Since early 2015, mule duck and Cherry Valley duck flocks have been suffering from short beak and dwarfism syndrome. This widely spreading infectious disease is characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. For better understanding, we identified and characterized virus isolates named AH and GD from diseased Cherry Valley duck and mule duck flocks and investigated the damage caused by novel parvovirus-related virus (NGPV) to tissues and organs, including kidney, brain, pancreas, liver, spleen, bursa of fabricius and myocardial tissues.AH and GD isolates shared high nucleotide identity with goose parvovirus (GPV). Alignment studies of AH and GD isolates showed 94.5–99.2% identity with novel parvovirus-related virus (NGPV), 98.7–91.5% identity with GPV and 79.9–83.7% with muscovy duck parvovirus (MDPV). Compared with other NGPV, classical GPV and MDPV sequences, a four 14-nucleotide-pair insertion in GD isolate was found in left open reading frame (ORF) (87–100 nt and 350–363 nt) and in right ORF (4847–4861 nt and 5122–5135 nt). However, in AH isolate, a five 14-nucleotide-pair deletions similar to other NGPV were found. The complete genome sequence comparison of eleven NGPV isolates from mule ducks and cherry valley ducks revealed no remarkable difference between them. Notably, the myocardium and bursa of fabricius of both disease and healthy animals are perfectly normal while other tissues have inflammatory cells exudation.The AH and GD strains are novel parvovirus-related virus that isolates from mule ducks or cherry valley ducks which DNA sequence has no remarkable difference. The histopathology of tissues and organs such as kidney, brain etc. revealed non-significant changes in experimental and control animals. Overall, this study has contributed better understanding of molecular biology of NGPV strains and will help to develop the candidate strain for vaccine preparation to get better protection against these viral infections. Parvoviridae [. GPV are etiological agents for Derzsy’s disease and results in huge economic losses to the duck industry [Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are members of the family oviridae , 2. GPV industry . The morindustry . The disindustry , 5, 6. MWaterfowl parvoviruses, including MDPV and GPV, contain a linear, single-stranded DNA genome (approx. 5.0–5.1 kb in length) . At bothDuring 1970s, Short beak and dwarfism syndrome (SBDS) was first reported in France in mule ducks, caused by a novel parvovirus-related virus (NGPV) , 15. SinIn order to have better understanding of SBDS disease, we characterized two NGPV namely AH and GD isolates, obtained from mule duck and Cherry Valley duck flocks in Anhui and Guangdong province, China. For detailed analysis, the complete genome of these isolates was sequencedand homology studies and phylogenetic comparison was carried out. The nucleotide sequences of the AH and GD genome and ITR were aligned for comparison with other GPV/MDPV/NGPV strains. The amino acid sequences of non-structural and structural proteins were also subjected to phylogenetic and homology analysis. The histopathological features were also studied in experimental and control groups.In 2014–2015, two mule duck and Cherry Valley duck flocks located in Guangdong and Anhui province, China were found to be suffering from SBDS disease. Liver and spleen samples were collected from diseased birds and processed for viral isolation.The AH and GD strains of variant GPV were isolated from mule duck and Cherry Valley duck flocks with typical signs of SBDS. Liver and spleen tissues were grinded and homogenized in physiological saline, freeze-thawed three times and centrifuged at 10,000 x g for 15 min at 4 °C. The supernatants were subjected to filtration using 0.22 μm sterile filters to remove contaminants and inoculated into chorioallantoic cavity of 10-day-old duck embryonated eggs (200 μl/egg). The allantoic fluid was harvested 5 days after inoculation and stored at − 80 °C for further testing.The allantoic fulid of AH and GD isolates was centrifuged at 5000 xg for 10 min to remove cellular debris and proceeded for DNA extraction using AxyPrep DNA Extraction Kit (ANYGEN). The stock DNA was dissolved in TE buffer for future use.To amplify the complete genome of GD and AH isolates, six primer pairs were designed, based on the conserved regions in the genome sequence of NGPV Table . The polThe amplified PCR products were purified using gel extraction kit (AXYGEN), and all fragments were cloned into pMD18-T vector (TaKaRa). The plasmids were sequenced by Sangon Tech . For ensuring accuracy of genomic sequences, triplicate clones of fragments were sequenced.Using BLAST sequence alignment, all six fragments were assembled to form a full–length complete genome. The sequence alignment and homology studies for whole-genome, NS  and VP  of GD and AH isolates was done by comparison with already reported sequences in the Genbank using MegAlign and MEGA 5.0 software programs  Table 22.Table 2Histopathological investigations were carried out in the diseased flocks by comparison with the healthy (Control) animals. At 14 days post infection (DPI), the animals were euthanized and necropsied. Intravenous anesthesia of high concentrations of pentobarbital sodium (150 mg/kg) was used in this study. Samples of kidney, brain, pancreas, liver, spleen, bursa of fabricius and myocardial tissues were fixed with 10% formalin; all the tissues were dehydrated, and immersed in transparent wax, cut into slices and stained with H & E dye. All of the animal protocols were performed in accordance with the ‘Guidelines for Experimental Animals’ of Ministry of Science and Technology .Two NGPV strains (GD and AH) were isolated from NGPV-positive mule duck and Cherry Valley duck samples respectively. These isolates were not able to cause death in duck embryonic eggs 5 days post infection (dpi). The main signs and symptoms in AH or GD-infected ducklings were retarded in growth with low mortality rate Fig. .Fig. 1ClThe complete genome sequence of mule duck isolate “GD” and Cherry Valley duck isolate “AH” was 5106 bp and 5053 bp respectively. The sequences were submitted to GenBank and their accession numbers are indicated in Table Phylogenetic analysis of NGPV, GPV and MDPV sequences revealed that all NGPV isolates have more similarity with GPV than MDPV Fig. . HomologIn order to understand the genetic diversity of Non-structural proteins (NS) in NGPV, GPV and MDPV isolates, phylogenetic tree was constructed and homology analysis was also carried out Fig. . ThroughHomology of NS proteins among NGPV isolates ranged between 93.7–100% at gene level and 95.4–100% at amino acid level. However, they share 93.4–97.9% identity with classical GPV and 81.0–82.4% identity with MDPV. At amino acid level, all NGPV isolates shared 96.3–99.5% identity with classical GPV and 89.0–90.1% identity with MDPV. As compared to classical GPV and MDPV, there are twelve synchronous specific mutations in NS amino acids (Table The genetic diversity of VP gene (Capsid protein gene) was also studied by phylogeny and homology analysis Fig. . ThroughHomology among NGPV isolates ranged between 94.0–100% at VP gene level and 96.9–100% at amino acid level. The VP gene of NGPV isolates has 90.9–97.5% identity with classical GPV and 80.9–91.5% identity with MDPV. At VP amino acid level, NGPV isolates shared 95.1–98.2% identity with classical GPV, and 88.0–92.6% identity with MDPV. As compared to classical GPV and MDPV, there are eight synchronous specific mutations in VP amino acids , therefore it might be possible that SimPlot software could not able to use analysis recombination.The histopathological examination revealed no remarkable difference in kidney, brain, pancreas, liver, spleen, bursa of fabricius, myocardium tissues between experimental group and control group. These findings suggested that NGPV causes non-significant pathological changes in these organs and tissues, which might be the reason of low mortality in infected animals (2%).In conclusion, the novel parvovirus-related virus is responsible for SBDS disease outbreaks in eastern China since 2014 , 26. TheAH and GD strains isolated from mule duck and Cherry Valley duck flocks are novel parvovirus-related virus. The comparison of complete genome sequence of eleven NGPV isolates from mule ducks and cherry valley ducks found no remarkable difference between them. Through histopathological examination, it was found that both experimental and control groups have perfectly normal myocardium and bursa of fabricius while other tissues have inflammatory cells exudation. The results suggested that NGPV has no significant influence on the tissues and organs. This study has contributed better understanding of molecular biology of NGPV strains and will help to develop the candidate strain for vaccine preparation in future.


Adrenocortical carcinoma (ACC) is a rare malignant endocrine tumor with a high tumor recurrence rate and poor postoperative survival. Recent studies suggest that CD276- (B7-H3) targeted therapy represents a promising therapeutic option for solid tumors. However, little is known about the expression status of CD276 or its association with progression and prognosis of ACC. n = 48). Archived, formalin-fixed, and paraffin-embedded samples were collected for immunohistochemical analysis, and the correlation between CD276 expression and clinicopathological parameters was evaluated. Kaplan–Meier and univariate/multivariate Cox regression methods were implemented to identify any prognostic effects. Data from The Cancer Genome Atlas (TCGA) ACC cohort (n = 77) were retrieved for quantitative validation analysis.  Clinical data were retrospectively analyzed from patients who underwent resection of ACC at our institution (P = 0.029) and advanced ENSAT stage (P = 0.02). Specifically, patients with a higher CD276-positive cancer cell density exhibited significantly worse overall survival and recurrence-free survival in our cohort  and in the validation cohort . The prognostic association remained significant in multivariate Cox regression analysis. Further analysis indicated that CD276 participates in regulating the immune response as well as in the malignant biological behaviors of ACC.  Positive expression of CD276 was detected on the cell membrane and in the cytoplasm of cancer cells or tumor-associated vascular cells in 91.67% (44/48) of ACCs. Vascular expression of CD276 was associated with local aggression (higher T stage,  These findings highlight the immune checkpoint factor CD276 as an independent prognostic factor and a potential therapeutic target in ACC. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy (0.5-2 cases per million per year) with a heterogeneous and often poor prognosis , 2. Pati+ T cells and expression of programmed death ligand 1 (PD-L1/B7-H1) were significantly associated with improved survival, indicating a potential role for the immune signature in the assessment of ACC prognosis [Both oncogenesis and immune status are poorly understood in ACC. In the tumor microenvironment, the immunosuppressive and immunostimulating signatures have a potential prognostic value for some cancer types , 10. Recrognosis . Howeverrognosis , 13. Givrognosis , identifCD276 (B7-H3) is one of the B7 superfamily molecules that correlates with prognosis in various cancer types , 16. As In the current study, we aimed to evaluate the clinical significance of CD276 as an emerging immune checkpoint in ACC. The relationship between CD276 and multiple clinicopathological parameters was explored. We demonstrated that differential expression patterns of CD276 were closely associated with tumor progression and prognosis in ACC patients. Herein, the regulatory relationships between CD276 and the immune signature are revealed to improve the understanding of the role of CD276 in the ACC microenvironment.Between 2009 and 2016, patients who underwent tumor resection at the West China Hospital that were pathologically confirmed as ACC were analyzed. A total of 48 patients were included in this study. Related clinical records were extracted as per our previous report [μm were subjected to immunohistochemistry (IHC) analysis following protocols. Briefly, sections were deparaffinized in xylene and rehydrated through a graded ethanol series, followed by placement in 3% H2O2 for 15 min at room temperature. After heat-mediated retrieval using sodium citrate or EDTA, slides were incubated with primary antibody overnight at 4°C. The primary antibody used was a rabbit antihuman B7-H3 (D9M2L) XP® monoclonal antibody . SignalStain® Boost IHC Detection Reagent  was applied for 30 min at room temperature according to the manufacturer's instructions.Serial FFPE tissue sections with a thickness of 4 The immunostaining results were independently evaluated by two investigators blinded to the clinical data (X.Y. and N.C.). The semiquantitative H-score of the cytoplasmic staining intensity was calculated as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Due to the limited number of ACC cases in this cohort, we next merged them into high expression (strong and moderate expression) and low expression (negative and weak expression) groups. Membranous and vascular expression status was evaluated as “positive” or “negative”. The cut-off proportion of positive expression was 5% in each specimen.http://xena.ucsc.edu). This study meets the publication guidelines provided by TCGA (https://cancergenome.nih.gov/publications/publicationguidelines). One-way ANOVA and the log-rank test were used in the GEPIA analysis [ACC clinical data and RNA-Seq data from the TCGA project were retrieved from the UCSC Xena project . The gene sets used in this work were downloaded from the Molecular Signatures Database (https://software.broadinstitute.org/gsea/msigdb/index.jsp). GO terms with a P value <0.05 and an enrichment score > 1.0 were considered significant [Gene expression relationships were evaluated using the R system, and the coexpression cut-off was Pearson ∣nificant .P value <0.05 were included in the multivariate Cox regression. P values <0.05 were considered statistically significant.Statistical analyses were performed using the R system (version 3.4.4) and GraphPad Prism version 6.02 . Overall survival (OS) was defined as the time elapsed from primary resection of ACC to death due to any cause. Disease-free survival  was defined as the time elapsed from primary resection of ACC to the first recurrence . As per our previous report, recurrent disease was diagnosed based on clinical, radiographic, and laboratory evidence, including local recurrence, peritoneal carcinomatosis, and distant metastases. The Chi-square test or Fisher's exact test was used to compare clinicopathological variables between two groups. Survival analyses were analyzed by the Kaplan–Meier method and log-rank test. Univariate and multivariate Cox regression analyses were performed to identify significant risk factors, and variables with a n = 48) from 2009 to 2016 were collected, and their clinical and pathological characteristics were analyzed , local tumor infiltration and invasion status were evaluated as well as other clinicopathological parameters. Next, the potential correlation between the differential expression patterns and the intensity of CD276 and pathological features was compared . ACC patP = 0.01 and P = 0.003, separately). Membrane expression of CD276 was lower in cases with larger tumors . Interestingly, expression of CD276 in the tumor vasculature was significantly correlated with gender, age, T stage, and ENSAT stage in our patient cohort, suggesting that the CD276-invasive rate in the tumor vasculature was higher in either male ACC patients (P = 0.003) or older ACC patients . Positive expression of CD276 in the tumor vasculature may also indicate a higher risk of local tumor infiltration, adjacent organ invasion or venous tumor thrombus (P = 0.029), and advanced ENSAT stage (P = 0.020). However, there was no association observed between hormone secretion, N stage, or Ki67 index and the CD276 expression patterns. In addition, mRNA expression of CD276 was also found to correlate with the disease stage in the validation cohort  and higher intensity of CD276 expression in tumor cells  were the only prognostic factors in the multivariate Cox model, suggesting that differential expression of CD276 in tumor cells is an independent OS factor in ACC, as well as the Ki-67 index.Next, we explored the association between CD276 expression and overall survival of ACC patients. In the Kaplan–Meier analysis, ACC cases with a higher intensity of CD276 expression in tumor cells exhibited significantly poorer overall survival compared to those with lower CD276 expression levels  and CD276 expression in tumor cells  were independent recurrence risk factors for ACC  from the TCGA-ACC dataset. As a result, we found that genes that highly correlated with CD276 expression were more involved in immune signatures, including “immune system,” “adaptive immune system,” “innate immune system,” and “cytokine signaling in immune system” . First, genes that strongly coexpressed with CD276 were selected  plays an important role in the microenvironment between tumors and the host immune system. Negative regulation by CD276 of the immune cell response, such as T cells and NK cells, has recently been reported , 24. MeaIn recent years, immunotherapy has been widely used to treat various cancer types. Accordingly, the expression of immune checkpoint factors is widely accepted as a predictor of response to immune checkpoint inhibitors. Given the limited expression of PD-L1 in ACC, exploring new targets for ACC patients is urgent. Increasing data suggest that CD276 represents a novel therapeutic immune checkpoint. Inhibition of CD276 is able to suppress tumor growth , and CD2The current study is a relatively large single-center cohort study of patients with rare ACC. However, due to the limited number of ACC samples available, full-quantitative experiments in our ACC cohort were not performed. To overcome this limitation, we retrieved external quantitative data from the TCGA cohort in this study, successfully validating the prognostic effects of CD276. The clinical pathological characteristics of the validation cohort were summarized in a previous report . ClinicaIn conclusion, in this study, we demonstrate for the first time the clinical significance of CD276 expression in ACC cells and the tumor vasculature. These findings highlight CD276 as an independent prognostic factor and potential immune checkpoint therapeutic target in ACC treatment.


PLEKHA7 and COL11A1 were genotyped for single-nucleotide polymorphisms (SNPs) to investigate the possible association of these two genes with primary angle-closure glaucoma (PACG) and disease severity. t-tests and chi-square tests were used to compare characteristics of PACG cases and controls. Chi-square tests were used to compare the distribution of allele frequencies in cases and controls and in MD-based subgroups with various degrees of glaucoma severity. Binary logistic regression was used to compare the distribution of genotype frequencies and calculate odds ratios (OR) with confidence intervals (CI).  A total of 51 PACG cases and 51 normal controls were recruited. Twelve SNPs in the PLEKHA7  and COL11A1  genes were genotyped by direct Sanger sequencing. Distributions of allele frequencies and genotype frequencies in cases and controls, as well as in mild, moderate, and severe subgroups, were compared based on mean defect . Independent Student's P=0.026, OR = 2.089, 95% CI = 1.092–3.996), rs3753841 , and rs12138977  were found to have a significant association with PACG. Furthermore, in the subgroup analysis, rs1676486 , rs12138977 , and rs3753841  showed statistically significant differences between moderate/severe groups and controls.  Three of the 12 SNPs in COL11A1, rs1676486 ( Our data suggested that COL11A1 rs1676484, rs3753841, and rs12138977 polymorphisms may be of value for further study as potential gene-dependent risk factors for developing PACG. Moreover, COL11A1 rs1676484 and rs12138977 polymorphisms might be associated with PACG disease severity. Primary angle-closure glaucoma (PACG) is a neurodegenerative disease characterized by elevated intraocular pressure (IOP) due to a mechanical obstruction of the trabecular meshwork by either apposition of the peripheral iris to the trabecular meshwork or a synechial closed angle . EpidemiPACG is a complex heterogeneous disease; causative molecular mechanisms leading to PACG are poorly understood, and genetic susceptibility for this disease is still under investigation. Genome-wide association studies (GWAS) have recently identified several new PACG loci and genes, including PLEKHA7, COL11A1, EPDR1, CHAT, GLIS3, FERMT2, and DPM2-FAM102 A, which may shed light on the molecular mechanisms of PACG . Among tThe contribution of the two most associated SNPs (rs11024102 and rs3753841) on PLEKHA7 and COL11A1 was confirmed in patients in Australia, Nepal, and China . MoreoveIn this study, we explored the association between 12 SNPs (in PLEKHA7 and COL11A1) and PACG. An analysis was also conducted to determine whether the SNPs are associated with PACG patients experiencing different degrees of disease severity. The association between ocular biometric parameters, such as anterior chamber depth (ACD) and axial length (AL), with 12 SNPs were also investigated. In sum, our work aimed at providing new insights and a better understanding of the association between candidate gene SNPs and disease severity and specific clinical features of PACG.The study was approved by the ethical committee of Eye and Ear Nose Throat Hospital at Fudan University in Shanghai, China, and all procedures were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects and normal controls prior to participating in this study.A total of 51 subjects with PACG and 51 matched healthy controls were consecutively recruited from Eye and Ear Nose Throat Hospital at Fudan University. Each subject underwent a standardized ophthalmic examination performed by glaucoma specialists that included the following assessments: refractive status, slit-lamp biomicroscopy, fundus, IOP, AL, ACD, visual field, and gonioscopy. MD and mean sensitivity (MS) were measured using an automated perimeter . We measured IOP using Goldmann applanation tonometry. Fundus photography was performed with a retinal camera . We used an A-scan ultrasound  to measure AL and ACD .PACG subjects were selected from hospital inpatients.P value less than 5% occurring in the normal population, and fulfilling the test reliability criteria . PACG was diagnosed in eyes meeting the following criteria: narrow angles and elevated IOP (IOP > 21 mm·Hg); at least 180 degrees of angle-closure obliterating the pigmented part of the trabecular meshwork, whether synechial or appositional, segmented or continuous; and eyes in which the degree of the peripheral anterior synechiae is too extensive to be managed by laser peripheral iridotomy [PACG was diagnosed on the basis of narrow angles with glaucomatous optic neuropathy and corresponding visual field loss. It was defined as a glaucoma hemifield test outside normal limits, including a cluster of three or more noncontiguous points on the pattern deviation plot, not crossing the horizontal meridian with a probability of less than 5% of being present in the age-matched normal (one of which was less than 1%), an abnormal pattern standard deviation with a ridotomy –19. SubjPACG and control subjects had no major systemic diseases  that could likely confound the results of the study. Participants with secondary angle-closure glaucoma due to uveitis, trauma, neovascularization, or any other optic nerve injury affecting either eye were excluded.Our previous study described the PACG inclusion criteria , which aWhole blood samples (2 ml each) were collected from each subject, which was used for DNA isolation and an analysis of the selected genetic polymorphisms. Before treatment, all the samples were stored in an EDTA tube  and stored in a −80°C refrigerator.http://hapmap.ncbi.nlm.nih.gov/, in the public domain).Genomic DNA was extracted from leukocytes of peripheral blood drawn from each participant. A RelaxGene Blood DNA System (0.1–20 ml) (DP349) was used to purify the blood. Twelve SNPs , including two SNPs previously reported as significantly associated with PACG through GWAS , were genotyped. The SNPs chosen in this study were either tagging or coding SNPs with minor allele frequencies greater than 10% in Chinese Han populations referenced to the HapMap database (μL of each primer (10 μmol/L), 1 μL of 10x buffer , 0.2 μL of MgCl2 , 0.2 μL of deoxyribonucleotide triphosphates , and 0.06 μL of HotStarTaq DNA polymerase . Amplified PCR products were separated by agarose gel electrophoresis and visualized by staining with YeaRed nucleic acid gel stain . PCRs were performed on an S1000™ Thermal Cycler . The PCR cycling program was 15 min at 95°C, 11 cycles × , 24 cycles × , 2 min at 72°C, and 4°C permanently. All the SNPs were genotyped via direct Sanger sequencing.Briefly, the polymerase chain reactions (PCRs) contained 50 ng of genomic DNA, 1 t-tests and chi-square tests were used to compare patient characteristics between groups. The chi-square test was used to evaluate the Hardy–Weinberg equilibrium (HWE) and differences in allele frequencies for each SNP between the case and control groups. Logistic regression was used to compare the distribution of genotype frequencies and calculate odds ratios (OR) with 95% confidence intervals (CI) and adjust for age and sex. A chi-square test was also used to analyze subgroups in which patients were divided into three groups with different degrees of severity based on perimetry: mild (MD ≤ 6.00 dB), moderate (6 dB < MD ≤ 12 dB), and severe (MD > 12 dB) [P < 0.05 was considered statistically significant.The data were analyzed using standard statistical software . Results are presented as mean ± SD. Independent Student's > 12 dB) , 21. A lP > 0.05). Demographics and clinical characteristics of cases and controls are shown in P > 0.05; A total of 51 subjects with PACG  and 51 normal controls  were enrolled in this study. The mean age of cases was 59.29 ± 11.9 years and 58.24 ± 6.49 years for controls. One eye was randomly selected if both eyes suffered from PACG. There were a total of 51 eyes in the PACG group. There was no statistical difference in the mean age, SBP, DBP, or sex distribution between PACG and control subjects , rs3753841  and rs12138977  in COL11A1, with higher minor allele frequencies found in cases than in controls . A significant association was also identified for GG genotype frequencies of rs3753841 . CT + CC genotype frequencies of rs12138977  also showed statistically significant evidence of genetic association in analyses comparing PACG and controls.Among the 12 SNPs, a significant genetic association was identified for rs1676486 , rs3753841 , and rs12138977 , showed a statistically significant association. Additionally, the following SNPs showed a statistically significant association between severe PACG and controls: rs1676486 , rs3753841 , and rs1213897 . However, when the mild PACG subgroup was compared to controls, there was no statistically significant association  using Sanger sequencing. In our study, rs1676486, rs3753841, and rs12138977 in COL11A1 showed significant differences in minor allele and genotype frequencies between cases and controls (see Tables P=0.024).Previous studies identified rs3753841 in COL11A1 and rs11024102 in PLEKHA7 as genetic risk factors for PACG . In a prMoreover, this study appeared to be the first to examine the association of susceptibility loci with MD-based glaucoma severity subgroups. It is well known that clarifying risk factors for different stages of a disease can be helpful for early diagnosis and prognosis in clinical practice. Surprisingly, we found that the allele frequency distribution of the three SNPs discussed above  was significantly different in moderate/severe PACG and controls, which suggested they may be associated with PACG disease severity. Thus, these three SNPs might be clinically useful in predicting PACG progression.COL11A1 encodes the alpha-1 chain of type Xl collagen, a member of the collagen family and a major component of the interstitial extracellular matrix (ECM) . ECM matOur study suffered several limitations. Due to the strict inclusion criteria, this study's sample size was relatively small. Additionally, although associations between PACG severity and COL11A1 polymorphisms were observed, the exact mechanism remains unknown and needs to be further investigated.Overall, our study validates the findings of GWAS implicating COL11A1 in the pathogenesis of PACG. The results revealed significant associations between rs1676486, rs3753841, and rs12138977 polymorphisms and PACG. Moreover, carriers of the minor alleles of these SNPs were shown to confer significant risk for PACG. We also demonstrated these three SNPs may be associated with disease severity. Further studies will be necessary to investigate how the candidate genes affect the pathogenicity of angle-closure glaucoma.


Accurate survival estimations in Ewing sarcoma are necessary to develop risk- and response adaptive treatment strategies allowing for early decision-making. We aim to develop an easy-to-use survival estimation tool from diagnosis and surgery. A retrospective study of 1314 Ewing sarcoma patients was performed. Associations between prognostic variables at diagnosis/surgery and overall survival (OS), were investigated using Kaplan-Meier and multivariate Cox models. Predictive accuracy was evaluated by cross-validation and Harrell C-statistics. Median follow-up was 7.9 years (95%CI 7.6–8.3). Independent prognostic factors at diagnosis were age, volume, primary tumor localization and disease extent. 5 risk categories (A-E) were identified with 5-year OS of 88% (86–94), 69% (64–74), 57% (50–64), 51% (42–60) and 28% (22–34) respectively. Harrell C-statistic was 0.70. Independent prognostic factors from surgery were age, volume, disease extent and histological response. In categories A-B, 5y OS increased to 92% (87–97) and 79% (71–87) respectively for 100% necrosis and decreased to 76% (67–85) and 62% (55–69) respectively for <100% necrosis. In categories C-E, 5y OS increased to 65% (55–75), 65% (52–78) and 52% (38–66) respectively for ≥90% necrosis and decreased to 38% (22–54), 11% (0–26) and 7% (0–19) respectively for <90% necrosis. We present an easy-to-use survival estimation tool from diagnosis in Ewing sarcoma based on age, volume, primary tumor localization and disease extent. Histological response is a strong additional prognostic factor for OS. Management rapidly evolved over the last decades, leading to a multimodality approach consisting of chemotherapy, surgery and/or radiotherapy that has become the standard of care. As a result of collaborating trials overall survival (OS) improved drastically, with 10-year OS rates of 55–65% for localized disease. Survival in metastatic disease, present in 20–25% of the patients and usually affecting the lungs (70–80%) and bone/bone marrow (40–45%), is still dismal with 5-year OS varying from 20–35%5. In primary non-metastatic disease 30–40% of patients experience recurrence, in metastatic disease this number increases to 60–80%. Relapse is mostly systemic (71–73%), followed by combined (12–18%) and local (11–15%) relapse7. 5-year post-relapse survival is poor, 15–25%, with local recurrence faring better than systemic9.Ewing sarcoma (EwS) is an aggressive bone and soft-tissue tumor predominantly affecting children and young adults10. Better selection of risk groups and thereby adjusted treatment allows for early decision making, will help improve future outcomes and assists in clinical trial design.Personalized medicine encompasses tailoring of treatment based on individual patient characteristics, needs and preferences to improve outcome. Accurate estimations of survival according to the individual patient’s risk profile at different time points are necessary to offer EwS patients the most appropriate treatment, balancing survival and prognosis with toxicity and quality of life. Especially in this young patient population, this balance is essential in our aim to provide the best possible care. Correct survival estimations are difficult and patients and physicians tend to be overoptimistic12 described combining these prognostic factors into risk groups. All three models present shortcomings. They are based on small homogeneous cohorts, that are either not validated or did not include all relevant variables in the model. Keeping these shortcomings in mind, our aim was to develop an easy-to-use survival estimation tool for EwS. Objectives are to: (1) Identify prognostic factors for overall survival from diagnosis and surgery; (2) Develop an accurate baseline prognostic model; (3) Validate the models’ predictive accuracy; (4) Develop a second prognostic model from surgery.Many studies evaluated the influence of various risk factors on survival in EwS. Only threeThis study was reviewed and approved by the Ethical Committee of the Leiden University Medical Center and granted a waiver for informed consent.A retrospective analysis of patients (randomized and non-randomized) from the EURO-E.W.I.N.G 99 trial database was performed. As detailed in Fig. 17. Cohorts often contain more variables than can reasonably be used for prediction. Therefore, the most predictive and sensible predictors should be selected. In order to provide all relevant risk factors for such a prognostic model a systematic review18 on the current known prognostic factors for overall survival (OS) and event-free survival (EFS) was performed. Based on this systematic review we selected the most predictive and sensible predictors to be included in the univariate analysis. Prognostic factors and outcome were collected prospectively. Patient characteristics included gender and age. Tumor characteristics included location, type, volume at diagnosis, skip lesions, disease extent and number of metastatic lesions. Histological response (percentage necrosis) and resection margins were assessed on the surgical specimen by local pathologists.For accurate risk group stratification large representative and contemporary datasets that closely reflect the target population are needed to enhance the relevance, reproducibility and generalizability of the modelThe outcome of interest was overall survival (OS) measured from date of diagnosis or date of surgery, until last day of follow-up or date of death. Prognostic factors were evaluated using univariate Cox regression analyses; significant prognostic factors were subsequently included into a multivariate Cox model.19. The prognostic value of the clinical risk categories was assessed by comparison with cross-validated risk categories. Details on cross-validation methodology and risk category classification are provided in Supplementary File 20. Observed survival probabilities of clinical risk categories and corresponding cross-validated counterparts were compared by Kaplan-Meier estimators.Significant risk factors at diagnosis from the corresponding multivariate Cox model were used to build a stratification scheme of prognostic groups. Prognostic groups were narrowed down into risk categories based on clinical expertise. Another set of risk categories was obtained from the same multivariate Cox model based on predicted survival; a leave-one-out cross-validation framework was used to form cross-validated risk categories on predicted 5-year survival probabilitySignificant risk factors at surgery from the corresponding univariate analysis were used to build a second multivariate Cox model. Associations were considered significant at a rejection level of 5%. All analyses were performed using SPSS version 23.0, R version 3.4.3, and Python 3.6.5.21, was 7.9 years  7.6–8.3 years); 531 patients died. Localized disease was present in 916 (69.7%), pulmonary metastasis alone in 182 (13.9%) and extrapulmonary metastasis with or without additional pulmonary metastasis in 216 (16.4%) patients. The 5-year OS was 73% , 53%  and 28%  respectively.Baseline characteristics and treatment details of the 1314 patients at diagnosis are presented in Table Univariate and multivariate Cox proportional hazard models were estimated to investigate the effect of risk factors on OS. Results are shown in Table Based on the independent prognostic factors at diagnosis , 13 prognostic groups were created and 5 clinically relevant categories (A-E) were estimated. Table Harrell’s C-statistic was 0.70. Discriminatory ability was further evaluated using cross validation. Detailed comparisons of OS in the clinical and cross-validated risk categories at 2, 3 and 5 years are presented in Table Table A multivariate Cox model with prognostic factors histological response, risk categories and an interaction term was estimated. The interaction between histological response and risk category was not significant, meaning that the effect of histological response does not vary significantly across the risk categories. The association between histological response and OS was therefore assessed by fitting a Cox model with risk category and histological response, details are presented in Table Figure To further improve survival in Ewing sarcoma development of risk- and response adaptive treatment strategies are necessary to allow decision making at different disease stages. Accurate survival estimations are challenging. We developed and validated an easy-to-use survival estimation tool for EwS, based on age, volume, primary tumor localization and disease extent. Furthermore, we show that during the course of treatment survival changes as more information becomes available.The model presented is based on a cohort of 1314 EwS patients with uniformity in diagnostics and treatment and availability of all relevant prognostic factors. The provided flowcharts are easy-to-use and based on assessable variables. The 13 prognostic groups provide detailed insight in expected survival and could assist in fine-tuning individual treatment. The prognostic groups were narrowed down to 5 risk categories (A-E) based on clinical expertise. The risk categories defined on clinical criteria are consistent with cross-validated risk categories defined on predicted 5-year survival probability. The information gained after surgery offers a second time-point for multidisciplinary decision-making, at this point histological response is an strong additional prognostic factor for OS.24; patients with extrapulmonary metastasis do significantly worse than patients with pulmonary metastasis alone26. Disease extent is also used to define risk groups in previous and current European EwS trials. We also found that primary tumors in the pelvic strongly affect survival, consistent with previously published studies27. Other studies suggested an adverse effect on survival for axial localizations (including pelvic) compared to tumours in the extremities30. Volume has also been used to design EwS trails31; research shows that larger volumes are associated with poorer survival. Cut-off points at 100 ml26 and 150 ml32 have been evaluated, but 200 ml seems the most appropriate34 and was therefore used in this study. Age is an independent prognostic factor for survival in the current study, but only shows strong impact on outcome in two prognostic groups. Cut-of points at 1830 and 14 years35 have been evaluated. Strong evidence for a specific cut-off point is lacking. All studies consistently show that older age is associated with poorer survival. We chose 16 years as cut-off, as it is at the interface of pediatric and adult treatment. Histological response, used to tailor treatment in European EwS, is considered of high prognostic value as confirmed in this study. According to literature patients with 100% necrosis have the best survival32, other studies showed similar results using cut-of points at 95%36 and 90% necrosis33.The prognostic significance of the variables in both models has previously been reported. Disease extent is the foundation of the model and strongest prognostic factor in this study. This is consistent with previous studies demonstrating that the presence of metastasis is a strong prognostic factor for survivalet al.8 used Cox proportional hazards models to identify four risk groups in 220 EwS patients based on age (</≥14 years), primary tumor site (pelvic/non-pelvic) and disease extent . Although based on a small cohort and not validated, our risk groups are similar, with the exception that we added volume to the model. Although they found that tumor size was an independent prognostic factor for survival, they did not include it in the final model. Biswas et al.11 developed a prognostic model for localized EwS based on 244 patients. Cox models were estimated showing that patients with axial tumors and elevated white blood cell count (WBC) (>11 × 109/L) had poor OS  and patients with symptoms >4 months, tumor size ≥8 cm and elevated WBC had poor event-free survival . These models were not validated and are based on a small unmixed cohort limiting its usefulness for clinical decision-making. Additionally, in the systematic review we performed before the start of the current study a consistent association between several biomarkers, such as neutrophil to lymphocyte ratio, hemoglobin and WBC count could not be found, in contrast to the model of Biswas et al.11 and another study37. Lastly Karski et al.12 derived prognostic groups from 2124 EwS patients in the Surveillance, Epidemiology, and End Results (SEER) database. Using Cox models for OS they constructed five prognostic groups: (1) Localized, <18 years, non-pelvic; (2) Localized, <18 years, pelvic or localized, ≥18 years, White/non-Hispanic; (3) Localized, ≥18 years, other ethnicities; (4) Metastatic, <18 years; (5) Metastatic, ≥18 years. Validation was performed on a cohort of 1680 EwS patients from the Children’s Oncology Group trials, which showed significantly different OS based upon this classification. Although validated, the primary model did not include all relevant variables as the SEER database lacks information on metastatic site. In addition tumor size was missing in 40% of the patients and therefore not included, limiting the strengths of the models.To our knowledge, only three studies described combining prognostic factors into risk groups. Rodriquez-Galindo 17. Cohorts often contain more variables than can reasonably be used for prediction and for sufficient power one needs at least 10 events per variable. We therefor choose to select the most predictive and sensible predictors to be included in the univariate analysis based on our systematic review18. Using a more extensive variable profile could have given useful insights, but we feel that by doing so we would lose simplicity while not improving predictability. Third, surgical margins and histological response were assessed by local pathologists and not by a reference pathologist. Differences between centers in analyzing specimens are possible. Last, the retrospective study design using data form a prospectively performed trial led to some missing data (11%), despite this, a large cohort of EwS patients was available for analysis.Limitation of this study include the fact that the local treatment choice was left to discretion of the threatening multidisciplinary teams and might have influenced the results discussed in this article. Secondly, a good prediction model should provide accurate prediction of events by using a comprehensive dataset. In addition, the model should be relatively simple and clinically easy to use. Inaccurate estimates of future events will mislead physicians to provide insufficient treatment. On the other hand, a model with high predictability but which is complex or has too many factors will not be useful. Achieving the optimal balance between predictability and simplicity is the key to a good prediction modelThis study presents an easy-to-use clinical tool to predict OS from diagnosis in EwS, based on age, tumor volume, tumor localization and disease extent. After surgery, the second multidisciplinary decision point, histological response is a strong additional prognostic factor for OS.Supplementary information


Fish intake has been postulated to reduce the risk of stroke. However, whether the beneficial effect of fish are mainly linked to fat content, as a source of omega-3 polyunsaturated fatty acids, remains unclear. We conducted a meta-analysis to compare the effect of fatty and lean fish intake on stroke risk.We performed a literature search on four database  through February 1, 2018 to identify prospective studies of fatty and lean fish in relation to stroke risk. A random-effects model was used to calculate the summary estimates.We identified five prospective studies, including 7 comparisons for fatty fish intake and 5 comparisons for lean fish intake. Compared with the highest category of intake with lowest category, the summary relative risk was 0.88  for fatty fish intake and 0.81  for lean fish intake. No heterogeneity across studies and publication bias were observed.Our findings demonstrated that fatty and lean fish intake has beneficial effects on stroke risk, especially lean fish intake. Additional prospective studies are necessary to confirm these observations. Stroke is the major cause of morbidity and mortality. In 2013, stroke was the second most common cause of deaths  worldwide, and the third most common cause of disability  [In 2002, the American Heart Association (AHA) published a scientific statement, “Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease” . AgenciePrevious meta-analyses showed that fish intake has a protective effect against stroke, especially in ischemic stroke , 7. HoweFour database  were search through February 1, 2018 using the key words fish and stroke without restrictions. Furthermore, we reviewed the reference list of retrieved articles. Prospective studies were included if they reported hazard ratio (HR)/relative risk (RR) with a corresponding 95% CI of stroke relating to fatty or lean fish intake in the healthy population. The data that we collected included the last name of the first author, year of publication, country of origin, study name, number of participants and cases, range or mean of participants’ age, duration of follow-up, covariate adjustments, and maximally adjusted risk estimates and 95% CI of lean and fatty fish intake. We used the Newcastle-Ottawa Quality Assessment Scale (NOS), with the highest scale is nine, to evaluate the quality of each cohort [I2 statistics [P < 0.1 was considered statistically significant; and for the I2 statistics, the following conventional cutoff points were used: < 25% (low heterogeneity), 25–50% (moderate heterogeneity), and > 75% (severe heterogeneity). We conducted a sensitivity analysis to investigate the influence of a single study on the overall risk estimates by omitting one study in each turn. To assess the potential publication bias, we performed Begg’s and Egger’s tests, and inspected the funnel plots as well [P value < 0.05 was considered to be statistically significant.We estimated the summary risk estimates of stroke for fatty and lean fish intake compared the highest with the lowest category using a random-effects model . Heterogatistics . For the as well . All staThe systematic literature search yielded 821 potentially relevant records. After the exclusion of clearly irrelevant publications by reading titles and abstracts, we obtained 41 full articles of potentially relevant studies for further evaluation. After full-text reviews, 29 articles were excluded because of lack of subtype of fish intake. Three studies were excluded because of meta-analysis of total fish intake and stroke risk , 7, 24. n = 2), Italy (n = 1), Spain (n = 1) and Sweden (n = 1). Numbers of participants ranged from 2710 to 41,020. The number of stroke events ranged from 66 to 1680. Except Atkinson’s study [Characteristics of the included studies are reported in Table ’s study , other 4’s study , 18, 19.’s study . Fish inP = 0.124), with low heterogeneity across studies  , without heterogeneity across studies   Fig. . The senP = 0.764 for fatty fish, P = 0.462 for lean fish) and Egger’s tests .There was no obvious dissymmetry in the shape of funnel plots for both analyses of fatty and lean fish intake. Publication bias was also not found using Begg’s test , tuna 16.4%) and salmon (14.2) in the UK. Meanwhile, hake/burbot (35.0%), tuna (15.2%) and cod (12.3) were typically consumed in Spain . In addi.4% and sOur findings demonstrated that fatty and lean fish intake had beneficial effects on stroke risk, especially lean fish intake. Considering the limited number of included studies and limitations mentioned above, additional prospective studies with improved methods of estimating the intake of different kinds of fish is warranted to investigate whether fatty and lean fish intake has different effects on stroke risk among the general populations.


Sharp increases in food production worldwide are attributable to agricultural intensification aided by heavy use of agrochemicals. This massive use of pesticides and fertilizers in combination with global climate change has led to collateral damage in freshwater systems, notably an increase in the frequency of harmful cyanobacterial blooms (HCBs). The precise mechanisms and magnitude of effects that pesticides exert on HCBs formation and proliferation have received little research attention and are poorly constrained.We found that azoxystrobin (AZ), a common strobilurin fungicide, can favor cyanobacterial growth through growth inhibition of eukaryotic competitors (Chlorophyta) and possibly by inhibiting cyanobacterial parasites (fungi) as well as pathogenic bacteria and viruses. Meta-transcriptomic analyses identified AZ-responsive genes and biochemical pathways in eukaryotic plankton and bacteria, potentially explaining the microbial effects of AZ.Our study provides novel mechanistic insights into the intertwined effects of a fungicide and eutrophication on microbial planktonic communities and cyanobacterial blooms in a eutrophic freshwater ecosystem. This knowledge may prove useful in mitigating cyanobacteria blooms resulting from agricultural intensification.The online version of this article (10.1186/s40168-019-0744-0) contains supplementary material, which is available to authorized users. In the past century, agricultural intensification supported by the use of agrochemicals increased human food supply to keep pace with a rapidly increasing population on Earth. However, this massive use of pesticides and inorganic fertilizers in combination with global climate change has led to extensive collateral damage in aquatic ecosystems, most notably an increase in the frequency of harmful cyanobacterial blooms (HCBs) , 2. HCBsFungicide residues in aquatic environments, which usually co-exists with human-induced eutrophication, can inhibit the growth of fungi, eukaryotic algae, and some zooplankton species –14. Cyanhttp://cn.agropages.com/, accessed on October 19, 2017), is a broad-spectrum strobilurin fungicide that protects food crops against many pathogenic fungi. The extensive use of AZ has led to contamination of nearby freshwater ecosystems at concentrations reaching 0.01–29.70 μg L−1 in streams, ponds, groundwater, and lakes in Denmark, Germany, France, Brazil, and the USA , where Aexposed and Acontrol are the cell densities at times 2 to 7 days in the AZ-treated and control cultures, respectively.Axenic strains of the green algae ytometer , 21. The−1 AZ (n = 4). In parallel, phycocyanin was extracted in a sodium phosphate buffer and measured by spectrophotometry according to the methodology detailed in Silveira et al. [n = 4).After 72 h, the microcosms’ chlorophyll a (Chl-a) contents were measured as described by Inskeep and Bloom  in microa et al.  (n = 4).n = 4). Five-milliliter microcosm samples were successively passed through a 0.45-μm aperture pinhole filter and a solid-phase extraction apparatus with WondaSep C18 column. The column was eluted with 1 mL of acetonitrile three times. The eluates were collected in a 10-mL centrifuge tube and diluted with acetonitrile. AZ was finally quantified by HPLC using a 250-nm UV detection wavelength at 25 °C with Acetonitrile:Water (68:32) as the mobile phase.Residual AZ concentration was determined in the dissolved phase of the batch cultures or microcosms  by solid-phase extraction-HPLC (high-performance liquid chromatography) using the following methodology  and N and P were added using NH4NO3 and KH2PO4 autoclaved stock solution . Cell number was calculated using a hemocytometer (n = 20).−1 AZ   were harvested after 7 days of culture for the meta-transcriptomic work. Microbial biomass in the six aquatic microcosms was centrifuged at 6500 g for 5 minutes at 4 °C and then the supernatant was processed by gentle filtration on a 0.2-μm filter. The microbial community collected on the filter as well as the centrifugal precipitation were used for RNA extraction. Total RNA was purified using TRIzol Reagent  and the RNeasy Mini Kit . RNA was quantified and characterized using an Agilent 2100 Bioanalyzer , and a NanoDrop (Thermo Fisher Scientific Inc.). Gel electrophoresis  was used as an RNA integrity test. One microgram total RNA with a RIN (RNA integrity number) value above seven was used for library preparation. The ribosomal depleted mRNA was then fragmented, reverse-transcribed and the sequences were processed and analyzed by GENEWIZ . Transcriptome sequencing (RNA-seq) was performed on an Illumina HiSeq 4000 platform. The raw data was quality trimmed, assembled, and annotated as detailed in Additional file Three control microcosms  as well as three other microcosms with 2.5 mg Lp < 0.05.We used FPKM (expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced) to calculate relative transcript level of genes. All experiments were repeated three times independently. Data are presented as means ± standard errors. Significant differences among treatments were tested with one-way ANOVAs (StatView 5.0). The ANOVA assumptions of normality and homogeneity of variance of residuals were validated with the Kolmogorov-Smirnov one-sample test and Levene’s test, respectively (StatView 5.0). Differences were considered statistically significant when M. aeruginosa as well as a common green alga C. pyrenoidosa were used to investigate the toxicity of AZ on green algae and cyanobacteria. Growth of M. aeruginosa was not suppressed by the range of AZ concentrations during the 7-day treatment  and at low initial algal cell density , which are conditions representative of naturally contaminated environments [p < 0.05), while M. aeruginosa growth remained unaffected at the same tested AZ concentrations. Previous laboratory studies also showed that AZ inhibitory toxic effects varied dramatically between green algae and cyanobacteria; for instance, the toxicity of dissolved AZ on the growth of the chlorophyte Pseudokirchneriella subcapitata was nearly 500-fold higher than that reported in the cyanobacterium Anabaena flos-aquae [Model cyanobacterium ent Fig. a, while ays Fig. a. Growthronments , 16, 17,os-aquae , 25.Fig−1 in the control microcosms after 3 days of culture in the medium and a phytoplankton bloom occurred  for 3 days decreased Chl-a concentration, indicating that AZ can have toxic effects on the phytoplankton populations comprising the green algae  (pH 7) between 8.7 and 13.9 days [C. pyrenoidosa than in M. aeruginosa cultures  in C. pyrenoidosa.The rate of azoxystrobin decomposition in the aquatic environment is stimulated by light and AZ has an aqueous photolysis DT3.9 days . InteresMeta-transcriptomic sequencing was carried out to investigate the changes in transcription of the whole plankton community after AZ exposure. A summary of the meta-transcriptomic sequencing results is provided in Additional file Monoraphidium sp. (a Chlorophyta genus) , while the abundance of Bacillariophyta increased by ~ 6-fold  dramatically increased by more than 20-fold, i.e., from 1.7% in the control group to 38.3% in the AZ group. The sequence abundance ratio of eukaryota/prokaryota decreased from 3.1 to 0.9  Fig. . Howeverent Fig. c. The repetitors –29.Fig.Monoraphidium sp. was halved and the RAT of Synechococcus sp. sharply increased after AZ treatment, AZ toxicity bioassays in laboratory batch cultures of Synechococcus sp. and Monoraphidium sp. were carried out to strengthen our hypothesis that AZ addition in the microcosms could preferentially benefit a cyanobacterium (Synechococcus sp.) over a green algal competitor (Monoraphidium sp.). Interestingly, the growth of monocultures of Synechococcus sp. in BG-11 medium as inhibited by ~ 28% after 7 days  to an initial AZ concentration of 2.5 mg L−1, although it was not significantly influenced after 4 days of exposure (p = 0.21), indicating that Synechococcus sp. growth could be inhibited by long-term exposure to high AZ concentrations. In contrast, the growth of Monoraphidium in laboratory monoalgal cultures was always inhibited throughout the entire cultivation process; with a cell yield inhibited by ~ 45% after 7 days of exposure to 2.5 mg L−1 AZ   Fig. b. The abSynechococcus sp. over Monoraphidium sp. in both filtered eutrophic lake water  was enhanced by the AZ treatment, from 1.2 in the control to 2.3, 3.6, or 7.7, for the treatment with 25 μg L−1, 250 μg L−1, or 2.5 mg L−1 AZ, respectively  after exposure to AZ compared to the control  (p < 0.05) in the AZ-treated microcosms. Although total dissolved oxygen concentration (DO) remained super-saturated in the AZ-treated microcosms compared to the control , fungi (belonging to Chytridiomycota), heterotrophic bacteria (Cytophaga and Bdellovibrio), and viruses  significantly decreased (p < 0.05) in AZ-treated microcosms compared to that in the control  are unable to synthesize de novo sterols and have to find them from their food. For instance, chytrid zoospores are an essential food complement for Daphnia when grazing on filamentous cyanobacteria [Daphnia grazing on cyanobacteria decreases, which would in turn contribute to decreased Daphnia growth , including carotenoid biosynthesis (+ 1326% compared with control), photosynthesis-antenna proteins (+ 1305%) and photosynthesis (+ 758%). We also noted that many pathways with a low relative abundance (< 0.05) related to antibiosis, vitamin metabolism, and polysaccharide synthesis were differentially expressed in response to AZ  coding for the enzyme F-ATPase, in the mitochondrial and chloroplastic membranes, were significantly under-expressed in both eukaryota and bacteria (decreased by around 40~80%) (p < 0.05). This decrease in F-ATPase transcription could be explained by the toxic action of AZ, which inhibits electron transfer complex between cytochrome b and cytochrome c1, decreasing mitochondrial respiration and ATP production [apcD, apcE, cpeC, cpeZ) were strongly over-expressed (by around 4–32-fold), while in eukaryota, the transcription of genes coding for proteins of the light-harvesting complex  remained mostly unchanged , which encode an enzyme protecting the cells against environmental stress [Modulation of oxidative phosphorylation and photosynthesis by AZ. The relative expression level of genes related to oxidative phosphorylation in both eukaryota and prokaryota decreased dramatically in the AZ-treated microcosms, and the degree of inhibition in the two kingdoms was equivalent, i.e., ~ 42% compared to control. Genes , but the pathway “other glycan degradation” was over-expressed by ~ 10-fold in the AZ-treated microcosms compared to the control. Assuming that the generalized predicted decrease in polysaccharide synthesis in prokaryote occurred in cyanobacteria, a putative decrease in polysaccharide ballast in cyanobacteria could help maintain cyanobacteria at the lake surface and help exploit light, which is strongly attenuated with depth during the bloom. Several cyanobacteria including those of the genus Synecocchococus have indeed been shown to modulate buoyancy through polysaccharide synthesis [Polysaccharide synthesis and degradation. Transcription of several genes related to polysaccharide biosynthesis in eukaryotes increased  were over-expressed (by 14~243%) in the AZ-treated microcosms compared to the control  were significantly under-expressed  in the AZ-treated microcosms compared to the control  transport  decreased by 88% and 57%, respectively, in the AZ-treated microcosms relative to the control  group at KEGG level 2. Table S1. Chemical composition of the Modified BG11 medium. Table S2. Taxonomic proportions of FPKM normalized transcript counts at the phylum level in the control and AZ-treated microcosms. Table S3. Variation of pathways related to glycan synthesis and degradation. Table S4. Variation of pathways related to vitamin synthesis and consumption. (DOCX 701 kb) (DOCX 672 kb)Additional file 2:Dataset 1. Detailed statistics of clean data, sample transcript and FPKM interval from six microcosms. Dataset 2. Taxonomic proportions in each microcosm. Dataset 3. Annotation and relative abundance of KOs in each microcosm. Dataset 4. Comparison in abundances between control and AZ groups at KEGG level 3. Dataset 5. Comparison in abundances between control and AZ groups at KEGG level 4. (XLSX 1138 kb)


Cannabissativa L., is often hindered by their limited bioavailability and undesirable psychoactivity. We conducted an observational study in December 2016 and another one in February 2018 to investigate respectively: (i) the effectiveness of Trokie® lozenges, a standardized formulation containing cannabis extracts, to deliver cannabinoids via buccal absorption and (ii) its long-term safety. Participants were members of the Palliative Care Corporation health clinic, registered California cannabis patients, and had a diagnosis of chronic non-cancer pain. For the effectiveness study, 49 participants were asked to self-report pain perception before and after 1–12 weeks of taking Trokie® lozenges, using an 11-point pain intensity numeric rating scale (PI-NRS). A mean reduction in PI-NRS score of 4.9 ± 2.0 points was observed. Onset of analgesia typically varied between 5 and 40 min, which seems consistent with, at least partial, buccal absorption. In the safety study, 35 participants were asked to complete a questionnaire about adverse events (AEs) associated with Trokie® lozenges. AEs were reported by 16 subjects (46%), the most common being dizziness/unsteadiness (N = 7), bad taste (N = 5), and throat irritation/dry mouth (N = 4). None of the self-reported AEs resulted in a serious medical situation and most of them had limited impact on daily functions. Despite the AEs, 90% of participants reported being “satisfied” or “very satisfied” with the product. These observations suggest that buccal administration of standardized extracts via Trokie® lozenges may represent an efficacious and safe approach to cannabis administration.Therapeutic use of cannabinoids, the main active ingredients of  Cannabis sativa L., poses to their absorption and distribution in the human body (9-tetrahydrocannabinol (THC), the primary active ingredient present in cannabis, need to be: (i) minimized for patients to maximize the therapeutic index of cannabis medications, and (ii) carefully controlled for adult consumers in order to reduce both the risk of acute intoxication and the impact on public health and safety. Economic reasons also play a role since increasing the bioavailability of cannabinoids could allow for a reduction in the amount of active ingredient per dose, thus lowering the cost of the final product. Current efforts to achieve efficient, reliable dosing aim at exploring different routes of administration  and delivery vectors  to optimize the pharmacokinetic profile of cannabinoids , an ethanol-based oromucosal preparation with 2.7 mg of THC and 2.5 mg cannabidiol (CBD) per spray, was the first cannabis product to attain regulatory approval as a pharmaceutical in 29 countries, having met the required standards of safety, efficacy and consistency (® lozenges) to deliver cannabinoids via the buccal/oral mucosa as well as associated AEs.Sativexsistency . Neverthsistency . An altesistency . Here, w® lozenges. Admission criteria for the health clinic include: (i) willingness to incorporate cannabis into current medication regime; (ii) not having known allergies to cannabinoid drugs ; (iii) not having a cardiac arrhythmia; and (iv) not being pregnant. At the time of enrollment, members were asked to rate their pain on a pain intensity numeric rating scale (PI-NRS). This study was carried out in accordance with the principles of the Declaration of Helsinki and followed the relevant institutional and national guidelines. The study met the criteria set forth in 45 CFR §46.101(b) (4) and was therefore exempted from institutional review board (IRB) approval by Quorum Review IRB .We conducted two observational studies with members of the Palliative Care Corporation (PCC), a health clinic in Huntington Beach, CA, which distributes Trokie® lozenges, embracing the “start low, go slow” motto proposed by clinicians for the use of herbal cannabis , were instructed on how to properly self-administer Trokiecannabis , startincannabis .® lozenges were prepared as described in U.S. Patent No. 62/018,484 (The following 4-dose lozenges were available to members at PCC: 50 mg (only CBD), 40 mg (1:1 ratio CBD:THC), 64 mg (1:15 ratio CBD:THC), 120 mg (1:15 ratio CBD:THC), and 120 mg (only THC). Trokie/018,484 . After p® lozenges. Participants using opiate medication were questioned about any modifications on their intake from the time of enrollment.On December 2016, members of the PCC with a diagnosis of chronic non-cancer pain who had been enrolled for at least 12 weeks were invited to participate in a phone interview conducted by KC. The time frame (1–12 weeks) was selected to maximize the number of members actively enrolled in the clinic at the time of the study. Participants were asked: (i) to score their current pain on a PI-NRS from 0 to 10 , (ii) an® lozenges, we conducted a survey on February 2018 among PCC members with chronic non-cancer pain that had been enrolled for a sustained period of time, between 4 and 60 weeks. This time frame was selected to capture AEs associated to mid- to long-term use of Trokie® lozenges. Participants were asked to complete a questionnaire to report adverse drug reactions . Differences were considered significant if P < 0.05. Statistical analyses were conducted using GraphPad Prism Version 7.0 . Patient-reported AEs were analyzed descriptively using IBM SPSS Statistics version 23 .Pain intensity was calculated as the difference between PI-NRS values reported on December 2016 and those reported at enrollment, and it is expressed as mean ± standard deviation of the mean. Significance of differences was determined using a paired Supplementary Table 1). An average reduction in PI-NRS score of 4.9 ± 2.0 points (from 7.4 ± 1.3 to 2.4 ± 1.8) was observed . Also, among 31 participants using opiates, 26 (84%) voluntarily reduced or discontinued their use of opiate medication . These reductions were completed by participants with no reported symptoms of opiate withdrawal. Lastly, all participants reported feeling an improvement in their condition (100%) and an onset of analgesia between 5 and 40 min.A total of 49 participants  with an average age of 59.9 years completed the study (descriptive statistics in Supplementary Table 1). Twenty AEs were reported by 16 participants (46%), with most reporting one single AE  and four reporting two AEs. Descriptions given by participants corresponded to 7 AEs (with some of them relating to more than one AE) which were: dizziness/unsteadiness (N = 7), bad taste (5), throat irritation/dry mouth (N = 4), drowsiness/fatigue (N = 3), impaired consciousness/high feeling (N = 2), nausea (N = 1), and palpitations (N = 1). There were no reports of damage to the oral mucosa, gums, or teeth. None of the self-reported AEs resulted in a serious medical situation and most of them had only limited impact on daily functioning. Of notice, AEs associated with CBD-only Trokie® lozenges were not related to any psychoactivity and were restricted to bad taste (4) and throat irritation/dry mouth (2). The remainder of AEs were associated with THC-containing lozenges, alone or in combination with opiates or other THC-containing products. Despite these AEs, 90% of participants reported being “satisfied” or “very satisfied” with the product (Table 1).The adverse drug reaction questionnaire was completed by a total of 35 participants  with an average age of 65.3 years  and Puerto Rico. Two observational studies were conducted with California-certified cannabis patients to assess the effectiveness and safety of Trokie® lozenges. First, we focused on the effectiveness of Trokie®lozenges to deliver cannabinoids, namely THC and CBD, through the buccal mucosa: does it work in practice?  and the rest being common to other cannabis products containing THC, such as dizziness or dry mouth, none of which resulted in a serious medical situation and only had limited impact on daily functioning. Interestingly, our results are in strong agreement with a recent study performed in Israel on a large cohort of elderly  reporting a reduction of pain levels from a median of 8 to a median of 4 on a scale of 0–10 after 6 months of cannabis treatment. Further, most common AEs were dizziness (10%), dry mouth (7%), and 18% of participants stopped using opiate analgesics or reduced their dose (Trokieractice? . Our finescribed . Of noteescribed . Howevereir dose . In our eir dose . This ma® lozenges and the improvement in participants well-being. However, the findings support the need for conducting a phase 1 clinical trial to formally characterize the pharmacokinetic profile of Trokie® lozenges in humans.Legalization of medical and recreational cannabis has bypassed the usual drug regulatory procedures in jurisdictions worldwide. Pending sound evidence for its effects in many conditions, physicians face the challenge of continuing to provide competent, compassionate care with an emphasis in harm reduction. Nevertheless, this regulatory scenario creates unprecedented opportunities to study the clinical impact of cannabinoids in human health and behavior. An illustrative example of how commodities can revolutionize the way we perform biomedical research are consumer physical activity monitors, which have raised enormous interest to physiology and psychopathology research because of their ability to measure activity continuously under real-life conditions and because they are already widely used by consumers . We beliKC and GM-S designed the studies. KC coordinated the field research and conducted interviews. GM-S and SdV analyzed the data and generated results of the effectiveness and safety studies, respectively. GM-S wrote the manuscript with the aid of KC and SdV.® lozenges. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MDP and handling Editor declared their shared affiliation.KC is an inventor on U.S. Patent No. 62/018,484, describing buccal and sublingual cannabinoid formulations and method of making the same, which are the basis for the preparation of Trokie


In general population, resting heart rate (RHR) is associated with cardiovascular disease. However, its relation to chronic kidney disease (CKD) is debated. We therefore investigated the relationship between RHR and urinary albumin/creatinine ratio  in general population at different levels of blood pressure and blood glucose.  We screened out 32,885 subjects from the REACTION study after excluding the subjects with primary kidney disease, heart disease, tumor history, related drug application, and important data loss. The whole group was divided into four groups  according to the quartile of average resting heart rate. The renal function was evaluated by UACR . Ordinary logistic regression was carried out to explore the association between RHR and UACR at diverse blood pressure and blood glucose levels.  The subjects with higher RHR quartile tend to have a higher UACR, even multifactors were adjusted. After stratifying the subjects according to blood pressure and blood glucose, the positive relationship between RHR and UACR remained in the subjects with normal blood pressure and normal glucose tolerance, while in the hypertension (SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg) group and the diabetic mellitus (FPG ≥ 7.0 mmol/L and/or PPG ≥ 11.1 mmol/L) group, the relationship disappeared. In the subjects without hypertension, compared with the Q1 group, the UACR is significant higher in the Q3 group (OR: 1.11) and the Q4 group (OR: 1.22). In the subjects with normal glucose tolerance (NGT), compared with the Q1 group, the UACR is significantly higher in the Q3 group (OR: 1.13) and the Q4 group (OR: 1.19).  The population with higher RHR tend to have a higher UACR in the normal blood pressure group and the normal glucose tolerance group. Resting heart rate (RHR) is a well-recognized marker of autonomic nervous system tone and has been found to be a significant correlate of blood pressure, increased body mass index, and metabolic disturbances in many studies; this association is particularly striking in patients with hypertension or diabetes . ElevateThe kidney is one of the important target organs of metabolic diseases. Chronic kidney disease (CKD), primarily caused by hypertension and diabetes , which cA raised RHR has been found to be an independent predictor for the prevalence of microalbuminuria in hypertensive patients with high cardiovascular risk . And peoAn early subanalysis of the randomized olmesartan and diabetes microalbuminuria prevention (ROADMAP) study on patients with type 2 diabetes has shown that a lower RHR was associated with a reduced risk of developing microalbuminuria, indicating that a lower RHR is an independent predictor for a lower risk of new-onset microalbuminuria in type 2 diabetes . Our stuA total of 53,639 participants from the Risk Evaluation of cAncers in Chinese diabeTic Individuals a lONgitudinal (REACTION) study gave written informed consent, and the protocols were reviewed and approved by the research ethics committee at each of the participating centers . After excluding the subjects with primary kidney disease, heart disease, tumor history, related drug application, and important data loss, 32,885 participants were investigated.2.The baseline data were collected by trained health workers via a standardized questionnaire during interviews, including the general situation, the past medical history, the current medication situation, lifestyle, physical exercise, smoking and drinking habits, family history, and other basic information. All involved investigators have been formally trained. Participants were asked to take off shoes, hats, and coats before measurements. Waist circumference (WC) was measured at the horizontal level of the midpoint of the ligature between anterior superior spine and inferior margin of the twelfth rib. Hip circumference (HC) was defined as the horizontal of the most protruding part of the hip. Body mass index (BMI) = weight (kg)/[height(m)]An automated electronic device  was used to measure blood pressure and resting heart rate in the nondominant arm of seated participants three times consecutively at 1-minute intervals after a ≥5-minute rest. The three readings were averaged for analysis. The subjects were stratified based on RHR quartiles .Urine samples were collected in the morning (midclean urine) for UACR measurements. Because the kits for measuring UACR in each center and the range of normal values are different, the value of UACR, divided by quartiles of all data in the center which the subject belonged to, was used to estimate albuminuria.Blood samples were drawn in the morning after subjects had an 8-hour fasting the previous night. Participants without a history of diabetes underwent a 75 g oral glucose tolerance test, and their venous blood samples were drawn at 0 and 120 minutes. Biochemical index included triglyceride (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), creatinine (Cr), alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transaminase (GGT), fasting plasma glucose (FPG), postprandial plasma glucose (PPG), glycosylated hemoglobin (HbA1c), and fasting blood insulin was measured by the glucose oxidase-peroxidase method.2 by the formula eGFR = 186 × [serum creatinine × 0.011]‐1.154 × [age]‐0.203 × [0.742 if female] × 1.233, where serum creatinine was expressed as μmol/L and 1.233 was the adjusting coefficient for the Chinese population. This formula is according to the abbreviated Modification of Diet in Renal Disease (MDRD), which was recalibrated for Chinese population.The estimated glomerular filtration rate (eGFR) was expressed in mL/min per 1.73 mNormal glucose tolerance (NGT) was defined as FPG < 6.1 and PPG < 7.8; impaired glucose regulation (IGR) was defined as 6.1 ≤ FPG < 7.0 and/or 7.8 ≤ PPG < 11.1; diabetes mellitus (DM) was defined as FPG ≥ 7.8 and/or PPG ≥ 11.1. Normal blood pressure (NBP) was defined as 90 ≤ systolic blood pressure (SBP) < 140 and 60 ≤ diastolic blood pressure (DBP) < 90; hypertension was defined as SBP ≥ 140 and/or DBP ≥ 90.U test and the Kruskal-Wallis test were used, respectively, to compare the means of 2 and 3 or more groups of variables not normally distributed. The frequencies of categorical variables were compared using Pearson Chi-square or Fisher's exact test, when appropriate. A value of P < 0.05 was considered significant. Ordinary logistic regression was performed to explore the association between RHR and UACR.The data was analyzed by SPSS version 20.0 for Windows . The normality of distribution of continuous variables was tested by the one-sample Kolmogorov-Smirnov test. Continuous variables with normal distribution were presented as mean ± standard deviation (SD). The Mann-Whitney P < 0.001). The means of age, HC, SBP, Cr, and eGFR have a significant difference between RHR quartiles. The percentile of UACR > 75% is higher in the higher RHR group as A total of 32,885 participants, including 10,058 males and 22,827 females were accepted into the study. The general clinical characteristics of the study population are presented in P < 0.05). Ordinal logistic regression was performed to explore the association between UACR and RHR. P < 0.05). After adjusted for gender, age, BMI, WC, smoking, and alcohol drinking, the risk of having UACR increased progressively across the lowest to the highest quartiles of RHR with ORs of 1.07 (95% CI 1.01-1.13), 1.18 (95% CI 1.12-1.25), and 1.33 (95% CI 1.26-1.41), respectively . When adjusted for all factors, the third quartile  and the fourth quartile  still tend to have a higher UACR.Multiple factor analysis was carried out to screen out the risk factors related to UACR. The association between UACR and RHR at diverse blood pressure and blood glucose was analyzed by ordinal logistic regression. As Chronic kidney disease has insidious onset and slow progression and is easy to be ignored. UACR is a sensitive indicator reflecting early renal function injury. Resting heart rate is a reliable indicator of sympathetic nervous system excitation, and epidemiological studies , 20 showA previous study in Germany that included 4447 patients with type 2 diabetes showed that as the heart rate increases, the risk of microalbuminuria appearing increased ; this poThere was also a study extending the previous findings to a population with cardiovascular disease, showing that heart rate is associated with a renal disease risk and suggesting that a reduction in heart rate might be protective for kidney function . In thisOur study shows that hypertension (OR: 1.66) and diabetes (OR: 1.52) are more significant than RHR  related to UACR. In the general population, the positive relationship between RHR and UACR exists, while in the new diagnostic hypertension and diabetes group, it disappeared. In combination with previous results, we may guess that after effective hypoglycemic and antihypertensive therapy, the relationship between RHR and UACR will reoccur, but it remains further clinical trials. Anyhow, RHR may be a marker of UACR, which reflects early renal impairment.Several mechanisms may underlie these effects such as progression of atherosclerosis, and thus nephrosclerosis, due to changes in endothelial oxidative stress, which is sensitive to RHR reduction , 22. FurParticipants in our study belonged to 8 communities of mainland China, guaranteeing representative results. Compared to other studies, our participants included diverse blood pressure and blood glucose (after excluding the effects of related drugs). Therefore, we fully adjusted confounding such as gender, age, BMI, WC, liver function, and serum lipid. However, several deficiencies exist in this study. First, protein content intake in the previous day and the interval between last meal and sleep were not available; second, UACR levels were determined by a single measurement, but detection methods of the 8 centers were different; third, this is a cross-sectional study; we cannot get a cause-and-effect relationship. Its specific mechanism needs to be explored and confirmed by further basic experiments, but the conclusion still has a certain clinical sense in some aspect.Our study reported the association between RHR and UACR in general population. For the population with nonhypertension and normal glucose tolerance, higher RHR is associated with higher UACR. As for the population with hypertension and diabetes, the positive relationship disappeared. After adjusting blood pressure and blood glucose, people with higher RHR tend to have a higher UACR. That indicates RHR may represent an independent risk factor for chronic kidney disease.


Drowning is the third cause of non-intentional injury death worldwide. Beaches of Gironde, in southwestern France, are exposed to strong environmental conditions, leading to rip currents and shore breaks. Bathing season usually lasts from April to October and is supervised from June till mid-September. The objective of this study was to study the characteristics of drowning victims along Gironde surf beaches and to identify peculiarities compared to national figures.All calls originating from beaches to the emergency call center of Gironde from 2011 to 2016 were analyzed. Patient data, filled by a physician based on information given by pre-hospital care team , were extracted from the emergency call center database. We used Szpilman classification (0 = rescue to 6 = cardiac arrest) to assess severity. Rescues are patients without respiratory impairment who needed lifeguards or helicopter intervention. We compared our findings with national studies carried every three years (2012 and 2015).p < 0.001).We analyzed 5680 calls from beaches and included 4398, 576 of which were rescued from the water, including 352 without respiratory impairment (stage 0). Among drownings, 155 had cough only (stage 1), 26 pulmonary rales (stage 2), 9 pulmonary edema (stage 3) and 1 had pulmonary edema with hypotension (stage 4). Five rescued people were in respiratory arrest and 28 were in cardiac arrest. 77.5% were bathers, others were mainly surfers or body-boarders. Drowning victims median age was 24 (quartiles: 17–40), and sex-ratio was 1.44 Male/Female. Men were significantly older than women (34 vs. 26 years old), and severity from stage 1 to 4 was positively associated with age. Compared to national data, Gironde drownings had a higher proportion of 15–44 year-old victims, and the case-fatality was lower in Gironde (11.5%) than at the national level (27.4%, Along Gironde coast, drowning is rarely severe, concerns mostly young men; the age distribution could explain the different case-fatality. Further study is needed to identify environmental predictors of drowning. Drowning is defined in 2005 by the World Health Organization (WHO) as “the process of experiencing respiratory impairment from submersion/immersion in liquid” , registering all cases of drowning leading to hospitalization or death, from June, 1st till September, 30th. During 2015, this study reported 1266 drownings  in France, including 637 (50.3%) on coasts . For each call, a physician fills the database with information given by callers, and paramedics and pre-hospital care teams when involved. We excluded calls without victim, trainings and duplicates. Contents of all calls were reviewed by the first author, a junior epidemiologist with training in emergency medicine. We then classified all calls in two groups, drowning cases and other calls.We collected location, date and time of the call, gender and age of the victim for all medical emergency calls from beaches. For drownings, we collected the medical history of the victim, activity , initial status given by lifeguards report, and discharge status . The severity of drowning was rated, from the first clinical description reported, with the Szpilman classification Table . Stage zWe assumed that stage-0 and -1 victims without hospital transfer had a favorable outcome. Outcomes of the most severe victims (stage 2 and over) were collected from the files of Intensive Care Units were patients were admitted. National data, collected every three years, were extracted reports of the national institute of public health, Santé Publique France, carried in 2012 and 2015  and 1 (n = 6) patients. Missing data concerned helicopter interventions for air-lifting operations where, usually, only the number of involved patients, their severity, and sometimes sex are filled. Median age was 24 years  among people rescued from water and 23 years among other calls . Compared to other beach-related calls, the sex-ratio was not different (p = 0.88). Males rescued from water were older than for other beach-related injuries (p < 0.001), but female victims were similar in age (p = 0.06).We screened 5680 call files, of which 1282 were excluded  were mostly secondary to medical cause, beach-related trauma, but there were specific surf injuries such as shore-break related injuries (17.7%) and envenomation (5%) (Table p = 0.14). Males were significantly older than females . Age varied across stages, independently of sex (p = 0.001) .Sex was available for 427 cases (74.1%) and most victims were stages 0 (61.1%) and 1 (26.9%) (Table n = 17), asthma (n = 13), and psychiatric disorders (n = 5); one patient had a history of epilepsy. The absence of medical history was documented in 112 cases (19%). Therefore, 415 patients (72.0%) were without reporting medical history. Alcohol consumption was notified in 2 cases. No drowning occurred within bathing area during the study period.A medical history was explicitly noted for 49 cases (8.5%), reporting mainly cardio-vascular diseases . Areas G and H had a significant higher incidence than other locations . This study is the first step to prevention, analyzing existing data in a specific context , compared to other studies  neither by the amount of lifeguards (comparable to area H). As rip currents are driven by wave and tide conditions (Castelle et al., This observational study in an area with specific hazards identified the demographics of drowning victims and led to the development of a specific questionnaire to improve reporting. This study revealed notable differences with national data and suggests that drownings along coastline must be studied at a local scale. The studied area is at high-risk of rip-related drowning and there is a need to identify days at risk using meteorological, wave and tide parameters. Such a study could lead to a prevention tool for lifeguards and emergency managers.