__Fusobacteriaceae;g__Cetobacteriumk__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Rhodocyclaceae;g__Methyloversatilisk__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Plesiomonas;s__Ambiguous_taxa.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Methylophilaceae;g__Methylophilusk__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhizobiales;f__Devosiaceae;g__Devosia.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Chitinophagales;f__Chitinophagaceae;g__Terrimonas.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Burkholderiaceae;g__Sphaerotilus.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Aeromonadales;f__Aeromonadaceae;g__Aeromonas.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Rhodocyclaceae;g__Methyloversatilis.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Plesiomonas;s__Ambiguous_taxa.k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Betaproteobacteriales;f__Methylophilaceae;g__Methylophilus.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Sphingobacteriales;f__env.OPS_17;g__Ambiguous_taxa;s__Ambiguous_taxa.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Chitinophagales;f__Chitinophagaceae;g__Terrimonas.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Cytophagales;f__Spirosomaceae;g__Emticicia.k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Chitinophagales;f__Chitinophagaceae;g__SediminibacteriumS1 Data(DOCX)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.