Background: The aim of this study was to compare for the first time IL-6 (Interleukin 6), testosterone (T) and estradiol (E) levels, their ratio (E/T), micronucleus (MN), and nuclear bridge (NB) frequency between newborns with regard to their mother’s residency and diet. Our results should enable an assessment of the possible environmental endocrine effects and interaction between biomarkers, pointing to possible associated health risks. Methods: Fifty full-term newborns of both sexes, whose mothers were healthy and not occupationally exposed to any known carcinogen, were analyzed. All of the mothers filled in a detailed questionnaire. Results: The results showed significantly higher levels of E in newborns of mothers with agricultural residency than those born by mothers with urban residency. Significantly, lower levels of E were measured in newborns of mothers who drank milk and carbonated beverages more frequently. Testosterone was significantly higher in boys of mothers with agricultural residency than from mothers with urban residency. Residence and other parameters had no impact on the difference in MN frequency. IL-6 levels were higher in newborns of mothers with agricultural residency. NB levels were significantly associated with E. A significant association between E levels and IL-6 was found. Conclusion: Our results were the first to show a significant impact of the mother’s agricultural residency and diet on their newborns’ sex hormone and IL-6 levels and their association. The complex mechanisms and dynamics of intrauterine development, which, if disturbed, may have lifelong consequences on health and susceptibility to xenobiotics, represent the top priorities of research. Of specific interests are endocrine disruptors (EDS), which may interact with a large number of mechanisms in the human body that cause not just traditionally sex hormone-related diseases but are also involved in mechanisms of carcinogenesis, immunology disturbance, infertility, and brain development .A large number of contaminants in the air, food, and water cross the placental barrier. Although exposure to urban pollution has been for decades associated with a large number of diseases, an agricultural living environment may also be associated with exposure to different xenobiotics, especially pesticides ,3. EndocDiet, smoking, and residency of the mother are reflected in genome damage in newborns ,10,11. MEarly life disturbances in estradiol (E), testosterone (T) levels, and their ratios have been shown to be important not only in different infertility problems, such as polycystic ovarian or low sperm quality, but also in immunological and inflammatory mechanisms with lifelong health effects ,17,18,19Sex-specific health risks associated with transplacental environmental stressors are recognized, but still, the much more integrative investigation is needed. It is known that transplacental exposure of boys has shown significantly more disturbance of fetal growth than girls ,24,25,26Disturbances of cytokine levels in newborns are reported to be associated with transplacental exposure due to different environmental settings of the mother ,28. IL-6The aim of the current study was to find an association between mothers’ living environment and the level of MN, nuclear bridge (NB) frequency, estradiol, testosterone levels, the ratio between E and T, and IL-6 levels in their newborns. Parameters, such as smoking, place of residence, and newborn’s gender, were selected as the most important based on the hypothesis of their association with applied biomarkers. This was the first study ever to compare genome damage, cytokine, and sex hormones levels in cord blood with the mother’s environmental exposure.Fifty healthy mothers who gave birth to full-term newborns were informed about the study and, after signing a written consent, filled-in detailed questionnaires on their medical and family history, occupational exposures, diet , hobbies , residence, in-house environment during pregnancy (renovation), and smoking. In addition to the information collected in the questionnaire, the residence was defined by Google Earth. Urban residence was defined in case that residence was next to the highway (up to 50 m) or within the Zagreb city district. The agricultural residence was defined if the residence was next to wheat, corn vegetable, vineyard, or orchard field (50 m). The mothers with the help of a neonatologist filled in the questionnaires in order to avoid any unclear questions. Newborns with malformations were excluded from the study. The exclusion criteria were parental occupational exposure to chemical agents or radiation, parental chemotherapy or radiotherapy during life, parental addiction to drugs of abuse and alcohol, and malformations in the newborn or siblings. The questionnaire was based on experiences from the NewGeneris project  adjustedCord blood samples were collected within a 10-month period. Samples were frozen at −80 °C for sex hormones and IL-6 analyses within a few hours. Cell culture for MN assay was launched within 24 h after delivery.The Ethics Committee of the University Hospital Center “Zagreb”, Zagreb, Croatia, approved the study (021-1/41-18).2 in an incubator) in tissue culture flask  with a total culture volume of 7.9 mL. After 44 h, cytochalasin B  was added at a final concentration of 6 µl/mL. At 72 h, cell cultures were harvested, centrifuged, and 90 mM KCl hypotonic solution  was added at 4 °C. After 15 min, samples were centrifuged and fixed in methanol:acetic acid 3:1 twice. The suspension was dropped on clean slides and stained by Giemsa . Duplicated cultures were prepared for each subject. Slides were double coded.Umbilical cord blood was collected in heparinized and EDTA tubes  immediately after delivery. For micronucleus assay, heparinized blood was used. The cell culture for MN assay was initiated by phytohemagglutinin  within 24 h after delivery. Umbilical cord blood was diluted by RPMI 1640  1:3. Cultivation was carried at 37 °C  for the quantitative determination of testosterone (2P13) and estradiol .®ELISA, Catalog Number D6050, R&D Systems Inc., Minneapolis, MN, USA). Briefly, serum samples and standards were added to microplate wells coated with a monoclonal antibody specific for human IL-6. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for human IL-6 was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells, and color developed in proportion to the amount of IL-6 bound in the initial step. The color development was stopped, and the intensity of the color was measured.The serum concentration of IL-6 was measured by human IL-6 sandwich enzyme immunoassay according to the manufacturer  were assigned values of 0.5 LOD (LOD = 0.7).To assess associations between mothers’ age, residency, smoking, dietary habits, biocides and disinfectants usage, and wall painting/decorating, on the one hand, and hormone levels, IL-6, and markers of genome damage, on the other, multiple regression models were used.All regression models were controlled for the mother’s place of residence, smoking status, and newborn’s gender. In addition to these three confounders, only one independent variable was entered at a time in each regression model due to the small sample size. To control for multiple comparisons, Bonferroni correction was applied to models corresponding to the same dependent variable . In the case of testosterone and E/T ratio, where regression models were separated by the newborns’ sex, this correction was applied to models for each sex separately.Associations with estradiol, testosterone, E/T ratio, and nuclear division index (NDI) as dependent variables were analyzed in linear regression models. In order to approach the normality of residuals, these dependent variables were log-transformed prior to the analyses. For all other dependent variables, logistic regression models were used. For logistic regression models, dependent variables were dichotomized as follows: IL-6—above LOD vs. below LOD; MN/1000—greater than 2 vs. 0–2; bridge/1000—greater than 0 vs. 0; bud/1000—greater than 0 vs. 0. Since only two mothers with rural residence had IL-6 values below LOD, in this case, Firth’s correction for the logistic regression model was applied.N = 3), this variable was not used as a predictor in regression models. However, as a sensitivity analysis, results from regression models with statistically significant associations were compared to the results of models with the same variables on the subsample of mothers reporting no alcohol consumption.Due to the small number of mothers reporting alcohol consumption  were assessed using Spearman’s correlation.All statistical analyses were conducted using statistical programs STATA, version 14.2  and R version 3.5.0 .In the study, cord blood samples of 50 full-term newborns of both sexes (25 girls) were analyzed for E, T, IL-6 levels, MN, and NB frequency and associated with different exposure factors during pregnancy. All mothers were healthy during pregnancy and at the time of delivery. In total, 75% of newborns were born vaginally.A detailed questionnaire was used for data collected on mother occupational and environmental exposure, age, weight gain during pregnancy, delivery type, residency, diet, smoking and drinking habit, biocide usage, and wall painting/decorating. The residency was defined by the mother’s address during the pregnancy as agricultural in the case when the residence was surrounded with agriculturally-used soil during pregnancy. All mothers consumed vegetables and fruits more than three times per week, and therefore, these parameters were not included in the analysis.t-test, p = 0.341). The mean body mass gain for mothers was 13.80 ± 4.84 kg .The mean age of mothers was 32.10 ± 5.40  are shown in Associations that were statistically significant in multivariate logistic regression models  are shown in p = 0.012 for mothers who did not report alcohol consumption; statistically significant only if Bonferroni’s correction was not applied).All associations that were statistically significant in multiple regression models with Bonferroni’s correction remained significant after excluding three mothers who reported alcohol consumption, except for association of milk consumption and estradiol (p < 0.001); E and T ; E and IL-6 ; E and NB frequency ; for E/T ratio and NB frequency .When association of the dependent variables was assessed, a significant association was found for E and E/T ratio  bottles , althougons only . Similarons only ,49. Decrons only ,51.Significantly increased IL-6 levels in newborns of mothers with agricultural residency in comparison with newborns of mothers with urban residency was in concordance with only one other similar study that describes the association between pesticides and IL-6 in newborns . HoweverMicronucleus and NB frequencies were not significantly associated with newborns with any of the considered exposure types in multiple logistic regression models in the current study. Our result regarding the MN frequency in newborns was in concordance with published data ,57. SuchAlthough, apparent only, before adjustment for multiple testing, significantly increased levels of E in newborns of mothers who consumed dark meat more than three times per week deserve attention. The origin of E in newborns of mothers with such diets may be a consequence of meat contaminated by hormones in order to increase the production of muscle meat . AlthougOur study limitation was the small number of subjects, which is not rare in the case of biomonitoring of the newborn population. However, statistical analysis and interpretation were carefully reasoned in order to achieve the reliability of results. In addition, measurements of xenobiotics in cord blood are planned to be conducted in the future. However, the significant added value of this study is the groundbreaking integration of biomarkers in order to provide insight into the mechanisms known to be critical during intrauterine development with possible lifelong health consequences.In conclusion, our study confirmed the significance of sex hormones as biomarkers of transplacental exposure and their significant association, while the association with MN frequency was not detected. The need for sex-specific preventive measures and risk assessment had already been recognized. Levels of E, T, and IL-6 showed higher sensitivity as biomarkers of transplacental exposure in this study than the MN assay, pointing the significance of multiscale biomarker application in biomonitoring. The introduction of an integrated analysis of the crosstalk between genome damage, cytokine levels, and sex hormone levels might contribute to a personalized approach to a newborn just as lifelong medical care.


Acute abdominal pain is often caused by intestinal obstruction, with high morbidity, and mortality, so that the early diagnosis is particularly important. Currently, both spiral CT and ultrasound are common imaging diagnostic methods. However, the accuracy and practicality of the diagnosis are controversial. Therefore, the purpose of this study is to systematically evaluate the accuracy and practicality of spiral CT and ultrasound in the diagnosis of intestinal obstruction.Retrieval of English database  and Chinese database  by computers. From the establishment of the database to October 2020, a diagnostic experimental study on the diagnosis of intestinal obstruction by ultrasound and spiral CT was conducted. Two researchers independently conducted data extraction and quality evaluation of literature on the included studies, and Meta Disc1.4 and RevMan5.3 were used for meta-analysis on the included literature.Sensitivity, specificity, po-sitive Likelihood ratio, NE-Gative likelihood ratio, diagnostic odds ratio and other indicators were used to determine the diagnostic efficacy of ultrasound and helical CT.This study is aimed at providing an evidence-based basis for clinicians to choose an appropriate or optimal diagnostic method by comparison of the accuracy and practicality between spiral CT and ultrasound in the diagnosis of intestinal obstruction.The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences.DOI 10.17605/ OSF.IO / Q5RNS. Clinical manifestations include nausea, emesis, colicky abdominal pain, and cessation of passage of flatus and stool. If lacking effective and positive treatments, patients with severe pain will have serious symptoms such as intestinal ischemic and necrosis, even perforated and shock. All of these threaten the lives of patients. The key point of treating intestinal obstruction is performing effective diagnosis to make sure the lesion and pathogenesis in time. All of these measurements are important for providing an early treatment regimen. With no specificity and sensibility of the clinal manifestations, physical examination, and laboratory examination results, iconography is widely applied to diagnose and determine treatment methods.Intestinal obstruction is defined that intestinal contents caused by any cause that cannot function properly and pass through the intestine smoothly. With high clinical morbidity and mortality, intestinal obstruction is a common cause of acute abdominal pain, which the beginning is acute, the progress is quick. Studies have suggested that X-ray is no longer used to evaluate abdominal symptoms. In stark contrast, however, ultrasonography and spiral CT are more accurate in diagnosing intestinal obstruction and providing important differential diagnosis. Spiral CT has become the preferred imaging method in patients with known or suspected obstruction depending on its ability to provide an all-around perspective of the intestinal tract, vascular system, mesentery, omentum, retroperitoneum, and peritoneum.However, with the in-depth study of iconography, CT also has many problems such as delayed diagnosis, high cost, and radiation.A flat abdominal radiograph is often used as the first imageological examination for diagnosing acute abdominal pain and intestinal obstruction. Still, it lacks accuracy and sensitivity, especially in diagnosing a closed-loop, ischemic, or strangulating obstruction.,10 But some other studies suggested that the accuracy, obstruction site, cause of obstruction, and the coincidence rate of obstruction type diagnosed by spiral CT.,12Currently, there is no unified conclusion on the accuracy and practicability of the above methods in indiagnosing intestinal obstruction. This systematic evaluation aims to evaluate the accuracy and practicability of the 2 methods in diagnosing intestinal obstruction to provide an evidence-based basis for clinicians.In recent years, ultrasound is more and more widely used in the diagnosis of intestinal obstruction. Some studies suggested that ultrasound is more accurate than CT in the diagnosis of intestinal obstruction.22.1 Moreover, it has been registered on the open science framework (OSF) on October 23, 2020 (registration number: DOI 10.17605/ OSF.IO / Q5RNS).This systematic review protocol and meta-analysis have been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P).2.2Since this is a protocol with no patient recruitment and personal information collection, the approval of the ethics committee is not required.2.32.3.1We will collect case-control studies and cohort studies of comparing ultrasound and spiral CT in the diagnosis of intestinal obstruction, and the language will be limited to Chinese and English.2.3.2Patients diagnosed with intestinal obstruction by ultrasound or spiral CT and confirmed by pathological examination according to the gold standard, regardless of nationality, race, age, gender, course of the disease, etc.2.3.3Ultrasound examination was performed in the observation group and a spiral CT examination was performed in the control group, with no limitation on the type of inspection equipment. All patients accepted the accuracy of the imageological examination and pathological examination.2.3.4Sensitivity (SEN), specificity (SPE), po-sitive likelihood ratio (+LR), ne-gative likelihood ratio (-LR), diagnostic odds ratio (DOR), and confidence interval (CI) of intestinal obstruction by ultrasound or spiral CT.2.41.Republished paper;2.The published literature is abstract or data is incomplete after contacting the authors;3.Meeting abstracts, comments, abstracts, reviews, case reports, animal experiments, etc;4.Studies not validated by the Gold Standard.2.5“Ultrasound,” “spiral CT,” and “intestinal obstruction” in Chinese were searched in the Chinese database, including CNKI, WAN FANG, VIP, CBMDISC. “Ultrasound”, “Spiral CT”, “Spiral Computed Tomography”, “bowel obstruction”, “Intestinal obstruction” in English were searched in English database, including PubMed, EMBASE, Web of Science and the Cochrane Library. From the establishment of the database to October 2020, all Chinese and English literature on the comparison of accuracy and practicability between ultrasound and spiral CT in the diagnosis of intestinal obstruction. Take PubMed as an example, the search strategy is shown in Table 2.6Referring to the method of research selection in version 5.0 of the Cochrane collaboration Network system Evaluator Manual, according to the PRISMA flow chart, the 2 researchers used the EndNote X9 document management software to independently screen and check the literature according to the above inclusion and exclusion criteria, and check each other, if there were different opinions, negotiate with a third party to resolve the differences. According to the inclusion and exclusion criteria in literature, relevant data were extracted independently from all eligible studies and recorded in Excel 2013. ①The basic features of the included studies including the first author, published year, language, country, case number, imaging method, the gold standard, and so on.②Key elements of bias risk assessment.③The concerned outcome measurement data, such as true positive values, false-positive values, false-negative values, etc. The screening process of the literature is shown in Figure 2.7 The tool consists of bias evaluation and applicability, including case selection, tests remained to be evaluated, gold standard, the process of cases, and progress. “high risk,” “low risk,” or “unclear” was given for all areas covered. Evaluation of risk bias according to the performance of the included literature in the above evaluation items and cross-checked after completing respectively. A discussion was required if there was a difference. If no agreement can be reached, a third party researcher was required.Evaluated the bias risk of the included literature by QUADAS-2 evaluation standard of quality.2.82.8.1I2 index was used to judge the size of heterogeneity. If P > .10 and I2 < 50% indicated the heterogeneity is small among studies, then the fixed-effect model was used for the merger. If P ≤ .10 and I2 > 50% indicated the heterogeneity is large among studies, then random-effects model was used for the merger. Calculated and merged sensitivity (SEN), specificity (SPE), positive likelihood ratio (+LR), negative likelihood ratio (-LR), diagnostic odds ratio (DOR) and confidence interval (CI). Drew and merged the summary receiver operating characteristic curve (SROC), then got area under the curve (AUC). Meanwhile, sensitivity analysis was performed to evaluate the results stability by excluding the included references one by one.RevMan 5.3 and Meta Disc 1.4 was used in meta-analysis. 2.8.2If there is a lack of data in the article, please contact the author by email to supplement relevant information. If there is no way to contact authors or get relevant data, a descriptive analysis will be conducted instead of meta-analysis.2.8.3This study will carry out a subgroup analysis based on the different patient characteristics, index and reference tests, and outcome indicators.2.8.4 Moreover, Egger test and Begg test were used for the evaluation of potential publication bias.If there is more than 10 studies, the Deek funnel plot will be conducted to evaluate the potential degree of publication bias.2.8.5 The evaluation content includes bias risk, indirectness, inconsistencies, uncertainty, and publication bias. The quality of evidence will be rated as high, medium, low, or very low.Grading of Recommendation Assessment, Development, and Evaluation (GRADE) will be conducted to grade the evidence for outcome measures.3Intestinal obstruction is a common type of acute abdomen, mainly caused by intestinal contents that cannot passing through smoothly, which leads to pathological changes. Therefore, intervention treatment is needed in time. A clear diagnosis is important because different intestinal obstruction causes, pathogenic site, and degrees should be conducted with different therapeutic schedules.The characteristic ultrasonic images of intestinal obstruction include “gas-liquid stratification”, “images like a fishbone”, “images like keyboard”, “images like bread”, “images like a concentric circle”, “images like sleeve sign”, etc.,18The biggest limitations on ultrasound are obesity and the presence of large amounts of intestinal gas.,20 Some studies pointed out the difference between the 2 methods is that CT can clearly show the specific parts of the intestinal obstruction and have a high value on the judgment of localization and causes of the lesion site. Still, ultrasound can show the specific structure of the intestinal wall in the abdominal cavity by the acoustic window to clearly observe intestinal obstruction.,21During the examination of intestinal obstruction by ultrasound, the continuous and obvious dilatation of intestinal canal, thickened edema of the intestinal wall, hydrops and pneumatosis in the enteric cavity and enhancement or weak even disappearance of bowel peristalsis were observed. The blood supply changes of mesenteric vessels and intestinal wall and ascites characteristics were also observed.There are advantages and disadvantages in diagnosing intestinal obstruction by Ultrasound and spiral CT, which are controversial in the clinical choice. And there is no systematic review of the subject currently. This study will systematically and comprehensively evaluate the accuracy and practicability of ultrasound and spiral CT in intestinal obstruction diagnosis based on the summary of current studies. The results of this study will provide an evidence-based basis for judging the accuracy and practicality of ultrasound and spiral CT in the diagnosis of intestinal obstruction.However, this systematic review has some limitations. Differences in the frequency and thickness of spiral CT scanning of the ultrasound probes used in the included studies may result in certain clinical heterogeneity. Due to language competence limitations, we can only search for English and Chinese literature and may ignore studies or reports in other languages.Data curation: Qingya Yang, Fan Zhao.Funding acquisition: Fan Zhao.Investigation: Qingya Yang.Project administration: Long Su.Resources: Qingya Yang.Software: Junfeng Qi.Writing – original draft: Qingya Yang.Writing – review & editing: Qingya Yang, Fan Zhao.


This study calls for further focus on the role of IFITM3 variants in the mechanism of cellular invasion of SARS-CoV-2, their impact in COVID-19 severity and their possible implications in vaccination efficacy.Evidence was brought forward in England and the USA that Black, Asian, Latino and Minority Ethnic people exhibit higher mortality risk from COVID-19 than White people. While socioeconomic factors were suggested to contribute to this trend, they arguably do not explain the range of the differences observed, allowing for possible genetic implications. Almost concurrently, the analysis of a cohort in Chinese COVID-19 patients proposed an association between the severity of the disease and the presence of the minor allele of rs12252 of the Interferon-induced transmembrane protein 3 (IFITM3) gene. This SNP, together with rs34481144, are the two most studied polymorphisms of IFITM3 and have been associated in the past with increased severity in Influenza, Dengue, Ebola, and HIV viruses. IFITM3 is an immune effector protein that is pivotal for the restriction of viral replication, but also for the regulation of cytokine production. Following up on these two developments in the ongoing SARS-CoV-2 pandemic, the present study investigates a possible association between the differences in mortality of ethnic groups in England and the combined haplotypes of rs12252 and rs34481144. The respective allele frequencies were collected for 26 populations from the 1000 Genomes Project and subgroups were pooled wherever possible to create correspondences with ethnic groups in England. A significant correlation ( Emerging scientific evidence from international  and UK  COVID-19). However, race disparities in those diseases are not large enough to fully explain the COVID-19 death disparity , diabetes, hypertension, obesity, etc. are more common in minorities compared to Caucasian populations and have all been associated with adverse outcomes in COVID-19 . Howeverisparity . Factorsisparity . Neverthisparity .As the importance of genetic polymorphisms (SNPs) in the modulation of individual susceptibility to, and severity of, infectious diseases has been well established , we turnThe minor allele of rs12252  has been associated with rapid progression of acute HIV infection , with thSARS-CoV-2 uses primarily the ACE2 receptor as main point of entry and the host cell serine protease TMPRSS2 for viral spike (S) protein priming . Severe The sole incentive of the current research is to help improve the existing and future treatment protocols for severe COVID-19 patients, and in no case to provide DNA-based arguments that may be used to mask existing social inequalities or racism.https://discovery.ucl.ac.uk/id/eprint/10096589). The rs12252 (A>G) and rs34481144 (C>T) allele and haplotype frequencies were collected for all available 1000 Genomes Project ancestral populations, from LDlink (LDhap tool: https://ldlink.nci.nih.gov/?tab=ldhap), specifically five major groups, that is, African (AFR), Ad Mixed American (AMR), European (EUR), East Asian (EAS) and South Asian (SAS), comprising 26 subgroups in total  of ethnic groups in England, adjusted for age and region, were adopted from the study by in total  . Secondly, we considered only 5 aligned items, specifically the sequence of (a) African groups, followed by (b) South Asian, followed by (c) White Non-British, (d) Chinese, and finally (e) White British, a permutation event with 1/120 chance to occur randomly, or p = 0.008. In both cases the correlations proved highly significant. It is noteworthy, that subgroups EUR-IBS (Iberian Population in Spain) and EUR-TSI , representative of two countries that suffered higher death rates than other European countries, share the highest h1 ratio between all European subgroups. Subsequently, in order to assess the potential strength of the theorized association, the rankings of pooled h1 ratios and SMR, per group, were linearly and significantly correlated, with Pearson r = 0.9687, p = 3 × 10−4 (>3.5σ)  .The calculated level of correlation appears to be remarkable, considering the possible discrepancies in the pooling of the available ancestral groups, but also the expected multi-parametric causes of the observed COVID-19 SMR in England’s ethnic groups . On one hand, the alignment of ethnic group rankings between SMR and un-pooled h1 ratio, is less than 1/120 probable to occur randomly at the worst case, and less than 1 in 3.6 million probable to occur randomly at the best case. In other words, if one hypothesizes that the reported SMR rankings are solely due to socioeconomic factors, then one would conclude that socioeconomic factors would be in perfect alignment with h1 ratios. The possibility of the above to occur seems highly unlikely, thus pointing to the fact that differential allele frequencies play a potentially important role in the reported SMR. On the other hand, the level of correlation (>3.5σ) between SMR and pooled h1 ratio, confirms the previous alignment and appears strong enough to suggest a possible causal link, albeit in this case, the pooling process may have introduced some discrepancies. The potential introduction of pooling discrepancies is expected and is impossible to quantify given the available data. However, the purpose of the pooled analysis was mainly to reinforce the primary association between IFITM3 and COVID-19 severity rather than to (indirectly) infer causality. Considering the parametric uncertainty of the pooled analysis it is probably inadequate to suggest a causal link, based on the strength of the observed correlation alone. However, taken together, this set of results constitutes a clear and valid starting point for designing further investigations regarding the role of IFITM3 in COVID-19 severity and appears as one more piece of evidence towards this direction. Therefore, the main point of this analysis is that the two examined SNPs should preferably henceforth be studied under a combined haplotype and not separately, as was performed so far for SARS-CoV-2, and a great variety of other viruses.Before proceeding to the discussion of the implications of the above conclusion, it is interesting to also view the present observations in the context of data from the USA. A preliminary analysis of death rates from COVID-19 in New York City shows 92.3 deaths per 100,000 population among black or African American people, followed by Hispanic or Latino people (74.3), then by white (45.2) or Asian (34.5) people . The samThe fact that the proposed risk haplotype (A_C) involves the reference alleles of both studied SNPs, appears as counterintuitive. Especially so, since it has been suggested after analysis of a Chinese cohort, that it is the minor allele rs12252:G that is linked to increased COVID-19 severity . In factAs part of the IFITM family of proteins, one of the evolutionary ancient first lines of antiviral cellular defenses, the localization in endosomal or lysosomal membrane, or at the surface, for example, of CD4+ T cells, and the exact antiviral mechanism of IFITM3, is regulated by many different post translational modifications, mainly palmitoylation, ubiquitination and phosphorylation. It is shown that genotypic variants of IFITM3 play a role in diversifying a host’s potential antiviral repertoire, in conjunction with selective post translational modifications, and therefore should not be considered de facto as risk factors but rather as trade-offs in antiviral specificity . This isn = 80 COVID-19 cohort with Chinese patients  = 0.54 and q2 = 0.29 according to Hardy-Weinberg equilibrium, or borderline significant p = 0.04, when q (frequency of rs12252:G) = 0.47, with q2 = 0.22. Therefore, it is equally unsafe to associate homozygote rs12252:GG with COVID-19 severity.The recent suggestion that rs12252:G is the risk allele in a patients , appearsp = 0.0008), in agreement with our current outcomes , found that rs12252:G was negatively correlated with the SARS-CoV-2 mortality rate , vs H2 (G_C), vs H3 (A_T) rs12252_rs34481144 haplotypes in SARS-CoV-2 infectivity and viral spread. Related in silico investigations should include the examined combined haplotype in their analysis and consider non-additive interactions. It is notable how various ongoing GWAS studies  did not


The participants were 202 elementary school students (average age: 11.6 ± 1.5 years). The garden-based integrated intervention program was conducted during regular school hours for a total of 12 weeks. The program, based on a mediator model for improving children’s eating behavior, included gardening, nutritional education, and cooking activities utilizing harvests. In order to examine effects of the program, the mediating factors related to children’s eating behavior were evaluated using pre-post questionnaires. As a result of the program, dietary self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, vegetable preference, and vegetable consumption were significantly increased, and food neophobia was significantly decreased. In addition, there were positive correlations between most mediating factors. Thus, the garden-based integrated intervention developed in this study was effective in improving children’s eating behavior for vegetables.This study was conducted to develop and verify the effects of a garden-based integrated intervention for improving children’s eating behavior for vegetables. A pre-pos Nutrition in growing children is very important; dietary habits that affect the nutritional state established in infancy and childhood, are solidified in adolescence . The dieNutritional status at school age affects not only physical aspects but also emotional and intellectual aspects . AccordiAccording to the Korea National Health and Nutrition Examination Survey, the average daily vegetable consumption of children aged 6 to 11 years is about 148 g, which is about one-third (420 g) the recommended daily vegetable consumption for this age group. As a result, more than half of all children in South Korea consumed less than the recommended amount of nutrients such as calcium and vitamin C, which are derived mainly from vegetables .Accordingly, various methods such as school garden programs, nutrition education, or cooking activities have been attempted to improve children’s eating behavior worldwide ,12,13. IHowever, there are not enough studies to investigate the effect of garden-based integrated intervention and to analyze the mediating effects of factors affecting children’s eating behavior. Therefore, this study was conducted to develop and determine the effects of a garden-based integrated intervention program combining nutrition education and cooking activities, based on mediating factors, for improving the eating behavior to vegetables of elementary school students.For recruitment of participants, a letter regarding the garden-based integrated intervention program was sent to elementary schools in the Seoul area, South Korea. Based on school garden environment, available grade levels, and number of students who agreed to participate, elementary schools C and K were selected. Promotional materials about the study and the program were sent to all parents of 3rd in the school C and 6th graders in the school K. Finally, the parents of all students provided consent for them to participate in the study (95 3rd graders in the school C and 107 6th graders in the school K). The average age of the participants was 11.6 ± 1.5 years. With regard to gender distribution by grade, there were 45 (47.4%) boys and 50 (52.6%) girls in the 3rd grade and 54 (50.5%) boys and 53 (49.5%) girls in the 6th grade. Before the program, the researcher visited the schools and conducted an orientation session in which the purpose of this study and schedule were described, precautions were listed, and written informed consent was obtained from participants. This study was approved by the Institutional Review Board of Konkuk University (7001355-201805-HR-242).A 12-session integrated intervention program, incorporating gardening, nutrition education, and cooking activities using harvests, was developed . This prIn order to select mediating factors affecting children’s eating behavior for vegetables, related studies were identified by using the following keywords in databases such as Web of Science, ProQuest Dissertation and Theses, Academic Search Premier, and Korean National Assembly Digital Library: (1) gardening-related keywords , (2) eating behavior-related keywords , and (3) children-related keywords . As per the search results, 40 research papers were used as reference to verify the effectiveness of the garden-based program to improve children’s eating behavior. The five factors that mediated the association between garden-based activity and children’s eating behavior for vegetables and fruits extracted from 40 studies were “skills required to engage in gardening” ,24,25, “Moreover, sub-elements of the social cognitive theory were incorporated in each session of the program according to the relevant themes . Social Solanum tuberosum), lettuce (Lactuca sativa), tomato (Lycopersicon esculentum Mill.), eggplant (Solanum melongena), carrot (Daucus carota), and beet (Beta vulgaris var. cicla). Moreover, the gardening knowledge education was focused on the cultivation, harvesting, and management methods of each crop, referring to Unit 2-1 (Growing Plants in Everyday Life) of a 6th-grade Korean elementary school textbook [The activities in the 12-session program were focused on gardening, nutrition education, and cooking activities using the harvests. The gardening activities mainly included plant cultivation, such as making plant beds, transplanting, watering, weeding, and harvesting plants. The participants cultivated seasonal plants, such as potato  with their friends and teachers. An example of the 11th session in the garden-based integrated intervention is presented in The 12-session garden-based integrated intervention was conducted once a week and 40 min per session. It was run by researchers who are a horticultural therapist certified by the Korean Horticultural Therapy Association and two volunteers during regular class hours. Gardening activities were conducted in the schools’ outdoor gardens, with a size of about 6 mIn order to identify the mediating factors influencing children’s eating behavior, self-reported questionnaires were used before and after the program. A pilot test was conducted by 10 elementary students to determine the level of difficulty and suitable questions for elementary school students before starting the study.Self-efficacy was assessed with a dietary self-efficacy questionnaire on fruit and vegetable consumption for elementary students . A 24-itTo assess outcome expectancies for vegetable consumption, an outcome expectation questionnaire for children’s fruit and vegetable consumption, developed by Domel et al. , was modIn order to assess aversion to new foods, the 10-item food neophobia scale developed by Pliner and Hobden  and tranTo investigate vegetable preferences, a list of representative vegetables from the Dietary Reference Intakes for Koreans 2015 was used . Some ofTo assess nutrition knowledge, the textbook for Korean elementary students published by the Ministry of Food and Drug Safety and the Ministry of Education was referred to in order to select items that matched the educational contents of this program. The items were revised and supplemented after consultation with elementary school teachers. In order to classify the degree of difficulty by grade, 15- and 21-item were graded by referring to textbooks for 3rd graders and 6th graders, respectively . Each nutritional knowledge item had three response options: “Yes,” “No,” or “I don’t know.” Participants received 1 point for a correct answer and 0 points for an incorrect answer. The perfect score of the 3rd students’ questionnaire is 15 points, and that of the 6th students’ questionnaire is 21 points. Higher scores were indicative of greater nutrition knowledge.For the assessment of gardening knowledge, the 6th grade textbook of Korean elementary was referred to in order to select items that matched the educational contents of this program, and after consulting with an expert such as elementary school teachers and specialists in horticulture, the researcher revised and supplemented the scale. The final 10-item scale included questions about environmental factors necessary for plants to grow, knowledge about plant cultivation, and so on . For each question, the participant received 1 point for the correct answer and 0 points for an incorrect answer; the total score ranged from 0 to 10. Higher scores were indicative of greater gardening knowledge.To investigate daily vegetable consumption, a dietary record sheet was developed to note the amount of food consumed based on the 2010 Dietary Reference Intakes proposed by the Korean Nutrition Society. The contents of the record sheet included the type and quantity of foods eaten during the day and how to measure and record food consumption. Before and after the program, children were instructed to record two days’ meals with their parents. Based on the dietary record sheet, the type and amount of food consumed were entered into the CAN 4.0 program developed by the Korean Nutrition Society to calculate the frequency of vegetable consumption. The average vegetable consumption frequency two days before and after the program was calculated and the values analyzed.The researchers revised the questionnaires developed by Park et al.  and Lee We also developed a satisfaction questionnaire for parents and teachers. It consisted of the following items: overall satisfaction, satisfaction with education and newsletter contents, re-participation, possibility of recommending the program to others, whether the children’s eating behavior improved after the program, and so on.t-test was conducted. Cronbach’s alpha was also calculated to test the reliability of the evaluation tools. Pearson’s correlation analysis was conducted to examine the correlations between mediating factors for improving eating behavior. A correlation coefficient of less than ± 0.2 means little correlation, that of ± 0.2 to ± 0.4 means low correlation, that of ± 0.4 to ± 0.7 means somewhat high correlation, and that of more than ± 0.7 means high correlation [p < 0.05. Satisfaction with the program and demographic information were analyzed using spreadsheet software in Excel .To compare and analyze the mediating factors related to eating behavior for vegetables before and after the program, a paired relation . All stap < 0.001; p < 0.05).After the program, the self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, and vegetable preference scores of all participating children significantly improved , but there were no significant differences in the scores of 6th graders (p > 0.05).Grade-level analysis of gardening knowledge, nutrition knowledge, outcome expectancies for vegetable consumption, self-efficacy, and vegetable preference showed significantly improved scores for both grades  were satisfied with the overall program and more than half were satisfied with the per session and sessions per week. The most preferred activity was cooking with the harvests (68.0%). Harvesting was the next most popular activity, at 8.8%. The next preferred activities were watering (8.8%), planting (7.7%), making a garden plot (2.1%), managing the garden (2.1%), weeding (2.1%), and sowing seeds (0.5%). Additionally, 81.9% (158 students) answered that they hoped for a continuation of the program and 83.9% (162 students) responded that they would recommend the program to friends.According to parents’ overall program satisfaction, 75.3% (113 people) answered that they were satisfied. Regarding the program contents, 87.3% (130 people) responded that they were satisfied with the horticultural activities, cooking activities, and nutrition education. Regarding post-program changes, 84.4% (130 people) answered that there were positive changes in their children’s eating behavior.Regarding teachers, all indicated that they were very satisfied with the overall program. In addition, all responded that they were satisfied with the professionalism and attitude of the instructor, the per session, and sessions per week of the program. Additionally, all answered that there were positive changes in their students’ eating behavior after the program.This study was to investigate the effects of a garden-based integrated intervention for children’s eating behavior to vegetables and to identify the mediating factors that affect eating behavior for vegetables in children. As the result of the program, dietary self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, vegetable preference, and vegetable consumption were significantly increased, and food neophobia was significantly decreased . MoreoveThe garden-based integrated intervention significantly increased dietary self-efficacy of children who participated in this study . MoreoveOutcome expectancies related to vegetable consumption of children significantly increased after the garden-based integrated intervention . The outChildren’s food neophobia scores significantly decreased after participating in the garden-based integrated intervention . MoreoveChildren’s gardening and nutrition knowledge scores improved after the garden-based integrated intervention. Gardening can be an effective strategy to improve children’s knowledge of horticulture and increase their vegetable consumption by involving them in the process of planting, growing, harvesting, and preparing vegetables . PreviouChildren’s vegetable preferences and consumption significantly increased . This reIn conclusion, the purpose of the present study was to investigate the effects of the garden-based integrated intervention for improving children’s eating behavior for vegetables by measuring the mediating factors related to eating behaviors in children. The result shows the potential of garden-based integrated intervention for improving children’s eating behavior for vegetables. Future study of the topic would be informed by a larger cluster randomized controlled trial to verify the effects of the intervention. Moreover, it would be valuable to verify the lasting effects on eating behaviors through the intervention. Furthermore, it would be interesting to involve parents who have a direct impact on their eating behaviors in the intervention.


The animals were divided into ontogenetic groups: male calf, female calf, juvenile female, juvenile male, and adult male, and reproductive groups: pregnant female, lactating female, and pregnant plus lactating. The blood from all the animals was analyzed for general stress , metabolic stress (ACAT2 and THRA), and immunity-related genes  using qPCR. YFPs living in Poyang Lake showed an increased relative expression pattern for IFNγ, IL1ra, IL4, ACAT2, and CRPL across all the ontogenetic groups with significantly higher expression in adult males. In contrast, YFPs living in the TZO showed a significantly higher expression in 13 of 15 genes analyzed in the male calf group. Across the reproductive states for porpoises living in Poyang Lake, eight of the 15 genes in the pregnant female and three of the 15 genes in the pregnant plus lactating group had a significantly higher expression level. However, in YFPs living in the TZO, eight of the 15 genes showed significantly higher expression in the pregnant and lactating groups. There was significantly a higher expression of most of the genes in porpoises living in the TZO compared to the age-matched groups from porpoises living in Poyang Lake. The exception was the pregnant female group. The higher relative expression of stress and immune genes in the TZO porpoise population compared to porpoises living in Poyang Lake suggests the effects of worsening habitat quality, possibly indicating water pollution and lack of feeding resources.Increasing anthropogenic stressors are potential threats to biodiversity conservation and management of Yangtze finless porpoises (YFPs). The objective of this study was to indirectly compare the habitat quality of a natural reserve, Poyang Lake and a seminatural reserve, the Tian-E-Zhou Oxbow (TZO) in terms of anthropogenic stressors by investigating different stress and immunological parameters in the blood of YFPs. Samples from a total of 74 YFPs from the TZO ( The world’s aquatic environments are undergoing significant alterations because of anthropogenic activities. Currently, approximately 37% of all cetaceans are at risk of extinction . CetaceaNeophocaena asiaeorientalis ssp. asiaeorientalis) is a critically endangered freshwater cetacean  (The Yangtze finless porpoise (YFP) (cetacean . It is ncetacean . For conow (TZO) . Currentow (TZO)  and overow (TZO) . Unfortuow (TZO) . The aniow (TZO) . Since 1ow (TZO) , 2018a. ow (TZO) . Howeverow (TZO) . Despiteow (TZO) .Except for a few physiological studies , 2018a,bThe study was approved by the Ministry of Agriculture of the People’s Republic of China. The Research Ethic Committee of the Institute of Hydrobiology, The Chinese Academy of Sciences reviewed and approved the procedure for animal chasing, handling, and blood sampling (NNSFC 31430080). In this study, no surgical intervention, anesthesia, and euthanasia were used. The whole study strictly followed the Chinese law and ethical guidelines for wildlife.2, but in summer, it extends to 4,000 km2. This lake is fed by five different rivers including the Xiushui River, Xinjiang River, Ganjiang River, Fuhe River, and Raohe River. The maximum depth of the water is 25.1 m (average depth 8.4 m)  .ex situ reserve established in 1992 by the Chinese Government for the conservation of YFPs. It is in Shishou city, Hubei Province on the north bank of the Yangtze River at E112°31′–112°36′, N29°46′–29°51′. The total length of the reserve is 21 km with a width of 1–2 km  and Poyang Lake (n = 33). However, blood samples were only available from 74 individuals. Samples were collected from the YFPs living in the TZO in November 2015 and in March 2015 for the animals living in Poyang Lake. Both populations were classified into various life history categories including male calf, female calf, juvenile female, juvenile male, adult male, pregnant female, lactating female (only in the TZO), and pregnant plus lactating. The body length was used to categorize YFPs into calves, juveniles, and adults  according to the manufacturer’s protocol and procedures.A commercial Bioteke kit  was used according to the manufacturer’s protocol for whole-blood RNA extraction. RNA was then dissolved in 50 μl ribonuclease-free water and frozen at −80°C. The integrity and purity of the RNA extracted were checked by gel electrophoresis and then quantified by a Nanodrop 2000 spectrophotometer before use. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit . Three replicates were used to obtain each average Ct value using the ΔΔCt method.A total of 15 genes were investigated in both porpoise populations . Four gepost hoc test (Tukey test) was used to measure relative variations in gene expression across the age groups  and reproductive groups . An unpaired Student’s t test was used in the reproductive groups of Poyang Lake population to measure differences in the relative gene expression between pregnant and pregnant plus lactating females. Similarly, an unpaired Student’s t test was used to compare one group from one population to its respective group from another population. Normality was assessed using the Shapiro–Wilk test. The alpha (α) set at ≤0.05 indicates a statistically significant difference.All data are expressed as means ± SEM using Graph Pad Prism, version 7.04 . One-way ANOVA followed by a versus juvenile females. No statistical differences were detected for the expression levels of TNFα, FTL, SOD1, THRα, and TXN  in male calves across the age groups. Similarly, the expressions of nine genes were significantly higher in juvenile males across the age groups, while no difference was observed in IL8 and TXN expression .Across the reproductive states in porpoises living in Poyang Lake, 8/15 genes  were expressed in the pregnant vs. pregnant plus lactating females at statistically significantly higher levels. However, the expression of ACAT2, CRPL, and THRA was significantly higher in the pregnant plus lactating vs. pregnant females. No statistically significant difference was observed for the FTL, HSPA14, SOD1, and TXN expression levels . In porpIn porpoises living in the TZO, the expression levels of 11/15 genes in calves were significantly higher compared to calves living in Poyang Lake. However, no statistically significant difference was observed in ACAT2, HSPA14, SOD1, and TXN levels between the two populations .Similar to calves living in the TZO, most of the genes (10/15) were statistically significantly higher compared to the juvenile males living in Poyang Lake. Only ACAT2 levels were statistically significantly higher in the Poyang Lake YFPs, while no difference was observed in the IL8, FTL, SOD1, and TXN levels between the two populations .Unlike calves and juvenile males, most of the gene (9/15) expression levels showed no statistically significant differences. The relative expressions of COX2, IL1α, IL1ra, and THRA were statistically significantly higher in porpoises living in the TZO. However, IFNγ and ACAT2 levels were statistically significantly higher in juvenile females living in Poyang Lake .Similar to juvenile females, most of the gene (9/15) expression levels showed no statistically significant differences between the two populations. Only the expression level of ACAT2 in porpoises living in Poyang Lake and five genes  of YFPs living in the TZO were statistically significantly higher .Interestingly, most of the gene (9/15) expression levels were statistically significantly higher in YFPs living in Poyang Lake. No significant differences were observed in the expression levels of ACAT2, CRPL, HSPA14, and TXN. Two genes (FTL and THRA) were highly expressed in the TZO population .In this group, only the expression levels of 6/15 genes were statistically significantly high. The expression levels of COX2 in porpoises living in Poyang Lake and IFNγ, IL1ra, IL4, IL8, and TNFα levels in porpoises living in the TZO were statistically significantly high .The body-weight/body-length ratios of calves and adult males in the TZO were statistically significantly lower compared to the animals living in Poyang Lake. In other groups across the age groups  and reproductive states , body-weight/body-length was not statistically significantly different. However, the overall trend showed lowered body-weight/body-length in the TZO population .2+) from ferritin, further increasing the iron load in the body  in YFPs living in the TZO, indicating exposure to a potential stressor in their environment. A hallmark of a stressed organism is the high expression of heat shock genes which function as molecular chaperones for maintaining proteostasis . The heathe body . Howeverulations . We did ulations .3 and T4) and thyroid hormone receptors (THRα and THRβ) have an essential role in the regulation of cetacean metabolism. In cetaceans, diet restriction reduces circulating T3 levels but upregulates the expression of both THRα and THRβ levels (3) further activates the generally negative regulation of the gene coding for Thyroid Stimulating Hormone (TSH) which reduces T3 levels  provide information on systemic inflammatory trends . In ceta and IL1 . IL4 is  and IL1 . Instead and IL1 , several and IL1 . The pyr and IL1 . These p and IL1 . We also and IL1 . Chronic and IL1 . It has  and IL1 . IL12 is and IL1 , mostly  and IL1 . The IFN and IL1 . Oxidati and IL1 . The hig and IL1 . HoweverYoung animals are more sensitive to stress  as obserex situ reserves, which should include criteria for location, size, topography, water quality, feeding resources, wetlands, climate, and surrounding communities. At the same time, regular surveys or investigations on the ex situ populations should be conducted to monitor animal health. In cetaceans, inflammatory incidences and immune function impairments are difficult to detect, therefore, cytokine markers would be helpful to monitor the health of YFPs (In summary, our findings indirectly suggest a worsening habitat quality of the TZO both in terms of pollution and feeding resources. Younger animals, especially, are more affected in the TZO compared to animals living in Poyang Lake. The opposite trends we observed for different gene expression levels in both populations across the ontogenetic and reproductive stages could be related to the nature of the different kinds of environmental stressors. The populations of YFPs living in the TZO has increased, however, their persistent exposure to pesticides and declining feeding resources require urgent attention and proper management. Improving feeding resources, regulating the number of porpoises so as not to exceed the carrying capacity of the oxbow, managing poultry and sewage waste, and replacing chemical pesticides with biopesticides around the reservoirs could have a positive impact. Furthermore, detailed studies are needed to investigate feeding resources, fish mortality and morbidity, and quantification of the different kinds of pollutants in both habitats. Further studies are needed to develop the standards for establishing new  of YFPs . In termAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Ministry of Agriculture of the People’s Republic of China. The Research Ethic Committee of the Institute of Hydrobiology, The Chinese Academy of Sciences reviewed and approved the procedure for animals chasing, handling and blood sampling. In this study, no surgical intervention, anesthesia and euthanasia were used. The whole study strictly followed the Chinese law and ethical guidelines for wildlife.GN conceived the study, collected and analyzed the data, and drafted the manuscript. YL analyzed the data. RM, ZM, and KW critically reviewed the manuscript. YH, JZ, and DW supervised and funded the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene and its oxide are nanomaterials considered currently to be very promising because of their great potential applications in various industries. The exceptional physiochemical properties of graphene, particularly thermal conductivity, electron mobility, high surface area, and mechanical strength, promise development of novel or enhanced technologies in industries. The diverse applications of graphene and graphene oxide (GO) include energy storage, sensors, generators, light processing, electronics, and targeted drug delivery. However, the extensive use and exposure to graphene and GO might pose a great threat to living organisms and ultimately to human health. The toxicity data of graphene and GO is still insufficient to point out its side effects to different living organisms. Their accumulation in the aquatic environment might create complex problems in aquatic food chains and aquatic habitats leading to debilitating health effects in humans. The potential toxic effects of graphene and GO are not fully understood. However, they have been reported to cause agglomeration, long-term persistence, and toxic effects penetrating cell membrane and interacting with cellular components. In this review paper, we have primarily focused on the toxic effects of graphene and GO caused on aquatic invertebrates and fish (cell line and organisms). Here, we aim to point out the current understanding and knowledge gaps of graphene and GO toxicity. It is dnar axis . Howevernar axis . The idenar axis ,6.It is the strongest and thinnest known material known to man . GraphenThese advantages of graphene make it desirable for the development of graphene composites through the incorporation of different functional materials. Graphene-based composites have been achieved successfully with organic crystals ,31, inorOn the other hand, graphene oxide (GO) is an intermediate product obtained during the synthesis of reduced GO (rGO) ,44,45. IAt this increasing phase of commercial production, the presence of graphene is evident in the environment at a significant level ,51. IrreIn this present review, we will analyze the biocompatibility and toxicology of graphene and GO in order to evaluate the safety limits needed for the implementation of upcoming researches. We will also try to draw attention towards knowledge gaps to attain valuable insights on the fate and risks presented by graphene and GO on fish in the aquatic environment. Furthermore, we will discuss the key physiological factors involved in influencing the toxicity of graphene and GO in fish in the surrounding water medium, degradation pathways, and the exposure and administration pathways. We believe that the critical study in this field will lead to collection of current information leading to the production of better modified materials emerging in healthcare, diagnostic, and therapeutics.Graphene is inert in chemistry because of the immense delocalized π electron system . The graCrassostrea virginica), both 1 and 10 mg/L GO exhibited a dose-dependent elevated lipid peroxidation. No significant changes in glutathione S-transferase (GST) levels were observed, but a reduction in the total protein levels was found in tissues of the digestive glands at both concentrations (1 and 10 mg/L) of the GO. Overall, the study indicated that short-term GO exposures can induce oxidative stress, epithelial inflammation and adversely affected the overall health of Crassostrea virginica [Crassostrea virginica, elevated lipid peroxidation and changes in glutathione-s-transferase (GST) were observed in tissues of gills and digestive gland exposed at 2.5 and 5 mg/L. Reactive oxygen species (ROS) induced oxidative damage. Therefore, the study suggested that detoxification enzyme such as GST are thought to be involved in stress signaling, leading to harmful effects on cellular health [Invertebrates are important organisms utilized for environmental toxicological studies. They have a relatively short life span, fast reproduction, and high sensitivity towards pollutants. They are also cost-effective and contribute quick results. These organisms are regarded as convenient test species for ecotoxicity studies of new chemicals and nanoparticles . The invirginica . In a sir health .Diopatra neapolitana (a polychaete species) for 28 days. The analysis of physiological (regenerative capacity) and biochemical  depicted negative effects of GO on regenerative capacity of Diopatra neapolitana, the organisms exposed to higher concentrations of GO took long periods to complete regeneration. GO also altered the energy-related responses, such as glycogen content with high polychaetes, which might have resulted from decreased metabolism. The research work concluded that cellular damage happened despite higher activities of antioxidant and biotransformation enzymes in Diopatra neapolitana exposed to GO [Daphnia magna with and without the presence of humic acid (HA). The GO induced significant toxicity to Daphnia magna with a median lethal concentration of 48 h LC50 equal to 84.2 mg/L and 21-day LC50 as 3.3 mg/L. The HA mitigated the acute and chronic toxicity in Daphnia magna, as in the presence of HA, the decreased toxicity of GO was attributed to alleviation of oxidative damage by HA [Daphnia magna was done by Guo and colleagues. Their results demonstrated the accumulation of graphene at 1% of organism’s dry mass. During the time for depuration, these organisms bore roughly constant body burdens after 24 h regardless of initial graphene concentration, whereas, after addition of an algae and humic acid, the depuration resulted in significant release of accumulated graphene but leaving some graphene in the organism. The study concluded that the remaining graphene in Daphnia magna might possess a risk to be passed on to neonates and thus require further risk assessment and relevant evaluation [Ceriodaphnia dubia in acute and chronic assay. The mean effective concentration (EC50) estimated during acute exposure was 1.25 mg/L, whereas chronic exposure of GO resulted in significant decrease in number of neonates, feeding rates, and ROS generation. Their research group concluded that in the presence of GO, there was a shift in the available energy for self-maintenance instead of feeding and reproduction activities [Palaemon pandaliformis, results suggested that GO did not cause acute toxicity at concentration up to 5.0 mg/L after 96 h exposure. However, GO in association with Cd and Zn increased toxicity of the trace elements as suggested by 96 h LC50 of Cd associated with GO 1.7 times less than the 96 h LC50 of Cd alone and 96 h LC50 of Zn associated with GO 1.8 times less than 96 h LC50 of Zn alone. Likewise, co-exposure of GO with trace elements impaired the routine metabolism of Palaemon pandaliformis. The study recommended that more researches and evaluation of data need to be done to understand the toxicity criteria of GO in aquatic organisms or aquatic bodies [Further in a study by De Marchi et al., different concentrations of GO  toxicity were tested over ed to GO . In anotge by HA . Furtheraluation . In anottivities . Moreovec bodies .In determining the cytotoxicity of environmental pollutants, cell lines derived from fish are considered very suitable experimental model. Testing the toxicity of nanomaterials is more complex than other chemical compounds. In nanomaterials, various physiochemical properties such as size, shape, surface area, and surface modification need to be considered prior to experimentation. Other factors including agglomeration, aggregation, sedimentation, dissolution, concentration need to be studied as well their fate in the test systems. If these aspects of nanomaterials are not investigated prior to experimentation, they might interfere with the results and misleading conclusions can be drawn. A study by Lamel et al. was done by exposing topminnow fish hepatoma cell line PLHC-1 cells to a single layer GO and carboxyl graphene (CXYG) (16 µg/mL GO or CXYG) suspensions with an aryl hydrocarbon receptor (AhR). Their results demonstrated pre- and co-exposure of cells to GO and CXYG nanoplatelets induced cytochrome P450 1A (cyp1A) expression, suggesting that graphene nanoplatelets increased the effective concentration of AhR agonists by facilitating passive diffusion into cells by damaging the plasma membrane or transporting them over the plasma membrane via a Trojan horse-like mechanism. Based on their results, it was found that there is an existence of combination effects between nanomaterials and environment pollutants . In a siIn another study, bluegill sunfish (BF-2 cells) were exposed to GO at concentrations of 0, 10, 20, 40, 60, 80, and 100 μg/mL for 24 h. Two biological assays -2,5-diphenyl tetrazolium bromide) MTT assay and neutral red uptake (NR) resulted in cytotoxicity and oxidative stress in BF-2 cells. The tested biomarkers of oxidative stress such as lipid peroxidation, superoxide dismutase, catalase, reactive oxygen species, and 8-hydroxy-2′-deoxyguanosine levels increased, and glutathione level was decreased in BF-2 after treatment with GO. The GO induced a dose- and time-dependent cytotoxicity on BF-2 cells . In addip < 0.05) decreased at 100 mg/L at 48 hpf. Furthermore, rGO and MWCNTs decreased the length of hatched larvae at 96 hpf but no mortality and morphological malformation were observed, which further requires more results and analysis at various concentrations at different time periods [A reliable way of nanomaterials administration to an aquatic organism is oral and direct injection . For dirFurther in a different study, when the zebrafish embryos were exposed to a concentration of 1–100 μg/L to analyze the developmental toxicity, the results yielded impaired DNA modification, protein carbonylation, and excessive generation of ROS. The work also highlighted nonmonotonic response of zebrafish developmental toxicity to GO at μg/L to mg/L levels. On further analysis, transcriptomics revealed deficiencies in the collagen and matrix metalloproteinase (MMP) related genes, which in turn affected the skeletal and cardiac development of zebrafish. Moreover, to contribute to the results of developmental toxicity, metabolomics assay showed inhibition in amino acid metabolism and disturbance in the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs). The work, therefore, demonstrated developmental toxicity on the basis of involved molecular mechanism at trace concentration equal to 10 µg/L .In an interesting study of Hu et al. with parent and offspring zebrafish when GO nanosheets were administered to parental zebrafish at a concentration of 0.01–1 µg/L, GO translocated from water to the brain of parent fish and the offspring with major loss of claudin5a . However, GO did not induce neurotoxicity in the parent fish and significant neurotoxicity occurred in the offspring, exhibiting loss of dopaminergic neurons and reduction in acetylcholinesterase (AChE) activity. Moreover, endoplasmic reticulum damage, autophagy promotion, ubiquitin down-regulation, and increase in β-galactosidase activities were observed, attributing to the failures in the carbohydrate and fatty acid metabolisms. Their work suggested that more researches and data compilation for the toxicity of GO on fish offspring at environmentally relevant concentration are required to do reliable analysis and to establish conclusions . Similar∙OH, and changes in the secondary structure of protein, which, in contrast, were relieved by HA. On the other hand, humic acid reduced interactions between GO and chorion mitigating chorion damage by regulating the morphology, structure, and surface negative charges of GO, indicating a feasible antidotal mechanism for GO in presence of HA [In a study by Clemente and co-workers, zebrafish embryos were analyzed after 5 and 7 days of GO exposure at 100 mg/L and humic acid (HA) at 20 mg/L either individually or together. Regardless of the presence of HA, the larvae exposed to GO for 5 days demonstrated increase in the locomotor activity, reduction in yolk sac size, total length, and inhibition of AChE activity. The larvae exposed to GO for 7 days did not show any significant differences in the locomotor activity, but the RT-PCR gene expression analysis depicted an increase in AChE expression. The work suggested that HA is associated with toxicity risk modulation by GO and some compensatory homeostatic mechanisms might be involved in the effects observed in zebrafish . In a sisynapsin IIa expression at 5 mg/L with down-regulation of dat expression were observed, showing potential compensatory mechanism. At 10 mg/L GO exposure, increase in heart rate, absolute turn angle, brain cell damage, and decrease in dopamine levels were observed. However, no changes were observed on higher concentrations of GO exposure, which is attributed to GO agglomeration. The work concluded that the results of the toxic effects of GO were not dose-dependent and are pre-eminent at lower concentrations, and hence more studies are required to gather relevant data and analysis [hcrt and hcrtr mRNA, while graphene did not obviously disturb the sleep behavior and gene expression patterns [In another study, zebrafish embryos were treated with GO at 0, 0.01, 0.1, 1, and 100 mg/L for 24, 48, and 96 hpf. Results revealed that the GO adhered to and enveloped the chorion of zebrafish embryos via the hydroxyl group interactions and it blocked the pore canals of the chorionic membrane causing hypoxia and hatching delay. Furthermore, GO penetrated the chorion of embryo via endocytosis, damaged the mitochondria, and primarily translocated to the eye, heart, and yolk sac regions involved in circulatory system of zebrafish. In these organs, GO induced excessive generation of reactive oxygen species, thus increasing oxidative stress, DNA damage, and apoptosis. The work highlighted specific adverse effects of GO on embryogenesis and emphasized the potential ecological and health risks of GO . Furtheranalysis . In the analysis . The gropatterns .Fusobacteria and the genus Cetobacterium and Lactobacillus and decreasing the abundance of Firmicutes and the genus Pseudomonas; GR caused marked shifts in the diversity of the gut microbiota. The GFMs also altered the intestinal morphology and antioxidant enzyme activities by inducing more vacuolation and generating more goblet cells [Acute toxicity, oxidative stress, and immune-toxicity of GO were analyzed at 1, 5, 10, or 50 mg/L for 14 days were done in a study using the zebrafish adult model. Histological analysis of the liver and intestine showed cellular alterations including vacuolation, loose arrangement of cells, histolysis, and disintegration of cell boundaries. Malondialdehyde (MDA) level and superoxide dismutase (SOD) and catalase (CAT) activities were increased and glutathione was decreased in the liver after treatment with GO. Moreover, levels of tumor necrosis factor alpha (TNF-α), interleukin-1B, and interleukin-6 depicted induction in immune response. Hence, the work suggested GO exposure in aquatic system caused oxidative stress and immune toxicity in adult zebrafish . Moreoveet cells .Anabas testudineus (the climbing perch) revealed accumulation of cellular lipid peroxides specifically in the mitochondria. Activity of SOD, CAT, and, GST was augmented in contrast to lowered level of reduced glutathione titer. The results also indicated oxidative stress in cell and mitochondria in fish after exposure to GO, thus suggesting compilation of more data for comparison of results and establishments [p ≤ 0.05 for RBC (0.770 cells/μL × 106) in T1 and 0.850 cells/μL × 106 in T2 compared with negative control (1.410 cells/μL × 106). There was no significant decrease p > 0.05 of each PCV in T1 and T2 (31 and 27%), respectively, compared with control negative (35%) and hemoglobin (10.30 and 9 mg/dL) in T1 and T2, respectively, compared with negative control (11.65 mg/dL), while the addition of graphene nanoparticles did not affect the number of WBC in T1 and T2 (15.1 and 19.1 cells/μL × 103), respectively, compared with negative control (15.6 cells/μL × 103) [The effects of GO were observed on the antioxidant metabolism of zebrafish; after 48 h exposure, SOD activity was significantly increased in 20 mg/L, CAT activity in 2, 10, and 20 mg/L, and lipid peroxidation (LPO) had an increase in 2 mg/L, whereas glutathione peroxidase (GPx) was inhibited at 20 mg/L. Later after 168 h recovery in clean water, SOD remained elevated in 20 mg/L, CAT activity remained unchanged and GPx activity was inhibited at 2, 10, and 20 mg/L, and LPO decreased in 2 mg/L. The study suggested that GO exposure disrupted antioxidant metabolism of adult zebrafish in which it was not restored even after 168 h recovery period in clean water . The toxishments . In a seL × 103) . The sumThe unique properties of graphene and GO along with other carbon nanomaterials such as carbon nanotubes and fullerenes have been used for various applications and can be released in environment at significant amounts, posing threat to aquatic species as well as humans. Once released in the bodies of water, graphene and GO can also interact with existing inorganic ions and natural organic matter (NOM), posing threat for significant adverse effects on the ecosystem. However, intentional exposure of graphene in biomedical applications is of interest. The literature cites approximately 60% graphene-related materials applications in biomedical engineering as stated by Mao et al. . This siHere in the present review, we have compiled, presented, and compared the current toxicological scenarios of graphene and GO in aquatic invertebrates and fish model organisms. We compared the paper published number  during the past 16 years (from 2004 to 2020). Results show the relative paper published number for graphene-based nanomaterial toxicity is around 50% less than their fullerene or carbon nanotube counterparts . In addiIn case of continuous waterborne exposure, the developmental stages of the embryo and the duration of exposure are very critical. The embryos exposed in waterborne graphene and GO might agglomerate in the exposure media or on the chorion depleting embryo of oxygen supply and may cause developmental deformities or worst, mortality. The chorion has been reported as a major barrier to prevent nanomaterial uptake by fish embryos . In thisIn this review paper, we hereby summarize that research of graphene and GO toxicity specifically on aquatic organisms is relatively limited. The research on graphene-based nanomaterials toxicity has started taking place progressively in the last decade. Thus, a comprehensive understanding of interaction of graphene and GO with environment, aquatic organisms, and other living systems in vitro and in vivo is essential for their safe usage and further development. The large part of available current literature indicates that graphene-based nanomaterials are cytotoxic. Although the particular mechanism for their toxicity has not yet been established, ROS elevation, lipid peroxidation, nutrient/oxygen depletion, and inflammation have been most widely recognized mechanisms for graphene-based nanomaterials toxicity in aquatic organisms.The applications and uses of graphene and GO are rising evidently in fields of biomedical sciences, supercapacitors, sensors, and construction materials. The release of waste material from these industries can prove to be harmful for environment, aquatic organisms, and humans. Graphene-based nanomaterials interact with natural organic material (NOM), aggregates, adsorbs, and colloids, which in turn can cause toxicity to aquatic organisms through many different body mechanisms. Furthermore, the physiochemical properties of graphene and GO such as particle size, surface functional groups, and oxygen content/surface charges may affect the toxicity upon interaction with aquatic organisms. The data we accumulated demonstrated a lot of information gaps that does not allow establishing a concrete statement regarding the toxicity criteria guidelines. It is also difficult to compare the toxicological effects of graphene and GO between different studies due to diversity in size, shape, surface modification, synthetization techniques, and model organisms. It is, hence, important to understand the toxicity caused by graphene nanomaterials for aquatic organisms in order to facilitate the practical applications of these promising new graphene-based nanomaterials.