Objective: This study aims to characterize the abnormal changes in placental DNA methylation associated with conotruncal heart defects (CTDs) and the level of methylation as epigenetic biomarkers for CTDs detection.Methods: This was a prospective study involving 28 fetuses diagnosed with CTDs in the second trimester at Beijing Anzhen Hospital between September 2020 and June 2021. These cases were classified into four groups based on their subtypes. 12 normal fetuses were used as controls. Placental tissue was obtained after inducing labor in fetuses. To identify differential methylation sites (DMSs) and regions (DMRs) in cases vs. controls, an Infinium Human Methylation 850 k bead chip was used. Differential methylation was assessed by comparing the β-values for individual CpG loci. Based on the p-value (<0.05), the most discriminating CpG sites were identified. The area under the receiver-operating-characteristics curve (AUC) was used to determine the predictive accuracy of CpG loci with significant methylation changes for CTDs. The function of genes was assessed through KEGG enrichment analysis, Gene Ontology (GO) analysis, and KEGG pathway analysis.Results: In comparison to the control group, the DNA methylation of the placental tissue is significantly different in fetuses with CTDs. We identified the most significantly different methylated loci and they demonstrated excellent individual predictive accuracy for CTDs detection with AUC >0.9 in cases compared with controls. HOXD9, CNN1, NOTCH1, and ECE1 were identified as CTDs-detection candidate genes.Conclusion Our study established the abnormal changes in placental methylation associated with CTDs and potential epigenetic biomarkers for CTDs detection. Conotruncal heart defects (CTDs) are a diverse subtype of congenital heart diseases (CHDs) that account for approximately 25–30% of all non-syndromic CHDs. Although fetuses with CTDs typically require catheter-based or surgical treatment in the early postnatal period, the mortality rate remains high. . The diaGATA4, GATA6, TBX18, TBX20, NKX2-5, and SAMDS, as well as unique copy mutations and chromosomal aneuploidy such as trisomy 13, 18, or 21, which account for 20–30% of CHDs. . Fetuses were classified according to the subtypes of CTDs which included TOF, double outlet right ventricle (DORV), transposition of the great artery (TGA), and pulmonary atresia with ventricular septum defect (PAVSD). All pregnant women consented to participate and signed an informed consent form. After inducing labor in fetuses, placental tissue was obtained. To ensure consistency in sampling, the placental tissue with the umbilical cord insertion part was chosen. Until DNA purification, the separated tissue blocks were stored in a refrigerator at minus 80°C. To avoid the effects of genetic malformations, Gene whole exon sequencing (GWES) analysis and copy number variation (CNV) detection were applied in all fetuses. We excluded fetuses with known or suspected genetic syndromes. As controls, 12 labor-induced fetuses with extracardiac anatomical abnormalities but no syndromic defects were used. Other obstetric complications were not noted.α, where α = 100). Between the two groups, the average β-values of the promoters were compared.QIAamp Mini Kit was used for the purification of DNA from placental tissues according to the manufacturer’s protocol. Genome-wide DNA methylation was determined using the Illumina Infinium Human Methylation 850K BeadChip , which provides genome-wide coverage with >850000 CpG methylation sites per sample according to the manufacturer’s instructions. It is capable of producing high-quality data for DNA extracted from the samples. The genomic DNA from both the cases and the controls was subjected to bisulfite-conversion according to the manufacturers protocol using the Zymo EZ DNA Methylation kit . The fluorescence signals from the BeadArrays were measured using an Illumina ‘iScan’ and then analyzed using the Genome Studio software . The software assigned β-values to CpG probes based on the ratio of the methylated alleles (C) fluorescence signal to the sum of the methylated (C) and unmethylated (T) alleles’ fluorescence signals. Before performing detailed bioinformatics and statistical analysis, data preprocessing and quality control were performed, including examination of the background signal intensity in the affected cases and negative controls, the methylated and unmethylated signals, and the ratio of methylated and unmethylated signal intensities. The processing was carried out entirely according to the manufacturer’s protocol, and 99% of the CpG loci were unequivocally determined. The methylation status of all probes was denoted by the β-value, which is the ratio of the methylated probe intensity to the total probe intensity  was used to correct probe type bias to obtain the methylation level  that could be used for different analyses.We determined differential methylation by comparing the β-value of individual CpG loci in two groups. Based on the p-value (<0.05), the most discriminating CpG sites were identified. The R-package Limma was used to analyze the differential methylation sites, and the moderated T-statistics and empirical Bayes methods were used to test the significance of the differential sites in Limma. Bumphunters method was used to find differential methylation regions. The method first clustered according to site distance information, and then selected continuous candidate regions with consistent methylation levels in each cluster. Bumphunter constructed a linear statistical model according to candidate regions and filtered the regions with significant differences (p < 0.05). The methylation level, or β-value, of each CpG locus (one per gene), was used to calculate its predictive accuracy for CTDs and its subtype’s detection on an individual basis. The area under the receiver-operating characteristics curve was used to determine the predictive accuracy (AUC). Continuous variables are expressed as the mean ± standard deviation values. Normally distributed data were analyzed by using student’s t-test. A p value < 0.05 was considered statistically significant. The clinical data were statistically analyzed using the SPSS 22.0 software .KEGG Orthology Based Annotation System (KOBAS) software was used to analyze pathways, diseases, and functions at the same time. The whole analysis process consists of two steps. The first step is to map the input gene sets to the database genes and then annotate the pathways, diseases, and functions in which these gene sets are involved. In the second step, the results obtained in the previous step are compared with the background , and the statistically significant enriched pathways, diseases, or functions are mined. Statistical tests can be performed using the binomial test, chi-square test, Fisher’s exact test, or hypergeometric distribution test. Multi-hypothesis testing methods were used to adjust the p value. Genes associated with DMSs and DMRs were enriched in the Gene Ontology. Additionally, we identified overrepresented canonical pathways, biological processes, and molecular processes. We performed literature research to determine the known or plausible roles of differentially methylated genes in cardiac development.In comparison to the controls, we identified 670 DMSs corresponding to 401 genes in CTDs. 444 (66.3%) of all DMSs were hypomethylated, while 226 (33.7%) were hypermethylated. The heat map in NR2E1 and ECE1 were identified in more than two groups of gene-related sites.Furthermore, when comparing CTDs and controls, we identified 29 DMRs. Three DMRs were hypermethylated and 26 were hypomethylated. The majority of the DMRs were located on the CpG island. In comparison to the controls, we identified 20, 6, 27, and 17 DMRs in TOF, DORV, TGA, and PAVSD, respectively. We discovered that the majority of DMRs in these cases were hypermethylated. PPAR signaling, gap junctions, salivary secretion, circadian entrainment, and endocrine resistance. CNN1 was found to be involved in smooth muscle contraction; NOTCH1 was found to be involved in the NOTCH signaling pathway.Between CTDs and controls, disease ontology analysis, go enrichment analysis, and pathway analysis of the DMRs were performed. Even with today’s highly developed diagnostic methods, early detection and classification of CHD remain a significant clinical challenge. Epigenetic biomarkers of CHD are of great clinical interest and the predictive value of DNA methylation changes in CHD has been demonstrated in numerous studies. Since the placenta and the heart develop in the same way, the placenta may be an ideal tissue for both understanding the mechanism of CHD and developing biomarkers for it. Several studies  have demHOXD9 and CNN1. To gain a better understanding of the function of HOXD9 and CNN1, we investigated the human protein atlas  was achieved by in atlas  databasearcinoma  and artearcinoma . Howevermyopathy , placentmyopathy . Furtheret al. reported that methylation changes in ARHGAP22, CDK5, TRIM27, and IER3 in the placenta are excellent individual predictive markers of TOF, a finding that was not replicated in our study. Different gestation weeks and tissue preservation in the cases may be the primary reasons. PCDHB15, SSTR2, and PRR5L were identified in TOF in our study. PCDHB15 has a low tissue specificity and its specific functions are unknown, but it is highly likely to involve in the establishment and function of specific cellcell neural connections  mRNA is found in the placenta, a recent study  1500 and ECE1 located within the gene body were found in more than two groups. NR2E1 mRNA has been detected in a variety of organs, including the placenta, but its precise function is unknown. Endothelin-converting enzyme-1 (ECE1) is required for the development of a subset of neural crest lineages, including cardiogenesis, and a case-control study . In our study, we also discovered differential methylation of ECE1 in TOF. As a result, additional research is required to elucidate the mechanisms. Consequently, the subtypes of CTDs may share some common differential methylation genes. However, distinct subgroups may have distinct differential methylation genes that influenced the development of the heart. Due to the interaction of all of these genes, distinct subtypes of CTDs exist.In comparison to the controls, our study identified 29, 20, 6, 27, and 17 DMRs in CTDs, TOF, DORV, TGA, and PAVSD, respectively. The majority of them had been hypermethylated. Among the genes with differential methylation regions , NR2E1 lol study  suggesteOur study had some limitations. To begin, only a few cases were included, especially in the subtype group. Additional research is required in a large number of cases. Second, one should consider the effect of gestational age on methylation. Additionally, we did not examine the levels of gene expression (mRNA or protein), which is the ultimate determinant of the biological consequence of DNA methylation. Moreover, our specimens were all derived from fetuses undergoing labor induction, so we could not observe their efficacy in predicting pregnancy outcome or even post-birth prognosis. These are the things we need to improve next.In conclusion, our study established that abnormal changes in placental methylation are associated with CTDs and may be predictive of CTDs. Numerous altered genes are already known or suspected to play a role in cardiovascular or placental development, additional research is planned to corroborate our findings.


Cardiovascular disease has become a growing global and public health concern among non-communicable diseases (NCDs). The purpose of the study was to focus on the increasing prevalence of the risk factors of cardiovascular diseases (CVD), irrespective of age and gender, and its effect on public health worldwide. A literature search was done in the electronic database: Medline, PubMed, Web of Science, Google Scholar, and the World Health Organization (WHO) website, based on recent research and the prevalence of the risk factors of cardiovascular diseases. Moreover, a manual search for published work has also been done. The coronary heart disease studies were not restricted during the search by sample size because of the limited number of studies in selected countries. The study reviews the potential risk factors responsible for coronary heart disease globally. Smoking was highly prevalent among the United States and Pakistani populations, but hypertension and diabetes were more common in Tanzania and the United Kingdom. However, dyslipidaemia and obesity were common in almost all the selected countries. CVD risk factors are highly prevalent in some countries, varying socioeconomic, gender, and educational levels. Furthermore, there has always been a need for awareness in the public and educational programs for a healthy lifestyle, intake of nutritional food, and increased physical activity to improve health conditions and reduce the risk of cardiovascular diseases. Atherosclerosis is the process of formation of plaque composed of cholesterol, fat, calcium, and other substances in the wall of large and medium-sized arteries causing diminished blood flow to an area of the body ,2. MoreoCoronary artery disease, also known as ischemic heart disease, occurs due to the formation of plaque, which is caused by the accumulation of cholesterol particles, and if the process continues, it may eventually reduce and block blood flow and decrease oxygen supply to the heart muscle -5. MoreoAtherosclerotic heart disease can be managed through medicines, surgical treatments, and lifestyle modifications, such as eating healthy, physical exercise, maintaining body weight, avoiding smoking and less salt intake -12. The This systematic review focuses on the prevalence of the potential risk factors of cardiovascular diseases, irrespective of age and gender, and its impacts on global public health. Furthermore, the study aims to draw attention to the need for health practitioners to ensure early interventions to prevent cardiovascular disease and its complications.Methods and materialSearch Strategy A literature search was conducted in the electronic database Medline, PubMed, Web of Science, Google Scholar, and the World Health Organization (WHO) website on all recent research work in the last five years (2015-2019) based on risk factors of CVD patients from a global perspective. Countries were selected from the European Union, Africa, Asia, and America to compare the global view of increasing risk factors of cardiovascular disease and gender differences. A strong association was found in those areas for cardiovascular disease burden among the non-communicable diseases (NCD) that significantly focus on global health strategy -18. The Study Eligibility CriteriaThe review included published papers covering both a healthy subject and a population with existing cardiovascular disease with different atherosclerosis risk factors during the literature search. All the abstracts were reviewed, and the selection of articles was made by the following eligibility criteria: This systematic review included cross-sectional and prospective cohort studies that evaluated major risk factors and family history associated with cardiovascular disease. The selected language was English for all the research articles, and each gender was given priority. Duplicate articles and papers with incomplete information, like conference proceedings, were excluded from the study.Data ExtractionThe initial search led to 506 publications from the existing published literature according to the eligibility criteria. After careful evaluation of the unique titles, abstracts, and availability of entire texts a total of 10 studies  statement and the Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guidelines for our search strategy. The medical subject headings (MeSH) terms were utilised to find the preceding search terms in PubMed and Embase databases. The literature search covers additional references from the thesis or dissertation repositories, preprint servers, and manual searching of the reference lists from the preferred articles. Furthermore, the reasons for exclusion were duplication, irrelevant and review articles, articles with inadequate information, and reports we could not retrieve the full text. The cardiovascular disease studies were not restricted during the search by sample size because of the limited number of studies in selected countries. A PRISMA diagram explained the study sampling Figure .The extracted data from each database covered details of the published paper-like year of publication, type of the study, number of cohorts, sociodemographic variables, risk factors, and a global view of heart disease with the relation of different ethnic groups in various countries. Table ResultsA nationwide survey of the rural population in Bangladesh evaluated subjects aged 20 to 69 and found a higher prevalence of CVD and coexistence of multiple risk factors than the Japanese and Chinese populations ,20. In PAccording to the researchers, potential risk factors for coronary artery diseases are hypertension, obesity, and physical inactivity -25. A stSmoking: The prevalence of smoking has been addressed in nine of the studies, and the rates of smoking were 45.9% , 90% 2424, 6.5% Diabetes: This study addressed the rates of diabetes in the ten existing studies, and the overall prevalence was 5-27% among different nations. Recent studies observed a higher prevalence of diabetes mellitus in Pakistan (18%) , India  than males (10.9%) [2, and based on frailty scoring high BMI, British men were subgroups into frail (24%), pre-frail (21%), not frail (16%) [Obesity: In Bangladesh, the mean BMI was 19.4 kg/m obesity ,25. Also obesity . In Indithan men . Nonethethan men . In Nige (10.9%) . In Roma (10.9%) . In Greay higher .2% than Family history: Albeit positive family history is a known risk factor for CVD, recent studies from Tanzania, Pakistan, and Bangladesh observed that ~53%, ~43%, and ~4% population had a positive family history of coronary heart disease ,21,23.DiscussionGlobal Health Impact2, overweight as BMI between 25 and 29.9 kg/m2, and obesity as BMI ≥30 kg/m2 [The World Health Organization (WHO) has defined average weight as a body mass index (BMI) between 18.5 and 24.9 kg/m30 kg/m2 ,32. More30 kg/m2 . In the 30 kg/m2 . Over th30 kg/m2 . A recen30 kg/m2 -22,30-35Future Directions in Measuring the Global Cardiovascular Disease BurdenIn a recent study, authors observed more influence of behavioural factors than preventive medicine in managing cardiovascular diseases -15. AccoRecent works show that health policymakers should not be waiting for a perfect epidemiological study that may guide them to certain decisions anymore -32. The Strength and limitationsThis study is based on a global population covering different ethnic groups of the population, which compares all age groups and gender. Although there has been some lack of presenting data at some national levels, there was an identical pattern of risk factors for CVD in most countries, especially the region that shared the same culture, environment, cuisine, and lifestyle. Moreover, we only included English-language papers, which may preclude important study findings published in other languages. Albeit, this systematic review was based on globally acceptable major cardiovascular risk factors, the availability of the papers was limited to some parts of Asia, Europe, Africa, and the United States. Moreover, we could not perform the sensitivity analyses due to insufficient data, and primary authors have not been contacted, which may give some unadjusted results to the review.The potential risk factors for CVDs are highly prevalent in different geographical regions, varying with the nation's socioeconomic, gender and educational levels. Furthermore, atherosclerosis and hypertensive heart disease are significant global health problems, and this review recommends a combined and intensive multi-disciplinary team approach at the population and individual level to diminish the burden of CVDs in all regions. Thus, this review could help to improvise practice and policy and provide the newest insight from a large group of data syntheses to reduce the global burden of cardiovascular disease morbidity and mortality.


The model accurately classified 89.9% of cases. For the lethal outcome, only two independent predictors contributed to the regression model: LUS score and patient’s age . The predictive model identified four key parameters at patient admission which could predict an adverse outcome.COVID-19 prediction models mostly consist of combined clinical features, laboratory parameters, and, less often, chest X-ray (CXR) findings. Our main goal was to propose a prediction model involving imaging methods, specifically ultrasound. This was a single-center, retrospective cohort observational study of patients admitted to the University Hospital Split from November 2020 to May 2021. Imaging protocols were based on the assessment of 14 lung zones for both lung ultrasound (LUS) and computed tomography (CT), correlated to a CXR score assessing 6 lung zones. Prediction models for the necessity of mechanical ventilation (MV) or a lethal outcome were developed by combining imaging, biometric, and biochemical parameters. A total of 255 patients with COVID-19 pneumonia were included in the study. Four independent predictors were added to the regression model for the necessity of MV: LUS score, day of the illness, leukocyte count, and cardiovascular disease (χ2 = 29.16,  Since the end of 2019, the world has battled the growing coronavirus disease 2019 (COVID-19) epidemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with more than 290 million confirmed cases worldwide .Thoracic imaging, including chest X-ray (CXR) or computed tomography (CT), is essential in the diagnosis of COVID-19 pneumonia. However, previous studies showed that CXR was less sensitive in the detection of COVID-19 lung disease compared to CT, with a reported baseline CXR sensitivity of 69% . The senIn addition to the aforementioned imaging methods that use ionizing radiation, bedside lung ultrasonography (LUS) is a rapid, non-ionizing, repeatable, and reliable examination technique that is increasingly used by clinicians in the COVID-19 pandemic, especially for hospitalized and critically ill patients reducing the need for transportation. Growing evidence shows that LUS sensitivity is close to that of chest CT and is much higher than that of CXR; its usefulness for the management of patients with COVID-19 pneumonia, from diagnosis to monitoring, follow-up, and even outcome prediction, is also demonstrated ,11,12. The vast majority of patients with more severe symptoms of the disease have one or more comorbidities, such as obesity and cardiovascular disease, with high mortality among elderly patients . BiochemIdentifying risk factors at hospital admission that can predict the clinical course of the disease would help physicians to provide appropriate and timely therapeutic interventions. The COVID-19 prediction models developed so far have mostly combined clinical features, laboratory parameters, and, less often, CXR findings ,18,19,20This was a single-center, retrospective cohort observational study.The study included a consecutive cohort of patients admitted with the diagnosis of COVID-19 pneumonia in the University Hospital of Split, Croatia, from November 2020 to May 2021, before the COVID-19 vaccine was widely available. Inclusion criteria were WHO diagnostic criteria for pneumonia COVID-19, SARS-CoV-2 infection confirmed by PCR . Fo. Fo22]. Chest X-rays were also acquired upon hospital admission, but the CXR score was assessed retrospectively. For CXR scoring, we used the Brixia score . Chest XAll patient records with an LUS exam were consecutively analyzed without exclusions to minimize selection bias. The only reduction in sample numbers was due to the availability of the other radiological scores, duplicate entries, or based on statistical tests .The radiological exams were performed by a single clinician , thus not as a two-person consensus. However, both authors  are highly experienced in their respective clinical fields. HRCT and LUS exams were blinded and performed without knowledge of laboratory parameters, current treatment, and further involvement in the treatment of the patient. However, the decision to mechanically ventilate the patient was up to the third party without knowledge (blinded) of the LUS score. The CXR and CT scores were assessed upon writing this manuscript with a clinician blinded to any of the observed predictors or clinical outcomes. This was a sample of consecutive patients collected during the 6-month pandemic peak in Croatia, and the expected enrollment was over 240 cases. We expected the area under the receiver operating curve to be above 0.7 with a ratio of positive outcome cases around 1:10. To accommodate statistical power of 80%, this required a minimum sample of 190 patients.t-test was used to compare the means and the Mann–Whitney U test to compare the medians between two groups, while the one-way Chi-square test was used to compare proportions for dichotomous variables. Logistic regression analysis  was used to analyze independent factors associated with the necessity for mechanical ventilation or lethal outcome. The continuous variables included in the models were added to the combined model with their respective slope coefficients as follows: combined score = β1 × var1 + β2 × var2 + β3 × var3 + … + βn × varn. The receiver operating curve (ROC) was used to determine the optimal threshold, the area under the curve (AUC), specificity, and sensitivity of the tested parameters. Regression analysis was used to describe the relationship between radiological scores. The type I error  was set to 0.05, and the type II error  was set to 80%. The statistical software used for analysis was MedCalc® Statistical Software version 19.6 .Descriptive statistics were performed: categorical data were presented by absolute and relative frequencies; continuous data with normal distribution were presented as mean and standard deviation (SE) when highly variable by the median and interquartile range (IQR). The outliers and far-out values were detected by the Tukey method . The norWe reported the study in line with the STROBE reporting guideline for cohort studies; the STROBE checklist is available in We examined 299 patient records hospitalized in the University Hospital of Split due to COVID-19 from November 2020 to May 2021. Eleven duplicate records or control LUS were excluded, and the rest of the 288 eligible records of patients who underwent LUS were analyzed . Out of Patient characteristics between groups not requiring and requiring MV differed only when cardiovascular disease, hemiplegia, or leukemia were present as a negative prognostic factor. Almost the same was noticed in the case of a death outcome . SimilarWhen combined in stepwise multivariate analysis, both CXR and CT scores were excluded from models for either MV or lethal outcome. Only the LUS score was retained in both models as follows . The model accurately classified 89.9% of cases . The model accurately classified 93.2% of cases ; however, there was significant variability around the regression line  and CXR score (AUC = 0.586 ± 0.054) showed no significant difference of 0.106,  = 0.136 A. p = 0.449  and CXR score (AUC = 0.645 ± 0.059) showed no significant difference of 0.052,  = 0.449 B.p < 0.0001); however, there was significant variability around the regression line  and CT score (AUC = 0.843 ± 0.077) showed no significant difference of 0.023,  = 0.819 C. p = 0.582  and CT score (AUC = 0.836 ± 0.080) showed no significant difference of 0.049,  = 0.582 D.The results of our study draw us to conclusions defining four independent predictors regarding the regression model for the necessity of MV: LUS score, day of the illness at admission, leukocyte count, and presence of cardiovascular disease. For the lethal outcome, the two independent predictors were the LUS score and the age of the patient. Although LUS’ predictive value was shown in several previous studies, as far as we know, it was not combined with demographics, clinical data, and laboratory parameters, creating prediction models for disease severity.The average CXR score was not previously shown to be different between the group of patients who needed MV and the group of patients who did not. Although it is a possible prognostic factor for death in the univariate model, the CXR score was dropped from the multivariate model as insignificant in favor of, for example, the patient’s age. Therefore, we can conclude that the CXR score is inferior to the LUS score in the prediction of the need for MV or death.For both the CT score and the CXR score, we showed a significant correlation with the LUS score by linear regression, although in both cases, there was significant variability. We attributed this to subjectivity in the scoring system based on the decision of the clinician interpreting the finding, the lower sensitivity and specificity of the CXR, and the time interval between CT, LUS, and CXR acquisition, in comparison with other studies that used shorter time delays among them . On the On the other hand, a much better correlation between LUS score and CT score, with significantly less variability and equal width of the scoring scale, speaks in favor of LUS as an excellent method of choice in monitoring patients with pneumonia; this was confirmed in other studies as well ,26. DespMultiple studies reported a significant association between elevated leukocyte counts and decreased lymphocyte counts among patients with severe cases of COVID-19 compared with those with mild cases ,29. HoweVarious biochemical markers were researched for their predictive usefulness, for example, LDH, CRP, and D-dimer ,31,32. IIn our study, a lower day of illness at hospital admission showed to be a predictive factor for disease severity (MV), as it might suggest a more rapidly evolving and progressive disease in these patients. Similar results concerning onset to hospitalization time were found as a part of the risk nomogram established to predict the incidence of severe or critical COVID-19 in elderly patients in the study by Zeng et al. .The percentage of smokers in the hospitalized population was significantly lower than in the overall population of Croatia, regarding which there is still controversy in the available literature, but most studies still link smoking to a higher risk of severe COVID-19 ,33,34,35The proportion of patients who ended up on MV or died was similar to our previous study . Only thOur study was conducted on a large sample of hospitalized patients, and data were analyzed without exclusion by clinical characteristics using only statistically relevant and transparent figures.As mentioned in the bias section of the methods, we did not do a double, independent check of the radiological scores, and this could be a limiting factor. Although we demonstrated a strong association between the LUS score and the CT score, the sample for the CT score was relatively small and did not reach the test strength of 80%.The predictive model obtained in our study identified four key parameters at the patient admission to the hospital, LUS score, day of the illness, leukocyte count, and presence of cardiovascular disease, that can predict an adverse COVID outcome. Hopefully, it will help physicians provide appropriate and timely therapeutic interventions.


Background: Cataracts are the main cause of visual impairment among the aging population, with a high impact on patients’ quality of life. It has been suggested that the antioxidant carotenoids lutein and zeaxanthin may play a role in cataract prevention. Recently, significant evidence has associated abnormal glucose metabolism with an increased likelihood of the development of cataracts.Aim: The current study aims to assess the relationship between nutrition and cataracts among adults in the Aseer Region, Saudi Arabia.Methods: A retrospective study was conducted targeting all accessible patients diagnosed with cataracts at Aseer Central Hospital during the period from August 8, 2019, to March 3, 2021, and at Khamis Mushayt General Hospital during the period from June 11, 2018, to March 3, 2021. Data were collected using a pre-structured data collection sheet that covered patients’ socio-demographic data, including age, gender, education, work type, and marital status. Also, the type of cataract diagnosed for the study patients was recorded with associated risk factors of cataracts, including chronic diseases, trauma, eye surgery, and family history. The last section covered patients’ dietary habits and frequency of dietary intake. Results: A total of 140 patients with cataracts who fulfilled the inclusion criteria were included in the study. Patients' ages ranged from 21 to 65 years, with a mean age of 54.2 ± 12.9 years old. Additionally, 12.1% of the study patients were smokers, 44.3% were diagnosed with hypertension, and 45% were diabetic, which was type 1 diabetes mellitus (T1DM) among 36.5% and type 2 diabetes mellitus (T2DM) among 52.4%. A family history of congenital cataracts was reported among 12.9%, and 12.9% had a history of eye trauma. Moreover, 37.1% of the study patients had vegetables once per week. Eating fruits or having juice was reported as once per week among 40% of the study patients and three times per week among 20%. Finally, 31.4% take nutritional supplements or vitamins.Conclusion: There is currently an inquiry to endorse or exclude a specific diet or dietary intake that may reduce or even prevent the development and progression of cataracts. It appears beneficial for people to have some antioxidants in their daily food. Cataracts are the main cause of visual impairment among the aging population, with a high impact on patients’ quality of life ,2. CatarNumerous studies indicate that there is a link between dietary habits and the development of cataracts. The antioxidant properties of vitamins C and E could have a protective role against the development and progression of cataracts -10. AlsoRecently, significant evidence has associated abnormal glucose metabolism with an increased likelihood of cataracts -16. ManyA retrospective study was conducted targeting all accessible patients diagnosed with cataracts at Aseer Central Hospital, which is the main tertiary hospital in Abha, the capital of the southern region of Saudi Arabia, during the period from August 8, 2019, to March 3, 2021, and at Khamis Mushayt General Hospital during the period from June 11, 2018, to March 3, 2021.Adults with cataracts who visited hospitals were tracked down by searching the medical record system with the term "cataract." Two authors extracted data from the medical record and patients. After the data have been extracted, the patient will be contacted via mobile phone to invite them to participate in the study.If the patient agrees to participate, an email link with an encrypted and high-security electronic signature will be provided to him or her to get his or her consent.After the consent is electronically signed, the patient will be asked about socio-demographic data, including age, gender, education, work type, and marital status. Also, the type of cataract diagnosed for the study patients was recorded along with associated risk factors for cataracts, including chronic diseases, trauma, eye surgery, family history, and all necessary clinical information, which will be recorded in a secured electronic data capture system (Castor EDC®). Patients aged less than 18 years and those who refused to participate in the study were excluded.Data analysisAfter data were extracted, they were revised, coded, and fed to statistical software IBM SPSS version 22 . Two-tailed tests were used for the statistical analysis. A P-value of less than 0.05 was statistically significant. A descriptive analysis using frequency distribution with percent was conducted for patients’ personal data, type of cataract, risk factors for cataracts, and dietary habits. Cross-tabulation was performed to assess the relationship between the type of cataract diagnosed among patients and their dietary habits. The significance of relationships was tested using a chi-square test and an exact probability test for small frequency distributions.A total of 140 patients with cataracts who fulfilled the inclusion criteria were included in the study. Patients’ ages ranged from 21 to 65 years, with a mean age of 54.2 ± 12.9 years old. Seventy-six (54.3%) patients were female, and 134 (95.7%) were Saudis. As for work, 103 (73.6%) patients were not working or retired; 33 (23.6%) were non-healthcare workers; and 4 (2.9%) were healthcare workers. Considering educational level, 100 (71.4%) had below a secondary level of education, 20 (14.3%) had a secondary educational level, and 20 (14.3%) had a university education. A total of 128 (91.4%) were married. As for patients’ BMI, 14 (10%) had normal weight, 13 (9.3%) were overweight, and 12.2% were obese, while 96 (68.6%) did not know their BMI  and were not working (mostly retired or aged). Regarding the type of cataract, the most diagnosed among one-third of the patients was nuclear cataract, followed by other, less frequent types, including cortical cataracts and posterior subcapsular cataracts. These types are the most common among patients worldwide ,24.Regarding risk factors reported among the study patients, the most reported were old age (>50 years); diabetes mellitus, especially T1DM; hypertension; a history of eye surgery; and eye allergy. Other less frequent risk factors were reported, including a family history of congenital cataracts, taking drugs containing corticosteroids, and vitamin C deficiency. The majority of patients did not know whether they had vitamin C deficiency, which explains the low percentage of those who said they had. The reported risk factors are the main factors reported globally for cataracts through different reviewed research studies -28.Considering the dietary habits of study patients with cataracts, the study showed that the vast majority of patients (91.4%) had vegetables at least once per week, 90% of the patients had a diet containing milk and dairy products, and 87.9% had fruits and juice at least once per week. Also, more than two-thirds of the patients took nutritional supplements or vitamins, and about 40% had a diet containing legumes and nuts. On the other hand, more than half of the patients did not have fish containing amino acids at all, which have a significant role in developing eye diseases, including cataracts ,30. AlsoLee et al.  reportedThere is currently an inquiry to endorse or exclude a specific diet or dietary intake that may reduce or even prevent the development and progression of cataracts. As to the positive association between nutrition and cataracts, it appears beneficial for people to have some antioxidants in their daily food. Eating more fruits and vegetables with more vitamin intake is recommended by the National Cancer Institute. The results should be considered carefully, and more studies are recommended to prove the proposed evidence. Also, prospective studies are recommended to identify the types of cataracts most affected by specific types of food, dietary patterns, or environmental influences, which will improve patients’ quality of life and help reduce the extensive load of cataracts on public health resources.


Holothuria glaberrima that can regenerate a full intestine over the course of 21 days after evisceration. We collected RNA-seq data from various intestinal tissues and regeneration stages and used these data to define the Wnt genes present in H. glaberrima and the differential gene expression (DGE) patterns during the regenerative process. Twelve Wnt genes were found, and their presence was confirmed in the draft genome of H. glaberrima. The expressions of additional Wnt-associated genes, such as Frizzled and Disheveled, as well as genes from the Wnt/β-catenin and Wnt/Planar Cell Polarity (PCP) pathways, were also analyzed. DGE showed unique distributions of Wnt in early- and late-stage intestinal regenerates, consistent with the Wnt/β-catenin pathway being upregulated during early-stages and the Wnt/PCP pathway being upregulated during late-stages. Our results demonstrate the diversity of Wnt signaling during intestinal regeneration, highlighting possible roles in adult organogenesis.Wnt signaling has been shown to play multiple roles in regenerative processes, one of the most widely studied of which is the regeneration of the intestinal luminal epithelia. Most studies in this area have focused on self-renewal of the luminal stem cells; however, Wnt signaling may also have more dynamic functions, such as facilitating intestinal organogenesis. To explore this possibility, we employed the sea cucumber  The Wnt gene family encodes ligands that bind to the Frizzled (Fzd) family of cell surface receptors. This, in turn, activates canonical and noncanonical Wnt signaling pathways that are known to modulate various cellular processes such as morphogenesis, cell fate specification and proliferation during embryonic/adult development, and regeneration ,2,3. TheWnt and Fzd, but some genes are unique to a pathway. For example, the Wnt/PCP pathway contains the so called “core” proteins, i.e., Celsr, Vangl, Prickle and Ankrd6 [The noncanonical pathway, known as the Planar Cell Polarity (PCP) pathway, can regulate cell migration and polarity by cytoskeleton rearrangements and activation of the transcription factors c-JUN and AFT2 ,9. Thesed Ankrd6 ,11. For Wnts have functions during gastrointestinal stem cell homeostasis, such as Wnt1 [Wnt2b [Wnt3a [Wnt5a [Wnt6 [Wnt9b [The cellular processes described above that are modulated by Wnt signaling can be species, organ, and cell specific. They also vary depending on developmental stage or whether the regenerative process is for cell maintenance (homeostatic) or injury response. One of the most studied functions of Wnt signaling is the self-renewal of the intestine luminal epithelium. Studies on distant groups, such as insects (Drosophila) and mammals, even hint at common mechanisms involving Wnt mediating the control of luminal epithelium regeneration ,13,14. I as Wnt1 , Wnt2b [1 [Wnt2b , Wnt3a [b [Wnt3a , Wnt5a [a [Wnt5a , Wnt6 [15a [Wnt6 , and Wnt6 [Wnt9b . In Dros6 [Wnt9b , but mor6 [Wnt9b ,23,24.Holothuria glaberrima [H. glaberrima  of certain cell types in the intestine, Wnt signaling is more complex and can be involved in the regeneration of a complete adult organ. Therefore, to obtain a holistic view of Wnt signaling during regenerative organogenesis, we employed a well-suited model organism, the sea cucumber aberrima . This ecaberrima ,26,27,28aberrima . To initWnt genes are seen as key molecular regulators [Wnt9 was upregulated at 3-, 7-, and 14-dpe during intestinal regeneration [Wnt9 in the luminal epithelium of early regenerates [Wnt6 upregulation during early intestinal regeneration [The molecular processes that define some of the cellular events during sea cucumber intestinal organogenesis were recently investigated. gulators . For exaneration . Moreoveenerates . More reneration . It was neration . Apostichopus japonicus and Eupentacta fraudatrix. In A. japonicus, it was shown by qPCR that WntA and Wnt6 were upregulated in early- and late-stage regeneration stages [Wnt7 and Wnt8, along with Fzd7 and Dishevelled (Dvl), were upregulated at early-stage regeneration but either lowly expressed or downregulated in the later-stages [E. fraudatrix showed the Wnt4, Wnt6, Wnt16, Fzd1/2/7, Fzd4, and Fzd5/8 genes to be differentially expressed by qPCR in early- and late-stage regeneration [Wnts and the genes involved in Wnt pathways during intestinal regeneration. However, they also present problems when trying to make comparisons. To name a few, some studies focused only on some Wnt or Fzd genes while neglecting downstream genes in the signaling cascade. In other studies, the exact developmental stage of the tissue remained unclear and, in some cases, even the organ or tissue composition of the sample could not be definitively determined from the authors’ description. The other major problem is that these studies often used inadequate tissue comparisons as controls. All these problems create a patched landscape of studies of Wnt signaling during intestinal regeneration.Wnt signaling is also upregulated during intestinal regeneration in two other holothurians: n stages ,35. A dir-stages ,37. Studneration . All theWnt gene identification and the expression of Wnt signaling genes during intestinal organogenesis from a holistic point of view. To do this, we first identified the Wnt genes found in the sea cucumber genome and compared them to those of other echinoderm species. We next characterized their expression at various regeneration stages and in different tissue types. Lastly, we tried to determine which Wnt signaling pathways were active at different timepoints. Our study adds to the growing field of research of intestinal regeneration in echinoderms by supplying new spatiotemporal timepoints with appropriate controls for early- and late-stage regenerates, as well as tying together the available data on Wnt signaling during intestine regeneration in echinoderms and other species. We hope that this extensive spatiotemporal RNA-seq data will aid in the identification of master genetic regulators of organ regeneration that can later be assayed for downstream functional analysis.Therefore, the present work focuses on The animal handling and dissection methods applied in this study have been described previously . In brieRNA extraction was done as published earlier . A combiWnt sequences using amino acid sequences from Strongylocentrotus purpuratus and Eupentacta fraudatrix, obtained from EchinoBase and NCBI. Sequences from these species were utilized as queries against the sea cucumber peptide transcriptomic database, available at NCBI (BioProject: PRJNA660762) [Wnt transcripts were then used to assess if further genes from the Wnt family were present in the genome. Additionally, these were manually annotated in the draft genome [Wnt genes identified. Based on the coordinates obtained, we extracted the sequences for each exon of all the Wnt genes using the getFasta feature of BedTools v.2.30.0. Exons of each Wnt were then joined together using a custom Perl script described in a previous study [Intestine transcriptomic data were first utilized for mapping all the potential A660762) . The ORFt genome  of the sus study .Wnt gene sequences obtained from the manual genome annotation were used to perform a phylogenetic analysis. Given that this annotation was done utilizing a draft genome assembly, we wanted to confirm that our characterization has been done properly and that each Wnt was grouped with those of other echinoderm species. This would also allow us to assess the conservation of the Wnt gene family with other echinoderms. Here, we followed protocols previously employed [S. purpuratus, Patiria miniata, Lytechinus variegatus, Apostichopus japonicus, and E. fraudatrix. All of these were obtained from NCBI or EchinoBase (https://github.com/devneurolab/HgWnt2023) (Accessed: 1 April 2023). The employed , startinemployed  to perfohinoBase . AlignedTo assess DGEs of the candidate genes, we utilized previously available data from 1- and 3-dpe timepoints deposited at NCBI (ID: PRJNA660762) , along wWnt genes sequences and annotated the transcriptome using UniProt database and predictions from S. purpuratus (NCBI ID: GCF_000002235.5). The complete script of the differential expression analysis can be found in the following GitHub: https://github.com/devneurolab/HgWnt2023 (accessed on 4 January 2023).DGE was performed based on previously reported protocols . Reads wH. glaberrima Wnt genes was done using Wnt gene sequences from the sea urchin S. purpuratus and the sea cucumber E. fraudatrix. These sequences were used to probe our H. glaberrima transcriptome database for sequences that showed considerable similarities. The obtained sequences were further characterized by BLAST against the NCBI non-redundant database .Our initial identification of database . CompariH. glaberrima [Wnt transcripts were located at distinct, non-overlapping regions in the genome, where we obtained their sequence from start to stop codon. Although most of the Wnts exons spanned across distinct scaffolds, the order of the exons with exact matches for nucleotide and amino acid sequences was maintained for each Wnt, providing sufficient information to map each exon to its respective Wnt gene. The annotations showed that all Wnt family genes in H. glaberrima ranged from four to six exons, with similar lengths compared to their intronic regions, which differed from gene to gene. To allow such comparisons, in The identification of these transcripts allowed us to manually annotate all of them in our recently published draft genome of aberrima   that no new Wnt genes were found in the draft genome that were not present in our transcriptome database. This was true for Wnt8 and Wnt11, which were not detected in the transcriptome or the genome, even after attempting to find a homologous sequence by using multiple sequences from other echinoderms.It is important to highlight that (i) all Wnt genes annotated in our H. glaberrima genome were compared to those found in NCBI or EchinoBase for species from other classes of the Echinodermata clade. Only sequences from three classes were reported: Asteroidea (sea stars), Echinoidea (sea urchins and sand dollars), and Holothuroidea (sea cucumbers). Therefore, Wnt sequences for the Crinoidea (sea lilies) and Ophiuroidea (brittle stars) classes were not included in our cross-species comparisons.The protein coding sequences of the Wnt sequences from sea cucumbers A. japonicus and E. fraudatrix (Holothuroidea), sea urchins L. variegatus and S. purpuratus (Echinoidea), and the sea star Patiria miniata (Asteroidea). The results showed that all our sequences were in the same clade of their proposed Wnt homologs, confirming the discovery and annotation of the Wnt genes mentioned above  analyses using various RNA-seq timepoints. These included previously published data from normal mesentery and from 1- and 3-dpe rudiments [To determine the differential expression of udiments , as wellWnt genes during intestinal regeneration was analyzed using all RNA-seq timepoints mentioned  when compared to the normal mesentery and in regenerating intestines at advanced stages when compared to normal intestine. An interesting finding was the difference in Wnt gene expression between 14-dpe anterior and 14-dpe posterior intestine. In the anterior intestine with luminal epithelium, Wnt6 was uniquely upregulated, while in posterior tissue, Wnt5 and Wnt10 were uniquely upregulated  and the Wnt signaling pathway protein Dishevelled (Dvl) . Our resGroucho (a repressor of TCF/LEF) and Kremen1 (an inhibitor of the Wnt/Fzd/LRP6 complex), were down-regulated from 12-hpe to 3-dpe when compared to the normal mesentery. DKK3 was upregulated only in the 12-hpe stage following evisceration, while Myc (a target gene) was upregulated during early-stage regeneration from 12-hpe to 3-dpe. Conversely, in the late regenerative stages, Kremen1 and Axin2 (a target gene and self-inhibitor) were upregulated when compared to the normal intestine. Additional genes associated with the canonical pathway that were found to be upregulated in some late-regenerative stages were Twist and Slug  and EDNRA, SP5, and BAMBI in anterior and posterior 14-dpe, as well as in 21-dpe.Two major branches of Wnt signaling pathways exist, i.e., the canonical and the non-canonical Wnt signaling pathways, which can mediate different functions in regenerative processes . A briefLamc1 and Ror1 at 12-hpe and c-Jun and Ctnnal1 from 12 hpe to 3-dpe  when compared to the normal intestine.As for the Wnt/PCP pathway, several associated genes were downregulated during the early regenerative stages, including to 3-dpe . Two genWnt genes in the genome of H. glaberrima: Wnt1, Wnt2, Wnt3, Wnt4a, Wnt4b, Wnt5, Wnt6, Wnt7, Wnt9, Wnt10, Wnt16, and WntA. Surprisingly, all these genes were shown to be expressed in the intestinal tissue, where they were initially identified, and no other Wnt gene was found in the genome. This is somewhat unexpected, as Wnt genes are involved in diverse developmental and biological processes; thus, we expected to find Wnt genes in the genome that were not expressed in the intestinal transcriptomes.In the present study, we found 12 H. glaberrima is similar across the gene family, with all containing between 4 and 6 exons, with a minimum size of 59 bp (mostly first exons) and a maximum of 494 bp. All the annotated Wnt genes, with exception of Wnt5, had a first exon smaller than the rest, and the last exon was the largest of the studied Wnts. Different from the exon structures, intronic regions were variable across the family, showing a minimum of 636 bp and a maximum of 21,268 bp, with an average of 7363 bp. While to our knowledge, there is no information available about the structure of Wnt genes in other echinoderms, a few studies have described the structure of some of these in other species, such as the zebrafish Brachydanio rerio and humans [Wnt1 in B. rerio and humans shows a similar structure to that of H. glaberrima, i.e., composed of four exons. Similarly, the structures of human Wnt5 and Wnt16, both with four exons, are the same as those found in H. glaberrima. Yet, when comparing the genomic structure of Wnt2, H. glaberrima contains six exons while humans have five exons.Further, the manual annotation and characterization of these genes allowed us to determine their genomic structure within the sea cucumber genome . The strd humans ,49. Wnt1Wnt genes by performing a phylogenetic analysis of Wnt of various echinoderms species  to assess the conservation of these genes within the Echinodermata phylum. Our phylogenetic tree shows adequate grouping of Wnt with the expected clades; however, when looking deep into each of these, there were several interesting results. For instance, as one would expect, Wnt from holothuroids was shown to be in the same clade sister to that of echinoids. However, in the cases where partial sequences were utilized, the arrangement of WntA and Wnt10 in E. fraudatrix was found to be different, resulting in an independent branch (WntA) or joining P. miniata as a sister clade to the echinoid clade (Wnt10). Other than this, which we attribute to the lack of a complete sequence, conservation was maintained, as all holothuroids and echinoid Wnt were maintained in distinct clades. Similarly, in almost all cases, Wnt genes of P. miniata appeared as a single branch throughout the tree.We extended the characterization of the  species . This waWnt genes  were 100% identical. Furthermore, the authors of that study suggested a duplication of Wnt9, but only one sequence could be found deposited. Additionally, we found four sequences identified as Wnt8 in NCBI, two of which seemed to be duplications  and another with homology to Wnt2 (PIK43830.1) in several species when performing a BLAST against the NCBI non-redundant database. Therefore, we only included the sequences for which we were certain of their homology, only one Wnt3 and Wnt9 sequences, Wnt8 duplicates, and the Wnt8 sequence that showed homology to Wnt2 of other species (labeled here as Wnt2). Similarly, we used the Wnt1 sequence from A. japonicus reported by Girich and colleagues (2019) [Wnt8 was not found in H. glaberrima. Additionally, the Wnt4 duplication appears to be unique to H. glaberrima compared to the species considered here. Lastly, the asteroid P. miniata is the only species that has all 13 Wnt subfamilies but no duplications.Furthermore, there are variations between and within the assessed echinoderm classes regarding the loss or absence of individual nt genes . Seven slication  that invs (2019) , althougs (2019) . Wnt8 waWnt1, Wnt6, and Wnt10 cluster in H. glaberrima. This cluster is known to also contain Wnt9 and Wnt3 in several other species [H. glaberrima, Wnt1, Wnt6, and Wnt10 are clustered together . All of this is consistent with clusters previously reported in other species, such as Drosophila melanogaster [Here, we also demonstrate the conservation of the  species . Nonethetogether . Our annriegatus , which anogaster . HoweverWnt7 is downregulated at 12-hpe when injury and/or wound healing occurs. This contrasts with what was found in A. japonicus, where Wnt7 was overexpressed during the injury/wound healing stage [Wnt3, Wnt6 and Wnt9. These genes may be involved from 1- to 7-dpe when the mesothelium dedifferentiates and migrates to the mesentery, where the cells redifferentiate and proliferate to form the intestinal rudiment [Although correlative in nature, DGE analyses provide insight into which genes may participate in or modulate specific cellular processes. As for early-stage regeneration, which spans from 12-hpe to 7-dpe, ng stage . Upregulrudiment . Wnts may facilitate cellular processes during early-stage regeneration, we referenced previous work from our lab and others. First, it has been proposed that Wnt signaling is involved in proliferation [Wnts in apoptosis and/or dedifferentiation has been discarded [Wnt6 as the most likely candidates to modulate the increase in proliferation since, in H. glaberrima, E. fraudatrix, and A. japonicus, Wnt6 is up-regulated between 3- and 7-dpe. Lastly, this upregulation precedes the large spike in cell proliferation (~7-dpe) observed in the mesothelium of regenerating intestines [Wnt3 and Wnt9 is stable throughout early-stage regeneration. Further evidence comes from other species. For example, Wnt6 expression is most abundant in the vertebrate intestine in the crypt epithelium, where proliferation mostly occurs [Wnt6 is also a known contributor to tumorigenesis and the development of colon cancer via its effects on cell proliferation, apoptosis, and migration [To determine which feration , while aiscarded ,51. Howetestines ,27, whily occurs . Wnt6 isigration . Wnt3 and Wnt9 as possible controllers of proliferation during early-stage regeneration or during the injury/wound healing phase, particularly since the Wnt9 protein is expressed in the regenerating mesothelium [Wnts are known to have redundant functions [Wnt3 and Wnt9 may supplement the effect of Wnt6. For example, Wnt3 is thought to time cell divisions in mammalian intestinal stem cells, where Wnt6 and Wnt9 are also expressed [Wnt3 can promote EMT in breast cancer cells [Wnts during early-stage regeneration. We cannot rule out othelium . Additiounctions . Thus, Wxpressed . Alternaxpressed . It is ter cells  while inWnt genes most likely associated with these stages are Wnt4b, Wnt5, and WntA, all of which are uniquely overexpressed in late-stage but not in early-stage regeneration. The emergence of these Wnts could be a result of the mesenchymal cells that surround the luminal epithelium. This is seen in mammals where mesenchymal cells beneath the basal lamina of the luminal epithelium uniquely secrete Wnt4 and Wnt5 [Wnts associated with the formation of the rudiment  may also contribute to intestinal growth and elongation, since their upregulation persists during late-stage regeneration. Late-stage regeneration is dominated by the formation of the lumen whereby at 14-dpe, luminal epithelial cells from the esophagus and cloaca proliferate and migrate into the intestinal rudiment . At 21-dand Wnt5 . HoweverFzd genes are differentially expressed during late-stage regeneration, namely Fzd1, Fzd4 and Fzd10. The high expression of Fzd genes is not fully unexpected since, in mammals, these receptors have been closely associated with the regeneration of the luminal epithelium and with stem cell modulation [H. glaberrima, i.e., differences in the spatial or temporal expression of genes and of cell proliferation and apoptotic events [Wnt expression can be found in other tissues and other species, mainly during embryonic development. For example, in mouse embryos, Fzd10 is found in the most posterior region of the epiblast and in the primitive streak but not in mesoderm migrating laterally and anteriorly [Wnt6 expression is not present posteriorly during embryonic patterning [Wnt5, as it is expressed at the caudal end of the embryo during gastrulation and eventually in the distal portion of structures that extend from the primary body axis [It is also interesting that several dulation . Additioc events . Additioteriorly . In sometterning . Howeverody axis .WntA during late-stage regeneration is also of particular interest. This Wnt is not found in vertebrates and is thought to have been lost during evolution. However, it is present in echinoderms, and its upregulation during late-stage intestinal regeneration has been confirmed in A. japonicus [WntA overexpression might be associated with the amazing regenerative abilities of echinoderms.The overexpression of aponicus . It is tWnts that are upregulated during early-stage regeneration have also been shown to be highly expressed in vertebrate paneth cells [Kremen and Groucho in the early-stage regeneration. Kremen normally prevents Wnt from binding to Fzd, thus disabling the signaling cascade, while Groucho acts as a repressor of the LEF/TCF. Other evidence comes from our lab, where pharmacological experiments targeting the Wnt/β-catenin pathway both in vivo and in vitro modulate cell proliferation in early intestinal regenerate [We have also used our DGE analyses to elucidate the dynamics of Wnt signaling during holothurian intestinal regeneration. As mentioned, Wnt signaling can occur via the canonical Wnt/β-catenin pathway and the noncanonical Wnt/PCP pathway. Our data suggest that the cellular proliferation observed in early-stage regeneration occurs via the Wnt/β-catenin pathway, while luminal epithelial events in late-stage regeneration occur via the Wnt/PCP pathway. Our proposition is supported by comparative data, e.g., the th cells ,20,62,63th cells ,64,65. Agenerate . Howevergenerate .Vangl, which is known to facilitate gut elongation and lumen formation [Wnt5, which can operate through the Wnt/PCP pathway to elongate the posterior gut, particularly when taking into account that, in mice, Wnt5 has been shown to work together with Vangl to orient cell division along the rostrocaudal axis to increase fore-stomach length [Fzd4, which is associated with the Wnt/PCP pathway, was highly upregulated in late-stage regeneration, while it was under-expressed in early-stage regeneration; finally, the upregulation of Kremen1, which is an inhibitor of the Wnt/β-catenin pathway, suggests crosstalk between Wnt signaling pathways. This would also suggest that Wnt/PCP regulates Wnt/β-catenin during late-stage regeneration, so that it can impose its effects on cellular processes. As for late-stage regeneration, the Wnt/PCP pathway is most likely involved in the regeneration of the luminal epithelium. This is evidenced by a few observations: first, the upregulation of ormation ; second,h length ; third, We recognize that most DGE-based discussions are purely correlational, and further experimentation is necessary to conclude that specific genes have or lack a specified function. Nonetheless, our analyses can be viewed as a series of hypotheses that can be tested in future investigations.Wnt genes found in the genome of our model organism, H. glaberrima, to a comparative expression analysis in normal and regenerating intestines. The results provide important insights into the molecular bases of intestinal regeneration and the role that Wnt signaling mechanisms might be playing in the process. They also provide investigators with a point of departure for molecular analyses, not only of regenerative organogenesis, but also of molecular evolution, signaling pathways, and organ homeostasis.In conclusion, the work presented here comprises an extensive analysis of sequence information ranging from the characterization of the


Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities  in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species,  Plant infectious diseases caused by phytopathogenic fungi are often coupled with the destruction of plant tissues by fungal enzymes. Most of these enzymes destroy plant cell wall (PCW) polymers—polysaccharides  and different proteins. Due to this, extracellular fungal enzymes involved in polysaccharide and protein depolymerization have been extensively studied ,2. In adBasidiomycota), especially xylotrophic ones that cause wood decay. The ligninolytic enzyme complex of basidiomycetes consists mostly of oxidoreductases that catalyze lignin degradation: lignin peroxidases , manganese (Mn)-dependent peroxidases , and polyphenol oxidases  [Ascomycota have been significantly less studied. By screening 610 strains of 16 species isolated from soil, compost, and decayed wood, more than 50 ascomycete strains have been shown to produce extracellular POs [Aspergillus flavus and Aspergillus nidulans, which belong to the Eurotiomycetes class of Ascomycota, have been shown to be able to degrade alkali lignin and exhibit Mn peroxidase, lignin peroxidase, and laccase activities [Leotiomycetes, Botrytis cinerea, a well-known causal agent of gray rot of different plants, laccases and peroxidases act as quenchers of free radicals and reactive oxygen species (ROS) during the plant defense response (hypersensitive response) [POs are extensively studied in basidiomycetes 14.18.1) ,13,14. T.18.1 ,13ular POs . Aspergiesponse) .Dothideomycetes class: Botryosphaeria rhodina [Botryosphaeria kuwatsukai [Clavariopsis aquatic [Coniothyrium sp. [Hortaea acidophila [Phoma sp. [Paraconiothyrium variabile [Paraconiothyrium variabile, a species found in wood-damaged stems and vines of different plants , has been demonstrated to produce Mn peroxidases and lignin peroxidases, and to decompose model and native lignin, guaiacol, polymeric dyes (phenol red and aniline blue), and antracen derivatives [Botryosphaeria kuwatsukai, a phytopathogenic fungus that infects a wide range of woody plants, had a negative effect on the virulence of this species [Laccases were revealed in many species from the  rhodina , Botryoswatsukai , Clavari aquatic , Coniothrium sp. , Hortaeaidophila ,23, Phomhoma sp. , and Parariabile . In addiivatives . The kno species .Sordariomycetes, laccases were revealed in the species of eight families: Chaetomiaceae—Chaetomium thermophilium [Melanocarpus albomyces [Hypocreaceae—Trichoderma atroviride [Trichoderma harzianum [Microascaceae—Petriellidium fusoideum [Magnaporthaceae—Gaeumannomyces graminis [Niessliaceae—Monocillium indicum [Sordariaceae—Neurospora crassa [Xylariaceae—Xylaria polymorpha, Xylaria hypoxylon [Glomerellaceae—Colletotrichum orbiculare [Coniochaeta ligniaria, laccase activity has not been detected; however, this fungus displayed lignin peroxidase and Mn peroxidase activities [Colletotrichum orbiculare, a causal agent of anthracnose of plants from the Cucurbitaceae family, as well as in many other fungal species, laccases (including LAC2) are involved in melanization that occurs, in particular, during the formation of appressoria necessary for the penetration of plant tissues. The lac2 mutant strain of C. orbiculare was unable to form functional appessoria and invade the leaves of the host plant [Within the class, ophilium , Melanoclbomyces ,28, Hyporoviride ,30, Tricarzianum , Microasusoideum , Magnapograminis , Niessli indicum , Sordaria crassa , Xylariaypoxylon ,37, and biculare . In Conitivities . In Collst plant .Xylariales order (class Sordariomycetes) mostly include xylotrophs, many of which cause wood soft rot [Xylaria polymorpha and Xylaria hypoxylon, have been shown to be able to decompose the lignocellulose substrate and produce extracellular laccases. Herewith, the extracellular activities of lignin peroxidase and Mn peroxidase have not been revealed in these species [Xylariales order contains the genus, Microdochium, which is represented, in particular, by the devastating phytopathogenic species, Microdochium nivale (Fr.) Samuels and Hallett, and M. majus (Wollenw.) Glynn and S.G. Edwards [M. nivale is one of the causal agents of snow mold disease, which develops under the snow cover in winter cereals/grasses, and often reaches the epiphytotic level. However, M. nivale-caused pathogenesis is not strictly coupled with the presence of snow cover, and this species may also cause foot rot, leaf blight, head blight, and other types of diseases throughout the growing period [The genera from the soft rot . The rep species ,37. In a Edwards . This sp Edwards ,43,44,45g period ,47,48.M. nivale species [M. nivale from a single territory (single field) and a particular crop (winter rye), and showed that these strains differed strongly from each other in terms of virulence and the level of production of different extracellular enzymes  [M. nivale. Therefore, our study aimed to analyze whether M. nivale—a pathogen of non-woody plants—produces different types of extracellular POs, to compare extracellular PO activities in the cultures of different strains, to check whether these activities are responsive to the presence of host plant metabolites, and to search for correlations between the levels of enzymatic activities and virulence.High genetic and phenotypic diversity is typical of  species ,49,50. IMicrodochium nivale collected at the field of the Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”, located in the forest–steppe area of the Volga region, Laishev District, Tatarstan Republic, Russia  north (N) 55.649 east (E) 49.3083), [Twenty-one strains of 9.3083),  were use2PO4—2; K2HPO4—3; MgSO4 × 7H2O—2.5; FeSO4 × 7H2O—0.03, pH 5.8, at 20 °C for 30 days in 100-mL flasks with 50 mL of the medium. As the inoculum, 5-mm mycelial plugs cut from the periphery of 10–14-day old cultures grown on 1.5%-agarized PSB or MM media, respectively, were used (1 plug per flask). The enzymatic activities were assayed at 10-, 20-, and 30-days post-inoculation in three biological replicates.For the determination of PO activities, all strains were grown submerged in two media: potato sucrose broth (PSB), containing (g/L) potato—200; sucrose—30, pH 6.2, and minimal medium (MM), containing (g/L) D-glucose—3; L-asparagine—0.3; KHM. nivale strain 1 was cultured in MM for 10 days. Then, 1/10 (v/v) of distilled water or 1/10 (v/v) of water extract of rye leaves was added to the fungal cultures, and the activities were measured prior to and 12, 24, 48, 96, and 168 h afterward in five biological replicates. To obtain the rye extract, 100 g of fresh rye leaves (grown in vermiculite under a 16/8 light/dark period for 14 days) were thoroughly ground in three volumes (w/v) of distilled water, and the obtained suspension was filtered through the gauze. The remaining debris was again thoroughly ground in two volumes of water and filtered. Two portions of the obtained filtrates were combined and centrifuged . The supernatants were collected, incubated for 10 min at 80 °C, and centrifuged again. Further, the extracts were sterilized through nitrocellulose filters with pores of 0.22 μm in diameter  under sterile conditions, and kept frozen (−20 °C) until use.To assess the effect of the host plant extract on the assayed enzymatic activities, g, 10 min, at room temperature). The activities were measured spectrophotometrically at 18 °C in 96-well polystyrol microplates on a Spark 10M . As a control, an equivalent volume of sterile medium (MM (with and without rye extract) or PSB) was used.To determine the extracellular lignin peroxidase, Mn peroxidase, laccase, and tyrosinase activities, supernatants of fungal cultures were collected and centrifuged  was measured at 310 nm.The lignin peroxidase activity was determined by the rate of oxidation of veratric alcohol to veratraldehyde . Here, 14 × 5H2O, and 0.1 mM H2O2, was mixed with 100 μL of the cultural supernatant. The accumulation of the stable cation radical (ε = 27.5 mM−1 cm−1) was measured at 468 nm.The activity of Mn peroxidase was determined by the rate of oxidation of 2,6-dimethoxyphenol  to a coerulignone  . Here, 1−1 cm−1) were measured at 436 nm.Laccase activity was determined by the rate of oxidation of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt  to a stable cation-radical ABTS+ [−1 cm−1) was measured at 475 nm.Tyrosinase activity was determined by the rate of oxidation of 3--l-alanine  to DOPA-quinone . Here, 1The time course of all reactions was 10 min. The amount of enzyme catalyzing the formation of 1 μmol of the product per min was considered an activity unit and expressed in μmol/min/mg of protein. The protein concentration was assayed by the Bradford method .M. nivale extracellular laccases, Mn peroxidases, and tyrosinases were analyzed by electrophoresis under non-denaturing conditions in a 7.5% polyacrylamide gel (PAGE) [M. nivale grown in PSB for 20 days were used. Cultural supernatants of M. nivale strains were separated from the mycelium and centrifuged at 10,000× g for 20 min. The supernatants were concentrated 20-fold by lyophilization, and 20 µL-samples were analyzed by electrophoresis.The patterns of l (PAGE) . For the2O2, and 0.2 mM MnSO4 [To visualize the proteins with laccase activity after PAGE, the gels were stained with o-dianisidine  . o-DianimM MnSO4 . TyrosinmM MnSO4 .M. nivale extracellular enzymes was analyzed for the supernatants of fungal cultures grown for 20 days in MM. Supernatants were separated from the mycelium and centrifuged at 10,000× g for 20 min. As a substrate for the reactions, nitrated lignin was used. To obtain the nitrated lignin, 1 mL of glacial acetic acid was added dropwise to 5 mg of native lignin from hardwood . The resulting suspension was stirred  and filtered through nitrocellulose filters with pores of 0.22 μm in diameter  to remove undissolved particles. Then, 0.2 mL of concentrated nitric acid was added, and the mixture was stirred for 1 h . After that, 2 mL of distilled water was added, and the mixture was titrated with 1 M NaOH to pH 7.0. The resultant solution (yellow-orange color) was diluted 100 times with 750 mM Tris-HCl buffer (pH 7.4) with 50 mM NaCl and used as a stock solution for the assays [The lignin decomposing capacity of e assays .2O2 or water; the total volume was 200 µL. The reactions were carried out both in the presence and absence of H2O2 [Lignin decomposition assays were carried out in a reaction mixture containing 160 µL of nitrated lignin solution, 30 µL of supernatant sample, and 10 µL of either 40 mM H of H2O2 . Lignin 2 + bx + c, and the determination coefficient was taken at a level > 0.95). For each approximated function, the derivative of the equation was determined. The equation of the derivative was used to calculate the values of the derivatives at the extreme points (1 and 20 min). The lignin decomposition level was expressed as a module of the difference in derivative values at the extreme points.To compare lignin decomposition intensity by the enzymes of different strains, the approximation of normalized  kinetic curves of lignin degradation product accumulation was performed , Mn peroxidase (MP), laccase (Lac), and tyrosinase (Tyr) activities were measured. Since the activity of these enzymes depends on culture conditions [To check whether nditions , we usedM. nivale strains were cultured in PSB, the levels of extracellular LP activity varied in different strains in the range of 57–542, 170–867, and 460–1278 U/mg of protein  (When ctively) A, extracctively) B, extracctively) C, and exctively) D. In mosctively) . In the M. nivale strains in MM resulted in a different pattern of the assayed activities compared to cultures grown in PSB , we found strong correlations between the activities of MP and Lac, as well as MP and Tyr . No corrM. nivale strains were analyzed by native gel-eletrophoresis and the subsequent staining of gels with chromogenic substrates of different enzymes. For most of the strains, the activities of extracellular MPs, Lac, and Tyr corresponded to a single band in a gel  of water extract of rye leaves or 1/10 (v/v) of water was added to the cultures. Enzymatic activities were measured at 12, 24, 48, 96, and 168 h after the addition of rye extract or water. No assayed enzymatic activities were detected in sterile rye extract.To check if the assayed enzymatic activities can be responsive to host plant metabolites, the effect of rye extract on the extracellular LP, MP, Lac, and Tyr activities of  studies , was choM. nivale cultures  (The most pronounced induction by rye extract was observed for the extracellular LP activity in the cultures A. The LPwo-fold) B,C. Tyr wo-fold) D.M. nivale strains, a correlation analysis was performed. The virulence of the strains was assessed in our previous study based on the weight of the roots of infected plants (compared to non-infected plants) [M. nivale strains in quantitative terms compared to other morphometric parameters of infected plants [To check whether the levels of the assayed extracellular PO activities corresponded to the level of virulence of  plants) . This pad plants . In our We proposed that a particular enzymatic activity might not correlate with virulence, but the combination of the activities might be important for virulence properties. To check this, we performed cluster analyses. Based on all assessed activities , the assayed strains were clustered into three groups . To compare lignin decomposition intensity by the enzymes of different strains, the derivatives for the equations describing the curve of lignin degradation product accumulation were calculated. The values obtained for the extreme points (1 and 20 min) were substituted into the equations of the derivatives, and for the resulting values, the modules of differences were calculated to express lignin decomposition levels.To check whether extracellular enzymes of M. nivale. The extracellular enzymes of the six analyzed highly virulent M. nivale strains differed in their lignin-decomposing capacity , a correlation analysis was performed. A correlation was found between lignin-decomposition intensity and the level of Lac activity . The activity levels of the other three types of enzymes did not correlate with lignin-decomposition intensity.M. nivale—a causal agent of different diseases of cereals and grasses [The POs are poorly characterized in plant pathogens that cause diseases in non-woody plants despite the potential role of these enzymes in disease development. In our study, we assayed the extracellular activities of different POs  in the strains of the phytopathogenic fungus,  grasses ,61,62. W grasses ,59.In terms of the pattern of PO activities, the assayed strains split into three clusters. The first cluster consolidated ten strains with relatively high extracellular PO enzymatic activities displayed in PSB and relatively low activities displayed in MM. The second cluster included three strains with rather high extracellular PO activities under both culture conditions. Within the third cluster, eight strains with rather low extracellular PO activities under both culture conditions were located.M. nivale strains, since correlations between these types of extracellular activities were found. In contrast, the LP activity did not correlate with any of the other three types of analyzed activities, indicating that the regulation of LP was probably carried out independently of MP, Lac, and Tyr activities. To the best of our knowledge, no correlation between the activities of different POs (and possible co-regulation of their production) has been previously analyzed in ascomycetes.We revealed that the co-regulation between MP and Lac activities, as well as between MP and Tyr activities, might occur in M. nivale strains.For most of the strains, we found that their extracellular MP, Lac, and Tyr corresponded to a single band in a gel after electrophoretic separation of the extracellular protein fraction, indicating that each strain produced either a single variant of each enzyme, or different variants with similar electrophoretic mobility. However, two bands, corresponding to the extracellular MP, Lac, and Tyr, were observed for some strains, suggesting that either the spectrum of isoforms or regulation of their production might vary in different M. nivale–plant interactions to varying extents. Although the inducing effect of plant metabolites on the PO activities of basidiomycetes has been shown previously [Among the assayed PO activities, the most responsive to the presence of host plant metabolites was LP: its activity increased five-fold after the addition of rye extract to the fungal culture. MP and Lac activities also increased in the presence of rye extract, but much less than the LP activity. The least responsive to rye extract among the analyzed extracellular activities was the Tyr activity. This likely means that different POs contribute to eviously ,63, as fM. nivale strains and the levels of their extracellular PO activities. None of the enzymatic activities correlated positively with virulence at any assayed culture condition or at any time point. The clusterization of strains based on all four, three of four, and two of four enzymatic activities  did not split the analyzed strains according to their virulence level, irrespective of whether two culture conditions and three time points were analyzed together or separately. However, this does not mean that POs are unnecessary for M. nivale–plant interactions, especially given the potential role of these enzymes in lignin decomposition and the destruction of antimicrobial plant metabolites. It is possible that different M. nivale strains, having high phenotypic and genetic diversity and utilizing different strategies of interaction with the host [M. nivale extracellular enzymes, breaking glycosidic bonds, including various PCWDEs, also had no correlations with virulence [M. nivale strain uses a unique “cocktail” of extracellular enzymes, and consequently applies a unique strategy of causing disease. Our present study shows that different POs are also likely to contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different strains of M. nivale.We did not find relationships between the virulence degrees of the host ,49,50, mM. nivale extracellular enzymes to decompose lignin was also unassociated with the virulence of M. nivale strains. The extracellular enzymes of six highly virulent M. nivale strains differed in their lignin-decomposing capacity. For the extracellular enzymes of one of these strains, the lignin-decomposing activity was not detected. The enzymes of two strains were the “leaders” in terms of lignin degrading capacity, whereas one and two strains produced extracellular enzymes causing medium and low levels of lignin depolymerization, respectively. An unequivocal role in lignin decomposition by the M. nivale extracellular enzymes was played by peroxidases, since no lignin degradation products were detected in the absence of hydrogen peroxide in the reaction mixture. Whether it was LP or MP that played a dominant role in the lignin decomposition by M. nivale was not absolutely evident. All six analyzed strains possessed a rather high LP activity, which differed among these strains by only 2.6-fold. Therefore, the minor differences in LP activity among strains cannot account for the high variability in lignin decomposition. In turn, the MP activity differed among the analyzed strains by 17.5-fold. Moreover, the “strains-leaders” in lignin decomposition were characterized by the highest levels of MP, whereas the strain whose extracellular enzymes were unable to depolymerize lignin produced the least level of MP, indicating that MPs might determine the lignin decomposition capacity. However, we did not find a correlation between MP activity and the lignin decomposition level. The correlation with lignin decomposition level was revealed only for Lac activity, indicating that these enzymes are also likely to play a significant role in lignin depolymerization by M. nivale. Thus, the decomposition of lignin is probably a result of the coordinated action of different types of M. nivale POs, namely, LP, MP, and Lac.The capacity of M. nivale strain 13 possessed the lowest  level of extracellular MP activity, and its extracellular enzymes were unable to cause detectable lignin depolymerization; although, this strain possessed the highest  level of extracellular LP activity under assayed conditions. Other studies concluded an important role of Lac in lignin decomposition, since strains displaying a high level of lignin-degrading capacity had high Lac and MP activities, but did not obligatory exhibit LP activity [M. nivale, Lac was also likely to play an important role in lignin degradation, since we observed a correlation between the Lac activity in cultural supernatants and their lignin-decomposing capacity. Some authors are of the opinion that the lignolytic enzymatic complex is variable, and its composition is not universal in different species; here, species with different patterns of PO activities can cause similar levels of lignin decomposition [The contribution of different POs to lignin degradation by different fungi remains debatable. In some studies, the crucial role in lignin decomposition was attributed to LPs and MPs, whereas Lac was considered an auxiliary enzyme in the degradation of this polymer ,14,64,65activity . The mosactivity ,68. In Mposition ,69.M. nivale strains, even if they inhabit a common crop in a common field. We suppose that the role of POs in plant–microbe interactions is highly underestimated not only for M. nivale, but also for other phytopathogens that cause diseases in non-woody plants. Given that the PO action may promote the destruction of the host plant tissues and plant defense metabolites, as well as the fact that the PO activities (at least some of them) can be induced by host plant extracts, these enzymes are obviously involved in the development of diseases, or at least contribute to pathogen fitness in planta.Our study supports the hypothesis of the differential behavior of different closely related


Although the ecological risks of antibiotics have been extensively researched globally, fewer studies have been conducted in sensitive and fragile plateau wetland ecosystems. To evaluate the ecological risk of antibiotics in plateau urban wetlands, 18 water samples, 10 plant samples, and 8 sediment samples were collected in March 2022 in the Xining urban wetlands on the Qinghai-Tibet Plateau. The liquid chromatography-electrospray ionization tandem mass spectrometry method was utilized to measure the concentrations of 15 antibiotics in three categories in three types of environmental media. Risk quotients were adopted to assess the ecological risk of antibiotics, and the principal component analysis–multiple linear regression model was used to analyze the source of antibiotics. The results showed that (1) the maximum concentrations of antibiotics in water samples, plants, and sediments reached 1220.86 ng/L, 78.30 ng/g, and 5.64 ng/g, respectively; (2) Tylosin (TYL), norfloxacin (NFX), ofloxacin (OFX), and ciprofloxacin (CFX) in water were at medium and high-risk levels, and OFX had the highest risk value, of 108.04; and (3) the results of source apportionment indicate that 58.94% of the antibiotics came from the Huangshui river and wastewater treatment plant (WWTP) near the wetlands. The current study may provide a reference for the risks and management of antibiotics in plateau urban wetlands. Antibiotics are widely used in medical, agricultural, and aquaculture industries . There iAntibiotics are widespread in the environment and have been detected in surface water, groundwater, plants, soil, sediment, and other environmental media around the world ,5,6. AntAntibiotic contamination has been studied in different types of wetlands, such as mangroves, lakes, rivers, and coastal wetlands ,22,23,24The Qinghai-Tibet Plateau is a significant gene pool for ARGs . ResearcPhragmites australis and Typha minima are the dominant species of aquatic plants [The HNWP consists of the Huangshui River course and four urban artificial surface flow wetlands, with a total area of 508 hectares. The four constructed wetlands are Haihu wetland, Huoshaogou Wetland, Ninghu wetland, and Beichuan wetland  Figure . It is ac plants .A total of 36 samples were collected from 20 sample sites in March 2022, including 18 water samples, 10 aquatic plant samples, and 8 sediment samples , Table 13-N), nitrate (NO3-N), orthophosphoric acid (H3PO4), and heavy metals were detected in the laboratory .pH, dissolved oxygen (DO), salinity (SAL) and oxidation-reduction potential (ORP) were determined by HQ40d multiparameter water quality analyzer . Total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD), total organic carbon (TOC), ammonia (NHAntibiotics were detected by the LC-MS/MS method . The expBCF) in accordance with Equation (1) [Antibiotics accumulative effects in aquatic plants were calculated using bioconcentration factors (tion (1) .(1)BCF=BSAFs) are contaminants in sediments and aquatic organisms and are used to describe the relationship between aquatic organisms and sediment contamination [Biota-sediment accumulation factors (mination :(2)BSAFskB is the equation regression coefficient, kFS is the factor score calculated by the PCA, and A PCA-MLR model was utilized to analyze the relationship between multiple variables through data dimensionality reduction ,44,45,46The formula for mean percentage contribution calculation is Huangshui River (A), Beichuan River (B), Jiefang Canal (C), and WWTP (D) were considered as four sources of pollutants.RQs are used to assess the ecological risk of antibiotic presentation in aquatic environments , calculaPNEC values were obtained after the literature review  to predicted no-effect concentration (PNEC) RQs value was stratified into four levels, ≤0.01, 0.01–0.1, 0.1–1, and >1, indicating insignificant, low, medium, and high risk, respectively.e review  or by diMicrosoft Excel 2016 was used to process the data; R software v4.2.1  and IBM SPSS Statistics version 26.0  was used for statistical data analysis, and Origin 2021 was used for mapping.Thirteen of the 15 antibiotics were detected in aquatic plants (chlortetracycline (CTC) and doxycycline (DOX) were not detected), and EFX, NFX, LFX, fleroxacin (FLX), DFX, and AZM were detected in 100% of plants. Macrolides (MLs) were the uppermost antibiotics in aquatic plants, and AZM had the highest mean concentration (68.98 ng/L).Six antibiotics were detected in sediments, and the detection rates of OFX, OTC, CTC, RTM, erythromycin (ERM), and TYL were 100.00%, 12.50%, 100.00%, 87.50%, 100.00%, and 62.5%, respectively. RTM was main antibiotic in sediment, with the highest average concentration of 5.46 ng/L.In the water samples, the distribution of antibiotics was the highest in the Ninghu wetland a, with tAZM was the most widely distributed in the wetlands b. QNs anRTM, ERM, and TYL were mostly distributed in the Beichuan wetland and Haihu wetland. TYL was not detected in the Huangshui River and Ninghu wetland, and OTC was detected only in the Huangshui River.The relationship between Phragmites australis and sediment contamination is presented in The ecological risk values of antibiotics in wetland water samples are shown in The results of the antibiotics source analysis are shown in The linear regression equation of Expanding the equation The highest mean concentrations of OFX (180.21 ng/L) and AZM (173.81 ng/L) were detected in water samples, similar to antibiotic concentrations in urban river systems , but dozThe cumulative proportion of antibiotics in wetlands was MLs > QNs > TCs; whereas the highest proportion of QNs was found in the water samples from Ninghu, which may be related to the fact that the water source of Ninghu mainly comes from the effluent of the WWTP, while QNs are the most consumed antibiotics in Chinese hospitals . TCs areCompared to natural wetlands, urban wetlands are more complex sources of pollution, and sediments are soaked for long periods, making a clear distinction between pollution sources difficult. Therefore, the PCA-MLR model was used to analyze the antibiotics sources. The Haihu wetland and Ninghu wetland, with the greatest intensity of human activities, was found to have the greatest source contribution, indicating that human activities had the greatest impact on antibiotics.The antibiotics were most distributed in the Haihu and Ninghu wetlands, which may be related to the fact that these wetlands are located in the most populated urban areas of Xining. The lower distribution in the Beichuan wetland may be attributed to the improved water quality of the Beichuan River over recent years . Althougp < 0.01), SAL and pH , TN and pH , NH3-N and SAL , NH3-N and TP , NO3-N and pH , NO3-N and TN , H3PO4 and SAL , H3PO4 and TP , H3PO4 and NH3-N , COD and ORP . These results show that the sources of these pollutants are consistent. ΣABs were positively correlated with anthropogenic pollution indicators ) (p < 0.01) and negatively correlated with natural property indicators (pH and DO) (p < 0.01). This indicates that the source of pollutants in the wetland is acquired and manufactured. Previous studies found that WWTPs and river inputs are the main sources of antibiotic pollution in wetlands [p < 0.01), and DO could affect microbial activity and reduce biodegradation [This study uses the Spearman correlation analysis method to explore the relationship between antibiotic concentration and conventional pollutants. wetlands , and thiradation .p < 0.01) and TYL and ERM  were significantly positively correlated, indicating that antibiotics with similar structures are more closely related. ΣABs were negatively correlated with TOC and TN, indicating that these indicators may be affected by more complex factors. ΣHm are significantly and negatively correlated with ΣABs, and although there are few studies on the effect of Hm concentration on antibiotic concentration, most studies have found that Hm pollution promotes antibiotic resistance [p < 0. 01) is consistent with the findings that pH affects the removal efficiency of antibiotics [In the sediment b, ERM ansistance ,61; thusibiotics .Pseudokirchneriella subcapitata) was moderate and low.The significant correlation between antibiotic concentrations in water and sediment and water quality indicators was similar. However, previous studies have shown that antibiotics are transient in water and that persistence in sediment is the dominant factor in antibiotic concentrations . In the (1)A total of 15 antibiotics were detected in water samples, of which OFX had the highest concentration (0.54–1220.86 ng/L), and 13 and 6 antibiotics were detected in plants and sediments, respectively, with the highest concentrations being that of AZM (68.31–78.30 ng/L) and RTM (5.27–5.64 ng/L).(2)There are significant spatial changes of antibiotics, which are related to wetland water sources and other factors. The highest average concentrations are detected in the Haihu wetland.(3)Compared to other antibiotics, TYL, ERM, and OFX have a higher bioaccumulation potential, and there is a high risk of NFX, OFX, and CFX in water samples.The current study investigated the multiphase occurrence levels of antibiotics in plateau urban wetlands, discussed the spatial distribution and source contribution of antibiotics, and evaluated the ecological risk of antibiotics and the enrichment ability of plants to antibiotics. The main conclusions are as follows:A limitation of the current study is the lack of seasonal data; the temporal variation of antibiotic ecological risk should be considered in future studies.