Trypanosoma cruzi presents great variability in morphology, virulence, pathogenicity, avoidance of the host immune system, and antigenic constitution, associated with different clinical manifestations of the disease. T. cruzi were cultivated in liver infusion tryptose to determine growth kinetics, morphometry and molecular characterization using restriction fragment length polymorphism polymerase chain reaction.  Two strains of th day. Morphologically, both strains showed short and thin forms and were classified as Group I.  The biological parameters showed sharp growth by the 7T. sherlocki is adapting to the home environment, requiring attention to future problems.Group TcI presents cardiac manifestations and  Trypanosoma cruzi (T. cruzi) (Chagas 1909). In recent decades, the epidemiological pattern of the disease has changed from rural to urban, affecting people living in metropolitan areas due to population mobility, urbanization, and emigration. Thus, an increasing number of cases have been reported in Canada, the United States of America, Africa, the Eastern Mediterranean, and Western Pacific. Due to the high number of undiagnosed and untreated people, along with areas with active transmission, approximately 75 million people are at risk of infection. During the chronic phase, up to 30% of patients suffer from heart disorders, and up to 10% have digestive, neurological, or mixed disorders. Years later, the infection can lead to sudden death, mainly due to arrhythmia or heart failure caused by the destruction of the heart muscle and its nervous system. Furthermore, patients with Chagas disease are at risk of severe manifestations of coronavirus disease 2019 (COVID-19), Therefore, they should be a priority group to be vaccinated because severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can cause myocarditis, and chronic Chagas disease usually leads to a prothrombotic state, cardiac alterations, and secondary thrombotic strokeChagas disease is a parasitic infection caused by the flagellate protozoan T. cruzi. However, characteristics such as defecating during or soon after feeding, adaptation to human dwellings, wide geographical distribution, and a high degree of anthropophilyPanstrongylus, Rhodnius, and Triatoma the main vectors. Triatoma sherlocki  is usually associated with rocks and is an endemic specie in Bahia, having medium vector importance. In addition, adults and nymphs infected with T. cruzi have been found in the domestic environment of mining communities in the municipality of Gentio do Ouro, indicating that this species is in the process of domiciliation in these areasCurrently, 18 genera and 157 species of triatomines have been admittedT. cruzi presents remarkably high levels of gene diversity and several genetic markers that allow its classification into seven subdivisions. According to the biomarkers large subunit (LSU) 24 Sα-ribosomal deoxyribonucleic acid (rDNA), heat shock protein 60 (HSP60), Histone H1, glucose-6-phosphate isomerase (GPI), and mini-exon loci using polymerase chain reaction (PCR)T. cruzi strains can be divided into six Discrete Typing Units (DTUs): TcI to TcVI and Tcbat. This molecular characterization is important and contributes to understanding parasite-host interactions and their evolutionary historyT. cruzi and its genetic variability.T. cruzi strains were isolated from nymphs and adult feces of Triatoma sherlocki captured in Santo Inácio, the municipality of Gentio do Ouro, Bahia. Growth kinetics studies were performed in triplicate by cultivating 1.5 × 106 parasites/mL in liver infusion tryptose (LIT) to evaluate the growth rate of the parasite in the first days of culture,In this study, the Tsh 4 and 18 T. cruzi genomic deoxyribonucleic acid (DNA) was extracted using the PureLink™ Genomic DNA Mini Kit (Invitrogen™). The Tsh 4 and 18 T. cruzi strains were characterized using PCR amplification of the D7 divergent domain of the 24 Sα ribosomal ribonucleic acid (rRNA) gene (LSU rDNA), according to Souto and colleagues (1996)HSP60 and GPI was performed using restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) with the following target/restriction enzyme combinations: HSP60/EcoRV and GPI/HhaI and incubated for 4 h at 37 ºC. The length and restriction profiles of the amplified HSP60 and GPI genes were analyzed by electrophoresis in 1-3% agarose gels stained with ethidium bromideAccording to the manufacturer’s instructions, for molecular characterization, Morphological characterization was performed using images analyzed under a light microscope  at x 1,000 using 30 epimastigotes from LIT and 30 trypomastigotes from artificial urine triatomine (TAU) of both strains. The morphological parameters used were total parasite length, body length, free flagellum, width, kinetoplast area, nucleus area, distance from anterior extremity to the nucleus, distance from posterior extremity to the nucleus, and nuclear index (IN=PN/NA) for trypomastigote forms, and width, total length, nucleus area, and kinetoplast area for epimastigote formsT. cruzi strains was verified during epimastigote multiplication in LIT medium, according to the results presented in The growth of HSP60 gene presented a 290 bp band, the GPI gene presented the formation of two bands (400 bp and 240 bp), and the LSU rDNA gene presented a 110 bp band gel for both strains To contribute to the morphological characterization of the Tsh 4 and 18 As demonstrated by Silva (1959)in vitro behavior of strains obtained from human cases and Triatoma infestans in the LIT medium differed by Brener and Chiari (1965)6 parasites/mL) and 18 (4.83 × 106 parasites/mL) T. cruzi strains displayed a growth phase on the 7th day, related to the parasite group TcI. A typical growth curve was observed with two distinct phases: log or exponential, and early stationary. From days 1 to 5, the log phase is defined by an exponential growth rate, whereas the early stationary phase is characterized by a significant decrease in growth rate (6-10 days)T. cruzi strains and help to improve the existing data.The T. cruzi I populations may be related to intrinsic characteristics of the parasite, such as kinetics of growth, and reinforce the importance of studying T. cruzi isolated from natural hosts.In combination, these results suggest that the maintenance of According to the Epidemiological Bulletin of Chagas Disease in the State of Bahia (2021)T. sherlocki has only been described in the district of Santo Inácio, Bahia. Although the species is considered wild, there are reports of its presence in homes, which may indicate that adaptation to the home environment is taking place,T. cruzi, which could result in vector transmission to the residents, a possibility that requires constant vigilance since infections by the TcI group present cardiac manifestations. In addition, the specimens used in this study came from the same area where T. sherlocki was collected and described, and both were found to be infected with T. cruzi, which proves that the epidemiological cycle of the disease persists throughout the regionTo date, the presence of


Periconia macrospinosa, Neocamarosporium goegapense, and N. chichastianum, isolated from roots of salt lake plants growing in the central desert of Iran, in alleviating the adverse effects of salinity and drought stresses on barley under greenhouse conditions. To perform this experiment, a randomized block design was applied with three factors: fungi , salinity , and drought . All plants were measured for growth characteristics, chlorophyll concentration, proline content, and antioxidant enzyme activities. A three-way analysis of variance indicated that all three fungal endophytes, to varying extents, induced the barley plants’ resistance to salinity and drought, and their combined effects. Additionally, we found that fungal endophytes were more effective when the barley plants were subjected to higher levels of salinity and drought. Under the stress of salinity and drought, a strong relationship between inoculation of fungal endophytes and enhancement of biomass, shoot length, chlorophyll concentration, proline content, and activity of catalase, peroxidase, and superoxide dismutase was indicated. We discussed that increased root growth, proline content, and antioxidant enzyme activity are the main physiological and biochemical mechanisms causing stress resistance in barley plants inoculated with endophytes. Our research findings illustrate that fungal endophytes have a substantial potential for increasing abiotic stress tolerance in barley plants, which can be applied in agricultural ecosystems.A characteristic trait of plants living in harsh environments is their association with fungal endophytes, which enable them to survive under extreme stress. Abiotic stress resistance in agro-ecosystems, particularly in arid and semi-arid regions, can be increased by inoculating these fungal endophytes on plants other than their original hosts. The present study is therefore focused on the possible role of three halotolerant endophytic fungi, i.e.,  Environmental challenges such as drought and salinity impose the greatest threat to plants growing on irrigated land which generates about 40% of the world’s food . MorpholArthrocnemum macrostachyum and Spergularia marina, ameliorate salinity tolerance of Vicia faba by improvement of enzymatic and non-enzymatic antioxidant accumulation , as a monocotyledonous plant species, is the fourth most commonly produced cereal after maize, rice, and wheat. Therefore, studies on the methods of how to increase barley plants’ resistance to abiotic stress are crucial for agricultural research. During the last decade, several studies have investigated the effect of fungal endophytes on barley plants’ increasing resistance to salinity and drought stresses  was used as a model plant for salinity and drought stress tolerance assays. Salt-sensitive barley seeds  were surface-sterilized for 10 min in 0.25 percent sodium hypochlorite, followed by rinsing with water  from Hoz-e Soltan  was used to induce salinity stress on endophyte-colonized plants as well as on the endophyte-free control plants. Salinity stress was applied at three levels of Electrical conductivity to the barley pots, including S1 = 8 dS/m (without yield reduction), S2 = 12 dS/m (25 percent yield reduction), and S3 = 16 dS/m (50 percent yield reduction). In order to attain different salinity levels, the salt lake water was diluted with distilled water. Barley plants were exposed to drought stress at four different levels, including D1 = 100%, D2 = 80%, D3 = 60%, and D4 = 40% of field capacity (FC). In order to determine the field capacity, soil samples of 200 g were taken at the time of filling plastic pots, stored in plastic bags, and transported to the laboratory. A 24-hour incubation at 105°C was then performed on these soil samples. By weighing and averaging these oven-dried samples, we determined the soil moisture content at the time of sowing the barley seeds. In order to estimate the saturation percentage of the oven-dried soil samples, distilled water was used to make a paste that was completely saturated.Barley plants were exposed to salinity and drought 10 days after inoculation. Salt lake water  for 2 min, followed by sodium hypochlorite/water (4:96) for 90 s, ethanol/water (70:30) for 2 min, and a final rinse in sterile distilled water for three times (1 min each). The inner layers of disinfected roots were cultured on PDA , and incubated at 28°C . A smallSix weeks after planting the seeds, the root wet and dry weight and shoot length of the plants were measured. Using a SPAD-502 Chlorophyll meter, we measured the chlorophyll content of all leaf samples .2CO3 (pH 10.2), was prepared. Then, using a spectrophotometer , one unit of superoxide dismutase activity was determined as the amount of enzyme needed to inhibit the 50% rate of NBT reduction measured at 560 nm. In a reaction mixture consisting of 500 μl of 10 mM H2O2, 1400 μl of 25 mM sodium phosphate buffer, and 100 μl of crude enzyme extract, catalase activity was measured. Following the decomposition of H2O2, absorbance at 240 nm declined. Catalase activity of the extract was expressed as CAT unit: min–1 mg–1 protein  containing 0.1 mM EDTA was added to the leaf powder. Supernatants (enzyme extracts) were used for chemical analysis once the final solutions had been centrifuged at 4°C at 16,000 rpm for 15 min . A photo protein . A spect protein . The reaFor the estimation of proline content, each leaf sample (0.2 g) was homogenized in 3 ml sulphosalicylic acid (3% w/v), and then centrifuged at 10,000 rpm for 15 min. A reaction mixture consisted of 2 ml supernatant, 2 ml freshly prepared acid ninhydrin solution, and 2 ml glacial acetic acid which was boiled at 100°C for 1 h. After completion of the reaction in an ice bath, the reaction mixture was extracted with 4 ml of toluene; absorbance was read at 520 nm .p ≤ 0.05 level. Analyses were conducted using statistical packages in R version 3.6.2 ; however, the interaction effect of these abiotic stress was not significant ; however, the interaction effect of these abiotic stress was not significant . Under the lowest levels of salinity and drought stresses, P. macrospinosa, N. goegapense, and N. chichastianum increased the CAT activity by 28.9, 30.9, and 28.8 percent, SOD activity by 1.7, 1.2, and 4.9 percent as well as POX activity by 48.2, 64, and 73.5 percent compared to the controls, respectively  are constantly produced in plants but maintained at a non-toxic level. However, ROS accumulation in plants exposed to abiotic stress, such as salinity, drought, high UV radiation, and extreme temperatures can cause significant damage to cell membranes, DNA molecules, and proteins . To prevt stress –6. Theseo plants . Barley t stress –6. Howevt stress . This fiP. macrospinosa under all levels of salinity and drought stress (Neocamarosporium species increase the proline content of cucumber and tomato plants only in higher levels of salinity and drought stress (N. goegapense, and N. chichastianum, improved the proline content of leaf under all levels of salinity and drought stress.Different levels of abiotic stress increase the accumulation of proline in plant tissues . Althougt stress . An incrt stress , here wePericonia macrospinosa increased the activity of antioxidant enzymes and proline content in both monocot and dicot plants under all levels of salinity and drought stress, two Neocamarosporiun species did not increase the levels of these biochemical markers in dicot plants under lower levels of the drought and salinity stress. It appears that identical fungal endophytes function differently in different plant species.Overall, the current study and our previous study  indicateP. macrospinosa, N. goegapense, and N. chichastianum, increased several physiological and biochemical markers, including plant growth parameters, chlorophyll concentrations, antioxidant enzymes activity, and proline content in barley plants exposed to salinity and drought stress. Overall, we observed that these fungi significantly improved plant performance under these abiotic stresses. Such endophytic fungi associated with desert plants have developed some strategies to thrive in the harsh environment of the desert and play a fundamental role in conferring resistance to their host plants against extreme environmental stress. The results of our study show that fungal endophytes might have the ability to increase the abiotic stress tolerance in plants other than their original hosts, which can be utilized in agricultural ecosystems. Different reactions were observed in two Neocamarosporium species in inducing tolerance to abiotic stress in dicotyledonous and monocotyledonous plants, highlighting the role of plant identity in such plant-microbe associations. We suggest studying the effects of endophytic fungal species on other major crops, especially in natural agricultural fields. Further in-depth studies are required to understand the molecular changes in the host plants induced by fungi under stressful conditions. Such studies are important regarding the future development of biofertilizers to reduce the excessive utilization of chemical compounds in agricultural fields.In the present study, three halotolerant endophytic fungal species, The original contributions presented in the study are included in the article/MH performed the experiments, analyzed the data, and wrote the manuscript. NS designed and supervised the study. NH-D and SR helped with data analysis and improving the manuscript. This research manuscript was accomplished with the collaboration of all authors.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to Beverages, median increases were higher for the MNP for vitamins C , E , and B12  from baseline to 37 wk; from baseline to 1 mpp, there was a higher median increase in B12  and folate ; and from baseline to 3 mpp, a higher median increase in iron  and folate . Conclusions: Intake of micronutrients was higher for MNP and Tablets, likely due to food displacement among Beverage consumers. Although iron bioavailability and absorption inhibitors were not considered for the present analyses, the distribution of Tablets or MNP had several advantages in this context where micronutrient deficiency remains high among pregnant women, but macronutrient intake is generally adequate or even high.Supplementation in malnourished pregnant women should not displace natural healthy foods. Objective: To estimate the differential effects of three nutritional supplements on macro- and micronutrient intake of pregnant women beneficiaries of the conditional cash transfer program Prospera (CCT-POP). Methods: Prospective cluster randomized trial. Communities were randomly assigned to receive a fortified beverage (Beverage), micronutrient tablets (Tablets), or micronutrient powder (MNP). Pregnant women (at <25 weeks) were recruited. The food frequency questionnaire was applied at 25 and 37 weeks of pregnancy and at one and three months postpartum (mpp). Differential effects of the three supplements on the median change in nutrient intake from baseline to each follow-up stage were estimated. Results: Median change in protein intake from dietary and supplement sources were significantly lower for MNP and Tablets than for Beverages (baseline to 37 w: −7.80 ± 2.90 and −11.54 ± 3.00, respectively; baseline to 1 mpp: −7.34 ± 2.90 for MNP,  Eating a balanced diet and maintaining a healthy weight during pregnancy is of the utmost importance for optimal maternal and fetal outcomes ,2,3. TheTo enhance its potential for addressing nutritional deficiencies, the conditional cash transfer program Prospera  in Mexico distributed a fortified beverage (Beverage) known as Nutrivida to pregnant and lactating women living in poverty from 1997 to 2018 ,6,7,8,9.In the poorest people, inadequate intake of essential micronutrients  affects several functional outcomes, including mental and motor development in newborns; nevertheless, supplementation should not replace healthy food consumption ,5.A cluster randomized trial involving CCT-POP beneficiary women was designed to assess the differential effect of three supplement groups: a fortified beverage, micronutrient powder (MNP), and micronutrient tablets (Tablets) on various outcomes. All three supplements reduced iron deficiency and there was no differential change in weight gain during pregnancy or weight retention up to 3 months postpartum among the women . At the www.clinicaltrials.gov (accessed on15 July 2020); NCT00531674). More details of the design, study population, intervention, information collection, and primary outcomes were published previously [This study is a cluster randomized trial of 723 pregnant women <25 weeks) beneficiaries of the CCT-POP in four Mexican states . The study was registered before recruitment began on the Clinical Trial Registry  ,7,8,9. TAll adult (>18 y) pregnant women  in the selected communities (18 communities) were identified through beneficiary listings at the corresponding health center and were recruited individually. Exclusion criteria for women included multiple pregnancies and hemoglobin <90 g/L. A sample of 238 women per group was estimated with 80% power and significance level of 5%. The final sample was as follows: Beverage = 220 women, Tablets = 226 women, and MNP = 236 women .Beverage, MNP, or Tablet supplements were delivered directly to women’s homes six days a week during the first six months of the project; subsequently, staff visits were performed on a weekly basis.®) with standard procedures [Baseline demographic variables were collected during home visits  . Weight ocedures . Ethniciocedures . At eachDietary intake was assessed at each follow-up visit by trained health personnel  during in-person interviews, using a previously validated (in Mexican women), semi-quantitative, 7-day food frequency questionnaire (SFFQ) ,21. The Foods were coded and transformed into daily energy and nutrient intake for each evaluation period, using the food composition table developed at the INSP . Energy Foods were categorized into nine food groups: dairy, fast food, legumes, drinks, snacks/candies/dessert, soups/cream/paste, fruits/vegetables, meat/sausage/egg, and cereals/root crop. For each woman and food group, total energy intake  was calculated at 37 wk, 1 mpp, and 3 mpp.2) was calculated and categorized according to the World Health Organization (WHO): underweight <18.5, normal 18.5 to 24.9, overweight 25 to 29.9, and obesity >30 [Body Mass Index  and was carried out in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki and its later amendments. Recruitment continued from November 2005 to January 2006 and field work was finished in October 2007.p < 0.05. Data were analyzed using Stata, Version 15.0 .General characteristics and baseline diet intake were compared between study groups using medians and interquartile intervals for quantitative variables and proportions for categorical variables. We compared subjects who were included in the analysis  and those not included (only 117 with sociodemographic information) through t-tests for means. Median changes of nutrient consumption from baseline to the other study stages were estimated by median regression with indicator variables of the study groups and design blocks as predictors. For covariate-adjusted analyses, unbalanced characteristics at baseline were identified between study groups and were added to the model predictor. Covariate-adjusted median changes by study group were obtained through predictive margins and pairwise contrasts between study groups were performed through Wald tests. The same model specifications were fitted using changes of nutrients plus supplement consumption as outcome variables. To assess median energy intake by food group and study group, median regressions were fitted for 37 wk, 1 mpp, and 3 mpp, adjusted by unbalanced characteristics at baseline. All standard errors were adjusted for data dependencies at the community level . StatistGenerally, the groups were well balanced at baseline in terms of maternal characteristics , with sop < 0.01), which was higher in MNP compared to Beverage (−33.9 vs. −62.5).Covariate-adjusted median changes in the consumption of carbohydrates, protein, vitamin C, vitamin E, and dietary folate equivalents showed that over 50% of women decreased their dietary intake from 25 wk to 37 wk in all study groups . The medMedian changes from baseline to 1 mpp were generally negative and statistically significant, although the median dietary folate change was higher (less negative) for MNP compared to Beverage. Median changes from baseline to 3 mpp showed a similar pattern, with higher median changes of dietary folate equivalents and iron for MNP compared to the Beverage group, and a higher median change of dietary folate equivalents for Tablets compared to the Beverage group.Median changes of macronutrient intake from dietary and supplement sources tended to be lower in general for MNP and Tablets compared to the Beverage group. With the exception of vitamin C, median changes of micronutrient consumption were all positive and statistically significant in the three study groups, i.e., more than 50% of women increased their consumption of micronutrients from baseline to any other study period . These cMedian changes without covariate adjustments are available in the p = 0.021).Mean adherence to supplementation during the week prior to the 37 wk SFFQ interview date was 85.6% ; for the Beverage group, 89.5%  for the Tablets group, and 92.5%  for MNP. During the week prior to the 1 mpp SFFQ, it was 89.0%  for the Beverage group, 88.6%  for the Tablets group, and 89.3%  for MNP. During the week prior to the 3 mpp SFFQ, it was 78.5%  for the Beverage group, 84.4%  for the Tablets group, and 83.3%  for MNP. There were significant differences in adherence between supplementation groups for MNP vs. the Beverage group only at 37 wk  at 37 wk. Our results suggest that the median energy intake of fruits and vegetables at 1 mpp was lower in the Beverage group compared to the Tablets  and MNP  groups, whereas it was higher at 3 mpp in the Tablets group compared to the Beverage  and MNP  groups  groups .The results of this cluster randomized trial illustrate that, in general, intake of energy and several micronutrients from home foods decreased among pregnant and lactating women consuming a fortified Beverage, compared to women consuming micronutrient-only supplements (MNP and Tablets). Even when considering nutrients from the supplements that were consumed, several differences persisted, leading to higher intake of several micronutrients among MNP consumers. Median changes of macronutrient intake from dietary plus supplement sources tended to be lower in general for MNP and Tablets compared to Beverage. These findings suggest that in the Beverage group, regularly consumed foods at home  were replaced by the Beverage. It is likely that the displaced foods were poorer sources of protein than the Beverage (given its milk base). Generally, however, protein consumption is not limited in this population. MNP showed a higher increase in micronutrient dietary intake alone, perhaps due to slightly higher consumption, although these differences were significant mainly in the first period ,27.n = 405). According to Suchdev et al. [Supplementation adherence in this study was very high, above 80% in all groups and study periods. This is likely due to the close follow-up and motivational approach taken to promote the supplement. In the CCT-POP program context, where promotion for consumption of the product was minimal, a cohort study found that only 25% of lactating women reported consuming the Beverage . Accordiv et al. , adherenInterestingly, in the current study, adherence to supplementation did not decrease even after daily visits were discontinued ,8. It isAccording to Neufeld et al. , no diffOur study has several limitations. Individual and group nutrient intakes obtained from the SFFQ instrument did not reproduce adequately true usual intake levels, as this instrument is limited to a determined number of food items. However, analyses of relative quantities  could be less prone to estimation bias. A better estimation of intake levels would require repeated 24 h recalls, which was considered infeasible as part of this study due to field logistics ,40. AnotThe study also has several strengths, particularly the longitudinal data on a relatively large group of women that permitted the assessment of individual changes between baseline and several follow-up points. This allowed us to control for both observed and unobserved factors with time invariant effects on the outcomes. On the other hand, potential reporting and instrument biases were not related to study groups by design, allowing us to compare differences between study groups. As an additional strength, continuous and rigorous monitoring of supplement consumption was performed by highly trained field workers in our study. Finally, the information reported here was used immediately to adapt the program in Mexico, considering the totality of evidence related to the supplements . That saIn many parts of the world, the provision of nutritional supplements forms a critical component of integrated care for pregnant and lactating women . The choIn this post-COVID area, the prevention and management of micronutrient deficiency urgently needs to be elevated as part of the public health agenda. Deficiencies can exacerbate the severity and consequences of several infections ,43,44. I


N6-methyladenosine (m6A) RNA, seemingly valuable biomarkers in CRCs. For ncRNAs, there exists a “molecular sponge effect” between long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). The detection of exosomes is a novel method in CRC monitoring, especially for predicting metastasis. There is a close relationship between exosomes and EMT in CRCs. This review summarizes the close relationship between epigenetic changes and EMT in CRCs and emphasizes the crucial function of exosomes in regulating the EMT process.Worldwide, colorectal cancer (CRC) ranks as the third most common malignancy, and the second most deadly with nearly one million attributable deaths in 2020. Metastatic disease is present in nearly 25% of newly diagnosed CRC, and despite advances in chemotherapy, less than 20% will remain alive at 5 years. Epigenetic change plays a key role in the epithelial-to-mesenchymal transition (EMT), which is a crucial phenotype for metastasis and mainly includes DNA methylation, non-coding RNAs (ncRNAs), and  Furthermore, it is the most common cause of concern and anxiety for patients with CRCs. Based on previous studies, the mortality of metastatic CRCs is much higher than that of primary CRCs, especially for liver metastasis. Metastatic cancers severely influence the 5-year survival rate and quality of life of patients  have become significant public health problems worldwide, and their incidence has increased to twice in the top 10 cancers . Monitorpatients . TherefoSNAI1, SNAI2, Zeb1, Zeb2, and Twist, called EMT-TFs, and some non-coding RNAs (ncRNAs) can influence the EMT process to regulate the migration and infiltration of tumor cells  is a vital phenotype in its metastasis. EMT is a reversible process that promotes tumor cells exfoliating into circulation and damaging the intercellular skeletal structure. These exfoliated tumor cells could reach distal cites and form metastatic loci. The transcription factors (TFs) or cells , 6. EMT The liquid biopsy (LB) technology is a novel method used to detect the genesis, progress of recurrence, and metastasis in CRC patients. Circulating tumor cells (CTCs), circulating tumor DNAs (ctDNAs), and cell-free RNAs are detectable biomarkers in serum . In addiExosomes are tiny vesicles derived from tumor cells that contain DNA, ncRNAs, epigenetic modulation, and metabolites. Especially in comparison to obtaining tissue biopsies, liquid biopsies, including the relatively noninvasive venipuncture, or sampling other bodily fluids is often safer and more convenient. According to previous studies, exosomes play a pivotal role in predicting cancer metastasis. Comparably, exosome detection is safer and more convenient in malignant cancers, especially for CRCs. LB has shown considerable accuracy in CRC testing. Its sensitivity and specificity can reach up to 60% and 90%, respectively . TherefoThe reverse EMT process is characterized by the downregulation of E-cadherin, desmoplakin, claudins, and β-catenin and the upregulation of N-cadherin, vimentin, fibronectin, and Snail1/Snail2 mediate the migration and invasive metastasis of cancer cells –13. Whetvia the detection of epigenetic biomarkers, such as DNA methylation, overexpression of ncRNAs, and N6-methyladenosine (m6A) modification. These epigenetic changes are regulated by genes and can be inherited by the next generation, but they cannot be transmitted into proteins to form phenotypes. Their influence is so strong that they have gained wide attention , microsatellite instability (MSI), and the CpG island (CGI) methylator phenotype (CIMP) pathways are three principle pathways involved in the genesis and development of CRCs. CIMP is related to the hypermethylation of promoters or the upstream regulator regions that usually contain CGIs, reported to be related to the detection of CRCs , 15. Thettention , 20. Sevttention –23.6A modification for RNA are two epigenetic modifications affecting the transcription and expression of critical genes. In addition, ncRNA is another group that cannot be transcribed, but has high regulatory ability for coding genes. These two mechanisms can undoubtedly regulate the EMT process in CRC metastatic events.Regarding epigenetic changes, 5-methylcytosine (5mC) methylation for DNA and mDNA methylation occurs when a methyl group is transferred to the carbon-5′ position of the cytosine base (5-methylcytosine) concentrated in CGIs under the catalytic action of DNA methyltransferase enzymes . The hypSDC2 methylation is a carcinogenic biomarker in CTCs tested in patients with CRC  and helicase-like transcription factor (HLTF), has prognostic value for therapy and metastatic location in CRC patients (SEPT9 and SDC2 (SEPT9 and OSMR (77.0%)  , SEPT9 aand OSMR , SEPT9 aYCD2/VHL , APC/MGMF2A/WIF1 , SYNE1 and FOXE1 , WIF1/NPNPY/PENK , THBD an C9orf50 , and the/TMEM90B  and MGMT1A/SEPT9  were rep markers , 58, wit markers , 59.Methylated DNA analysis in exosomes has been reported for gastric cancer, prostate cancer, lymphoma, breast cancer, and gingivitis . AlthougNcRNAs are a class of small RNA fragments or circulars in the non-coding regions. Although an association between DNA methylation in vesicles and CRCs exists, no study on their relationship has been conducted. The ncRNAs in CTCs originate from three main pathways, namely, membrane-bound vesicles, apoptotic bodies, and RNA-binding proteins (RBPs), which have been elucidated in detail in a previous review . The ncRMiRNAs were the first discovered ncRNAs that can regulate the functions of genes and proteins. The sequence of miRNAs is short and can form complementary pairs with mRNAs. Most miRNAs are inhibitory and can lead to the degradation of mRNAs, which decreases the expressions of target proteins. Many cytoplasmic miRNAs have been discovered that can regulate expression of N-cadherin, Vimentin, MMPs, E-cadherin, Claudin, and EpCAM, thereby influencing the direction of EMT through molecular regulation in targeted pathways –69. Go eSOX4 , which is produced by cancer-associated fibroblasts (CAFs) and released into serum, is associated with the metastasis of CRCs. The upregulation of UCA1 can change the expressions of the genes in EMT (BANCR is an oncogenic molecule that enhanced the aggressiveness of CRC metastasis (LNCRNA-ATB) can suppress the expression of E-cadherin in EMT to promote CRC metastasis  of m6A modification under the catalytic function of methyltransferase 3 (METTL3)  . The earand ZEB2 , 92. Theand ZEB2 . Most ci6A modification could alter its local RNA structure at the terminal loop region of primary miRNAs (pri-miRNAs) to promote their processing through nuclear transcripts and alternative splicing by modulating RALY binding  and the m6A/METTL-associated complex (MACOM). MAC consists of METTL3 and METTL14, which can form stable heterodimers  could increase export into the cytoplasm in the circNSUN2/IGF2BP2/HMGA2 ternary complex (6A modification enhances the metastasis risk of CRC cells (In total, 11 readers, 7 writers, and 2 erasers have already been found. The term “writers” often denotes methyltransferase as the beginning of mity. The methyltrarodimers . BesidesmiR-1246 . Contrar miR-375 –149. Fur miR-375 , 142. Th complex . In addiRC cells .6A modulation sites, such as YTHDF1, IGF2BP1, IGF2BP3, and EIF3B (FTO (GAS5 can reversely regulate YAP1 via m6A (6A can positively increase with the increased concentrations of circRNAs (RP11 by m6A methylation can accelerate the spread of CRC cells through the upregulation of ZEB1 (6A circRNAs are different from m6A mRNA sites. This indicates that disturbance in the m6A modification of mRNA is not equivalent to that of m6A circRNAs. Therefore, to abolish the effects of m6A modification, then the m6A of circRNAs must be considered as well (6A modification, then the m6A of circRNAs must be considered as well. NcRNAs can regulate the critical enzymes as the downstream target molecules or combined partners. Feedback of the essential enzymes affects the m6A modulation of ncRNAs, and this process can change the cancer-related phenotype and lead to metastasis (Beyond the methyltransferases, the “readers” that can recognize mnd EIF3B , are alsF3B (FTO . Moreove via m6A . SurpriscircRNAs . The upr of ZEB1 . Interes as well , 154. Thtastasis .via the addition of anti-miRNAs and exogenous exosomes containing miRNAs confirmed that ncRNAs negatively regulated the downstream target proteins or mRNA molecules to promote tumor metastasis via EMT (via increasing the permeability of vascular epithelial layers, and miR-27b-3p can regulate this process through the STAT3 pathway (What is an exosome? Exosomes are microvesicles with diameters from 30 to 150 nm. They are derived from normal intestinal cells, CRC cells, and other cancer-related stromal cells . Exosome via EMT –162. In  pathway . There i pathway . The eff pathway .6A methylation of RNA in CRCs could enhance the invasive and migration ability to promote metastasis. The mechanisms of the mutual effect between methylation and ncRNAs may provide a novel direction for future studies.Cancer metastasis has always been a hot topic for scientists and clinicians, and discovering the risk factors and suitable therapeutic targets is a critical part of preventing metastasis. Epigenetic change is a popular context associated with the progression and metastasis of CRCs due to its variability and vulnerability. NcRNAs plays a crucial role in regulating epithelial tissues and the mesenchymal components. The dynamic process determines the tumor metastasis tendency. In this regulation, the sponge effect between lncRNAs/circRNAs/miRNAs/mRNA is critical. In addition, epigenetic modification is a crucial link. The 5mC methylation of DNA and the mThe EMT is a vital regulator in metastasis, while the mechanism and influence factor of EMT is still unclear. As mentioned before, these epigenetic changes are not independent but interacting. The methylation of promoters can regulate the process of alternative splicing and production of ncRNAs. The review interprets a crosstalk interaction in epigenetic changes from DNA methylation, ncRNAs, and m6A methylation. The function of epigenetic changes is powerful and complex in regulating mechanisms in CRC metastasis. At the same time, EMT is a cross point that the DNA methylation, m6A methylation, and ncRNAs can all affect CRC metastasis. They can upregulate or downregulate the biomarkers in EMT such as N-cadherin, Vimentin, MMPs, E-cadherin, Claudin, and EpCAM to control the progress of the epithelial transition.Another novelty of the review is that the exosomes can regulate EMT as a carrier for transporting ncRNAs. The function of exosomes has gained widespread attention in cancers. The exosomes have been proved to be related to cancers detection. This review summarizes the effect of exosomes in CRC metastasis related to EMT. That indicates that exosomes are a vital factor in the tumor micro-environment. The tumor cells or other stromal cells can secret exosomes to regulate EMT. And this review indicates that DNA methylation, ncRNAs, m6A methylation, exosomes, and EMT can all act as potential therapeutic targets in CRC metastasis. The associated relationship and mutual interaction among them are elucidated thoroughly in this review. While prior studies have been published elucidating the mechanisms of exosomes and EMT, the field remains in its relative infancy, with the potential to discover important new therapeutic targets for CRC.The detection of exosomes in LB is a novel method to predict metastasis in CRCs. The variety of stromal cells can produce exosomes to participate in the progression, proliferation, migration, and EMT in CRCs. Epigenetic changes, ncRNAs, and exosomes are not independent of each other; on the contrary, there exists an intrinsic interrelation between them. Different from previous studies, this review elaborated on the potential links between epigenetic changes, exosomes, and EMT in CRCs regarding metastasis. It provided novel sights for the study of probable mechanisms of metastasis in CRCs. There have already been several studies on the mechanisms of exosomes and EMT, but more studies are needed to discover new therapeutic targets for CRCs.MY and HZ designed the study design and wrote the main manuscript. MS contributed to the final proof. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Japan Society for Blood Purification in Critical Care (JSBPCC) has reported survey results on blood purification therapy (BPT) for critically ill patients in 2005, 2009, and 2013. To clarify the current clinical status, including details of the modes used, treated diseases, and survival rate, we conducted this cohort study using data from the nationwide JSBPCC registry in 2018.We analyzed data of 2371 patients who underwent BPT in the intensive care units of 43 facilities to investigate patient characteristics, disease severity, modes of BPTs, including the dose of continuous renal replacement therapy (CRRT) and hemofilters, treated diseases, and the survival rate for each disease. Disease severity was assessed using Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores.P = 0.001). The survival rate was significantly lower in patients with multiple organ failure (MOF) (34.6%) compared with acute lung injury (ALI) (48.0%) and sepsis (58.0%). Multivariate logistic regression analysis revealed that sepsis, ALI, acute liver failure, cardiovascular hypotension, central nervous system disorders, and higher APACHE II scores were significant predictors of higher 28-day mortality.BPT was performed 2867 times in the 2371 patients. Mean APACHE II and SOFA scores were 23.5 ± 9.4 and 10.0 ± 4.4, respectively. The most frequently used mode of BPT was CRRT (67.4%), followed by intermittent renal replacement therapy (19.1%) and direct hemoperfusion with the polymyxin B-immobilized fiber column (7.3%). The most commonly used anticoagulant was nafamostat mesilate (78.6%). Among all patients, the 28-day survival rate was 61.7%. CRRT was the most commonly used mode for many diseases, including acute kidney injury (AKI), multiple organ failure (MOF), and sepsis. The survival rate decreased according to the severity of AKI (This large-scale cohort study revealed the current status of BPT in Japan. It was found that CRRT was the most frequently used mode for critically ill patients in Japan and that 28-day survival was lower in those with MOF or sepsis. Further investigations are required to clarify the efficacy of BPT for critically ill patients.Trial Registration: UMIN000027678.The online version contains supplementary material available at 10.1186/s41100-022-00445-0. Survival rates for patients admitted to the intensive care unit (ICU) have improved over the past decade –3. Reduc. JSBPCC conducted this cohort study using a nationwide registry of critically ill patients who were treated with BPT in 2018, with the aim of clarifying its current status, including the number of critically ill patients treated with BPT, the diseases treated, the mode of BPT, and survival rates.The Japan Society for Blood Purification in Critical Care (JSBPCC) has reported survey results for BPT in critical care in Japan in 2005, 2009, and 2013 , 12. In JSBPCC has created a registry for data from its nationwide surveys of critically ill patients. The details of the methods have been described previously , 12. BriData were collected on patients’ demographic and clinical characteristics, including age, sex, medical history, chronic dialysis status, the presence of sepsis, primary and secondary diseases treated with BPT, mode of BPT, dose of blood purification, Sequential Organ Failure Assessment (SOFA) scores , Acute PThis study was conducted in accordance with the Declaration of Helsinki, Japanese privacy protection laws, and the Ethical Guidelines for Medical and Health Research Involving Human Subjects published by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare in 2015. The study was approved by the ethics committee of Chiba University Hospital. No personally identifiable information is stored in the JSBPCC registry. The need for informed consent was waived due to the use of de-identified data. This study is registered with the University Hospital Medical Information Network (UMIN000027678).The following diseases are treated with BPT in Japan: 1) AKI, (2) sepsis, (3) congestive heart failure, (4) MOF, (5) acute liver failure (ALF), (6) acute electrolyte, fluid, and acid–base disorders, (7) acute exacerbation of autoimmune disease, (8) severe acute pancreatitis, (9) ALI, (10) thrombotic microangiopathy, (11) acute drug intoxication, and (12) others, including Guillain–Barré syndrome, toxic epidermal necrolysis, and acute metabolic disorders. The modes of BPT used in Japan are listed in Additional file  AKI, (2)Primary and secondary diseases requiring BPT and the modes of BPT for these diseases were recorded. For each disease, the number of cases was recorded as the cumulative number because many patients had multiple diseases at initiation of BPT. Similarly, for each mode of BPT, the number of cases was recorded as a cumulative number because some patients were treated with multiple modes.t-test. Comparison among three or more groups was performed using repeated-measures analysis of variance with Tukey’s honestly significant difference test or the Kruskal–Wallis test, as appropriate.Data are reported as numbers and proportions or as the mean ± standard deviation. Categorical variables were analyzed using the Chi-square test, and continuous variables were compared using the P < 0.05.For analysis of survival, patients were divided into eight age groups , which were defined a priori. Furthermore, we compared the survival rate according to the primary disease requiring BPT. Among patients with AKI, subgroup analysis was performed according to KDIGO classification   for compOverall, 3364 patients were registered and 993 were excluded, leaving 2371 patients for inclusion in the analysis . By disease, 28-day survival was significantly lower in patients with MOF (34.6%), ALI (48.0%), and sepsis (58.0%) (P < 0.0001)  were diagnosed as having AKI based on the KDIGO criteria. In a subgroup analysis according to AKI stage , 70.7% of these 2140 patients had stage 3 AKI (Table P < 0.0001). The hazard ratio (HR) of the PMMA group was significantly higher and that of other types of membrane group was significantly lower compared with the PS (reference) group. However, the significance was lost after adjustment for characteristics at baseline , and CHDF in particularly accounted for 48.0% of all BPT procedures. The next most common was intermittent renal replacement therapy (19.1%). SOFA score, which could not be investigated in the 2013 survey, was used to examine the effect of organ failure on 28-day survival in this cohort. Multivariate logistic regression analysis revealed that the presence of sepsis, ALI, ALF, cardiovascular hypotension, and central nervous system disorders in addition to higher APACHE II scores were significant predictors of lower 28-day survival in the 2018 survey.The JAKID study investigated the diagnosis, treatment, and prognosis of 2292 patients who were admitted to the ICU from June to December 2016 at 13 hospitals in Japan . Among tRRT is the mainstay of treatment for severe AKI. For dialysis in the ICU, CRRT has primarily been used because of its accurate volume control, acid–base stability, and electrolyte correction, as well as its ability to achieve hemodynamic stability. CRRT can improve the clearance rate of small, medium, and large molecules in blood by removing water via ultrafiltration and making hemodynamics more stable. Small molecule clearance rates are low, and hemofiltration requires large volumes of fluid replacement. In the previous JSBPCC surveys conducted in 2005, 2009, and 2013, the frequency of CHDF was 50.3%, 53.0%, and 50.6%, respectively , 12. In n = 224) and a sham hemoperfusion group (n = 226) [P < 0.0001), and median days of noradrenalin treatment, days of CHDF treatment, and ventilator-free days were significantly higher in the PMX group. However, considering the conflicting results and the low quality of evidence, Surviving Sepsis Campaign 2021 and The Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock 2020 recommend against using PMX-DHP for patients with septic shock [The frequency of using PMX-DHP is declining. It was performed in 15.0% of patients in the 2005 and 2009 surveys and 11.5% in the 2013 survey . In the n = 226) . No signn = 226) , 28-day ic shock .PS membranes and AN69ST membranes are now frequently used as hemofilters for CRRT outside of Japan, PMMA was frequently used in the 2013 JSBPCC survey in Japan . HoweverThe optimal dose of CRRT is not clear. For patients with AKI, the KDIGO recommendations are weekly Kt/V of 3.9 when using intermittent or extended RRT and an effluent volume of 20–25 ml/kg/h for CRRT . This wiThis study has some limitations that should be kept in mind. First, observational cohort studies and repeated surveys conducted every few years may have variations in mortality between centers due to changes in the practices and patient populations of each center. In addition, BPT for critically ill patients was not performed according to the same protocol. Variations in the therapeutic regimen may have affected the responses to therapy and outcomes. However, we consider the present results to be representative of the actual clinical setting for BPT in Japan because our survey was conducted nationwide at 43 centers in various urban and rural locations. Second, data were not available for some possible confounders, such as residual kidney function, diabetes status, and serum albumin levels, but it is known that diabetes and hypoalbuminemia are associated with mortality in patients with AKI , 38. ThiIn conclusion, this study has revealed that CRRT, especially the CHDF mode, was most frequently used among critically ill patients in Japan. The 28-day survival rate of patients with AKI was relatively high, but lower in cases with MOF such as ALI or ALF or cases with sepsis.Additional file 1. Supplementary Table 1. Modalities of blood purification therapy currently used in Japan and their common abbreviations


The continuous growth of the human population and the need for resources has led to expanding encroachment into wildlife habitats . IncreasTo meet the growing demand for meat and meat products, swine production has followed a similar growth trend . FollowiNext generation genome sequencing techniques are becoming routinely used in virus diagnostics, contributing to a better understanding of virus evolution and virus discovery . MetagenNovel viruses of pigs have been detected in the last 3 to 4 decades at a higher speed than ever before. Among the viruses that resulted in the highest economic losses are re-emerging porcine reproductive and respiratory syndrome (PRRS), porcine circovirus type 2 (PCV2), influenza A viruses (FLUAV), and porcine epidemic diarrhea virus (PEDv), which are endemic in many high intensity swine production systems and continue to challenge producers and veterinarians –11. ReceThis Research Topics comprises of six original research articles about characterization of ASFV and FLUAV, vaccines against pseudorabies virus (PRV), inactivation of ASFV, multiplex diagnostics of ASFV, CSFV, and PRRSV, and characterization of porcine parvovirus 7 (PPV7). Two brief research reports are also included about PRV.Tan et al. reported a naturally occurring recombinant event in Hunan province, China between PRV classical strain and its derived HB-98 live attenuated vaccine strain, highlighting safer vaccines and/or vaccination protocols are needed. The isolated HN-2019 the glycoprotein E (gE) and gG genes of the PRV classical strain but the TK gene of the HB-98 vaccine strain. The recombinant strain replicated to similar titers as the parental strains in vitro but resulted in intermediary level of replication and disease in vivo.Tan et al. investigated the seroprevalence of PRV in Hunan province in China during 2020 and 2021 and the awareness of the risks of this infectious agent among swine practitioners. In the study, 18,812 pig serum samples and 1,634 tissue samples were tested from 798 farms. The authors found that 8.9% of animals and 20.2% of farms were seropositive for PRV-gE antibodies, and 2.3% of tissues were positive for PRV nucleic acids, demonstrated PRV is prevalent in China. However, workers were mostly unaware of the risks of PRV to humans.Nuanualsuwan et al. investigate the thermal inactivation of ASFV in three swill formulae with different nutritive compositions at the temperatures of 60, 70, 75, and 80°C. The authors also defined a prediction model to predict decimal reduction time (DRT or DT) for other inactivation temperatures. The mean D60, D70, D75, and D80 of ASFV in three swill formulae were in the ranges 23.21–33.47, 5.83–10.91, 2.15–2.22, and 1.36–1.47 min, respectively. Based on the results, the estimated time for inactivation for the recommended temperatures by the World Organization for Animal Health  and Food and Agriculture Organization  are 4 and 119 min, respectively.Shi et al. characterized the ASFV strains circulating in Guangxi province, southern China in the ongoing outbreak. For this study, 336 tissue samples collected from domestic pigs that died with severe hemorrhagic disease in 86 farms during 2019–2020 were tested, and 66 ASFV strains were sequenced and genetically characterized. Phylogenetic analysis showed that genetically diverse strains circulated in the region in 2019–2020, including strains of the genotypes I and II, as well as serogroups 4 and 8, the majority belonging to genotype II and serogroup 8. Interestingly, both wildtype and gene-deleted ASFV strains were identified.Shi et al. reported the development and testing of a new multiplex crystal digital PCR (dPCR) for differential detection of ASFV, CSFV, and PRRSV, a modified version of the conventional PCR. The assay targets detection of p72, 5′ untranslated region (UTR), and ORF7 genes of each virus, respectively. The assay showed high specificity and sensitivity, capable to detecting low levels of ASFV, CSFV, and PRRSV, but no non-specific detection of other swine viruses was observed. This assay proved to be accurate and a reliable tool for screening for ASFV, CSFV, and PRRSV infection, and could aid in the rapid detection of outbreaks.Ren et al. compares the efficacy of a commercially available Bartha-K61 vaccine and an rPRV/XJ5-gI–/gE–/TK– prototype vaccine against lethal challenge with a PRV variant strain. Both vaccines were administered the same dose to 12 week-old pigs and resulted in protection, with all vaccinated pigs surviving challenge. Pigs were equally protected against weight loss, pathology, and virus shedding. These results differe from previously published studies when younger pigs were used, demonstrating the two vaccine can be effective in a less PRV susceptible age.Wen et al. reported the first detection of PPV7 in Inner Mongolia Autonomous Region, China. More than 28% of animals and 60% of herds were positive for PPV7, and co-infection with PCV2 and PCV3 was often observed. The authors sequenced and characterized four strains, demonstrating that all Mongolian PPV7 clustered together, and showed multiple amino acid mutations compared to reference strains.Osorio-Zambrano et al. characterized the influenza A virus circulating in swine in Colombia before, during, and after the 2009 H1N1 pandemic. Ten virus isolates were selected from a repository, subtyped, and sequenced. Strains isolated in 2008, prior to the pandemic, carried an HA segment from the North American clade 1.A.1 classical lineage. After the introduction of the 2009 H1N1 virus, all isolates contained the HA and NA genes from pandemic clade 1A.3.3.2, demonstrating the pandemic lineage replaced the previously circulating classical H1N1 lineage. Interestingly, evolution of the FLUAV gene segments within the studied strains followed a geographic pattern.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Klinefelter syndrome is the most commonly reported sex chromosome abnormality. It is heavily underdiagnosed due to the substantial variability of clinical presentations but is generally characterized by small, firm testes, hypergonadotropic hypogonadism, and the absence of spermatogenesis. Most patients with Klinefelter syndrome have a 47,XXY genotype. If they present with mosaicism, two different cell lines are usually identified, an aneuploid 47,XXY cell line and a normal male 46,XY cell line. There are very few cases of 47,XXY mosaicism with the additional female cell line 46,XX described in the literature. We report a case of an adolescent with the male phenotype and a rare variant mosaic 47,XXY/46,XX karyotype who presented with painless bilateral gynaecomastia. 47,XXY and 46,XX mosaic cell lines were identified with GTG-banding and further characterized using fluorescent in situ hybridization. We summarized the available clinical presentations of reported male patients with 47,XXY/46,XX mosaicism. To improve the clinical management and quality of life in individuals with rare and cryptic genomic imbalances, the genetic diagnosis would need to be extended to atypical cases. Klinefelter syndrome (KS) is the most commonly reported sex chromosome abnormality, with an incidence of 1 per 600 newborn males . In 1959KS affects the testicular function. The loss of germ cells, which begins in foetal life and accelerates through puberty, leads to fibrosis and hyalinization of seminiferous tubules and relative hyperplasia of Leydig cells. Those processes result in characteristic features of KS such as small, firm testes, hypergonadotropic hypogonadism, and the absence of spermatogenesis ,5.Despite the high prevalence, only 10% of cases are diagnosed before the age of 14, reaching approximately 25% in adulthood . The conKS is a result of either maternal or, in approximately 50% of cases, paternal meiotic nondisjunction of the sex chromosomes during gametogenesis . While mThe phenotype of aneuploid and different mosaic cell lines vary from typical KS male to disorders of sex development (DSD) from true hermaphrodite, ovarian hypoplasia, ovotesticular DSD, or normal female . In thisThe propositus, an adolescent with one-year history of gynecomastia, was referred to our clinic for evaluation. He was born as an only child, conceived on the second attempt of IVF to a 34-year-old mother. The reason for her infertility is unknown. The pregnancy was reported to be normal. He was born at term with a low birth weight of 2300 g. He presented with chordae, proximal penile hypospadias, and right-sided cryptorchidism, which were surgically repaired during the early childhood. At that time, the right testis and right spermatic cord were found to be hypoplastic, and the left testis appeared to be hypoplastic on palpation. In the first year of life, he had a febrile seizure. His psychomotor development was normal, as was his school performance. He had no known diseases or allergies.On first examination at our clinic, he was 14 years old, his height was 176 cm (87p), and his weight was 52 kg (48p). Apart from gynecomastia and asthenic constitution, there was nothing specific about his aspect. Physical examination showed normal male genitalia after surgical correction; both testes were estimated to be 4 mL in size by Prader orchidometer and positioned in the scrotum. The pubertal stage after Tanner was P2-3, A2. Palpation of the chest revealed 4 × 4 × 2 cm bilateral non-tender gynecomastia. On later check-ups, the testes were estimated to be 3 mL in size, and the right testis was found to be firm in consistency and atrophied.Our patient had breast tissue removed by a plastic surgeon. He was monitored by an endocrinologist, who started testosterone undecanoate depo therapy when his testosterone level dropped below a critical level ) at the age of 17.5 years. He was diagnosed with mild Hashimoto thyroiditis but remained euthyrotic during follow-up. His bone mineral density measured by dual energy X-ray absorptiometry (DXA) before the introduction of testosterone supplementation was within the normal range . 2—5P). He was reaching normal testosterone values with 250 mg of testosterone undecanoate every 3 months.At the last check-up, he was 20 years old, his final height was 182.13 cm (76P), and his weight was low  and luteinizing hormone LH; 23.4 IU/L (0.2–1.9 IU/L) levels, low total testosterone; 5.4 nmol/L (0.1–28.6 nmol/L) and free testosterone; 8.0 pmol/L (30.15–189.8 pmol/L) levels and oestradiol levels of 0.14 pmol/L. His serum β-hCG was in the normal range. Semen analysis was performed at 15 years of age, and no sperm were found.Cytogenetic analysis of peripheral blood revealed the presence of two cell lines: a normal female 46,XX cell line in 91% of the examined cells and an aneuploid 47,XXY cell line in 9% of the examined cells. Karyotyping was performed by GTG banding with a resolution of 400–500 bp in a haploid set. Mosaicism was also confirmed with fluorescent in situ hybridisation (FISH) using probe LSI SRY/CEPX . Combined dual labelled probe hybridizes on the centromeric region of the X chromosome and on the specific SRY gene section on the Yp11.3 chromosome .Among 121 metaphase cells from peripheral blood, 110 cells were 46,XX and 11 cells were 47,XXY, corresponding to 91% and 9% of cells, respectively. We determined the final genotype of our patient according to ISCN nomenclature 46,XX[29]/47,XXY[3].ish X[81]/X(DXZ1x2),Y(SRYx1)[8]. PCR-based Y-microdeletion analysis successfully identified five Y-specific loci , confirming the presence of a Y chromosome. Karyotyping of the chromosomes from peripheral blood lymphocytes of the patient’s parents showed a normal female 46,XX in his mother and a normal male 46,XY karyotype in his father. Gonadal tissues were not available for the study.47,XXY Klinefelter syndrome is a result of meiotic nondisjunction of the sex chromosomes during gametogenesis. The origin of a 47,XXY cell line from a 46,XX cell line is not easily explainable. In the case of our patient, the most likely hypothesis, therefore, is that the 46,XX cell line was derived from a post-zygotic loss of a Y chromosome in a proportion of cells of a primordial 47,XXY zygote. In contrast, the loss of an X chromosome would give rise to a 46,XY cell line. The possibility of gonadal mosaicism in the parents, which could result in our patient’s genotype, is less probable.There are 20 cases of 47,XXY/46,XX mosaicism with different phenotypical outcomes described in the literature. To our knowledge, eight cases of phenotypic male individuals with KS features ,22,23,24Previous research suggests that patients with higher-grade aneuploidies are more severely affected , whereasPhenotypic variations in non-mosaic KS could be explained by several factors. First is the X-linked androgen receptor (AR), which is linked to a polymorphic chain of cytosine-adenine-guanine (CAG) triplet repeats. The number of CAG repeats is inversely related to the activity of this receptor . An incrIn mosaic KS, the degree to which different tissues are affected by mosaicism could be the key factor of clinical variability in these patients . Tachon The development of the testes is determined by the presence or absence of a Y chromosome. In mosaics, the differentiation of gonads to testes or ovaria depends on the predominating cell line in the gonads . In our Klinefelter syndrome and other related anomalies are heavily underdiagnosed, likely due to their clinical variability and also due to possible occult and rare presentations. It affects the testicular function, which results in primary hypogonadism. Patients can present with a variety of symptoms due to insufficient testosterone levels: poor muscle bulk, sparse body hair, narrow shoulders and broad hips, underdevelopment or regression of secondary sexual characteristics, osteoporosis, depression, and diminished intellectual capacity. In adulthood, patients are most commonly affected by fertility problems and sexual dysfunction ,38. AnotThe identification of spermatozoa in sperm is extremely rare in KS patients. While in adolescents, spermatozoa are extracted from sperm anecdotally, in only 0–4%, a slightly better outcome (9%) is obtained in young adults . NeverthBased on our experience, active searching for Klinefelter syndrome symptoms is simple and effective and therefore sensible. Simple screening by palpation and volume estimation of the testes as part of a standard health examination of (pre)pubertal boys is neither difficult nor expensive. We believe that examination of the testes should be an essential part of preventive health checks during childhood and adolescence.In conclusion, there are very few cases of 47,XXY mosaicism with an additional female cell line 46,XX described in the literature. We report a case of an adolescent with male phenotype and a rare variant mosaic 47,XXY/46,XX karyotype who presented with painless bilateral gynaecomastia. 47,XXY and 46,XX mosaic cell lines were identified with GTG-banding and further characterized using fluorescent in situ hybridization. We summarized the available clinical presentations of reported male patients with 47,XXY/46,XX mosaicism. To improve the clinical management and quality of life in individuals with rare and cryptic genomic imbalances, the genetic diagnosis would need to be extended to atypical cases.


Objective: To describe the availability and nutritional composition of commonly available street foods in Almaty, Kazakhstan.Methods: 384 street food vending sites (in 10 public markets) were assessed; information on vending sites’ characteristics and food availability and samples of the most commonly available street foods  were collected for chemical analysis.Results: Fruit, beverages and food other than fruit were available in 1.0%, 47.4% and 92.7% of all vending sites. Homemade food other than fruit  were available in 63.4% of stationary vending sites, while industrial  in 45.6% of them. Industrial foods were the most energy-dense . Traditional homemade dishes were high in sodium, reaching 2,248 mg/serving (lagman) and major contributors of protein and fat to energy content . Industrial chocolate and homemade cake presented the highest saturated (14.6 g/serving) and trans-fat (3.20 g/serving) contents.Conclusion: These findings advocate for the implementation of health promotion strategies targeted at vendors, consumers and other stakeholders. Street food is widely available to several populations worldwide, especially in low and middle-income countries (LMIC) , 2. It iCountries in this region are undergoing a transition from traditional to more globalized eating patterns. Throughout the last 4 decades, a decreased availability of cereal, roots and tubers and increased availability of animal products, sugar, sweeteners and vegetables has been observed . In paraFood consumption away from home is increasing in the region, fostered by socio-economic phenomena, such as urbanization and women empowerment . Street Almaty, the former capital, is the largest in the country, with an estimated population of approximately 1.8 million inhabitants, corresponding to approximately 10% of the country’s population  or informal .The interviewers registered the Global Positioning System (GPS) coordinates and collected the following information, through direct observation at each vending site: sex of the vendor, mobility of the vending site and type of physical setup. Stationary vending sites were categorised into formal  complied with the limit of 2 g TFA/100 g fat defined by the Eurasian Economic Commission to be applied in fat products used in the manufacture or preparation of foods [lepyoshka, a typical flatbread in Central Asia, also presented 39 g TFA/100 g of fat. Even though this legislation was not in place when this study was conducted, these findings advocate for the need to address monitoring actions at large and small-scale production levels to attain compliance with national and international standards.Although traditional homemade dishes presented the highest mean energy content per serving, they were also the street foods with highest protein contribution to their total energy 11–23%). These foods are essentially meat-based, as observed in other settings , 28, 30,3%. These vendors , which mof foods . Althougplov and lagman overcame, respectively by 4% and 12%, the maximum daily WHO recommended intake of 2,000 mg [kurut, or fried pastries, such as samsa, piroshky, chebureki or belyashi. One of the possible reasons underlying these findings might be the “Silk Road” pattern, described by the use of salt for food preservation, which strongly remains in the food culture nowadays [Almaty population is more than triple the recommended . The same study concluded that the population was aware of the adverse health effects of high salt intake, despite only 10% knew that their intake was too high [The main sources of sodium were also traditional homemade dishes. One single serving of 2,000 mg . Similar2,000 mg . A high nowadays . The meatoo high . In additoo high . Adding too high , which akozhe, of considerable protein content, were concurrently available in urban Almaty. These findings suggest that, in contrast with other settings where westernised products, including ultra-processed options, were widely available [Almaty, Dushanbe and Bishkek [The low contribution to nutritional quality in the studied foods also extends to the availability of ultra-processed foods , namely vailable , 30, a m Bishkek , 28. The Bishkek , 38.Kazakhstan is committed to implement salt reduction and TFA elimination initiatives, product reformulation and labelling, standards for public provision of food, public awareness and improved availability of fresh fruits and vegetables . The curOne of the limitations of this study is the fact that it was conducted in the summer, and the observed number and diversity of street food vending sites could be higher than in other seasons, given that a substantial proportion of the informal vendors reported not working in adverse weather conditions. In addition, it has been previously reported that street foods may vary with agricultural seasonality . NeverthDespite the limitations inherent to a cross-sectional description of Almaty’s street food environment in the summer of 2017, it is noteworthy that this study adds comprehensive insight into the characterization of street food environments in more urbanized settings , and in Almaty seem to illustrate its relevance for this urban population. Industrial foods were more energy-dense than homemade foods, but no significant differences were found regarding energy per serving. Meat-based traditional homemade dishes and pastries were predominant sources of protein and particularly high in total fat, namely SFA and TFA and sodium. The findings of this study call for the implementation of health promotion strategies targeted at vendors, consumers and several stakeholders, in order to improve the nutritional profile of street foods, ultimately addressing NCDs and associated health inequities in the urban context.The wide availability and variety of street foods found in


Diplodus sargus , exposed to realistic levels of MeHg in feed , comprising exposure  and post-exposure  periods. Total Hg levels increased with time in gills and liver during E and decreased significantly in PE (though levels of control fish were reached only for gills), with liver exhibiting higher levels (2.7 times) than gills. Nuclear magnetic resonance (NMR)-based metabolomics revealed multiple and often differential metabolic changes between fish organs. Gills exhibited protein catabolism, disturbances in cholinergic neurotransmission, and changes in osmoregulation and lipid and energy metabolism. However, dietary MeHg exposure provoked altered protein metabolism in the liver with decreased amino acids, likely for activation of defensive strategies. PE allowed for the partial recovery of both organs, even if with occurrence of oxidative stress and changes of energy metabolism. Overall, these findings support organ-specific responses according to their sensitivity to Hg exposure, pointing out that indications obtained in biomonitoring studies may depend also on the selected organ. Mercury (Hg) is a dangerous and persistent trace element. Its organic and highly toxic form, methylmercury (MeHg), easily crosses biological membranes and accumulates in biota. Nevertheless, understanding the mechanisms of dietary MeHg toxicity in fish remains a challenge. A time-course experiment was conducted with juvenile white seabreams,  Mercury (Hg) is recognized as a dangerous metallic pollutant because of its persistence and high toxicity to organisms , and in A major concern is the ability of Hg to be easily transferred along trophic chains through bioaccumulation and biomagnification processes and then be accumulated in wildlife at high trophic levels ,9. In paAlthough Hg toxicity has been the topic of several studies ,18,19, cDiplodus sargus , is a common demersal fish that inhabits infralittoral and circalittoral rocky habitats in the Mediterranean Sea and eastern Atlantic Ocean [D. sargus accumulates dangerous elements in its tissues, such as Hg [D. sargus a valuable bioindicator species for monitoring aquatic contaminants. In previous studies [1H NMR) allows for the simultaneous analysis of a wide range of metabolites involved in different metabolic pathways, thus offering numerous advantages for elucidating organism–environment interactions. In addition, it allows for the identification of novel metabolic biomarkers in response to stress in organisms provoked by changes in abiotic factors, diseases, or environmental pollutants [The white seabream, ic Ocean , and it ch as Hg , even if studies ,30, whit studies . Lin et  studies  identifillutants ,39,40,41Although previous studies have highlighted the detrimental impact of MeHg on the brain of white seabream ,30, so fD. sargus, through the evaluation of Hg bioaccumulation and metabolomic profiling. In detail, fish were exposed by diet to a realistic concentration of MeHg (8.7 μg g−1 dry feed) over 14 days. Successively, a post-exposure period of 28 days was also taken into consideration in order to estimate the recovery ability of the two selected target organs in fish. In order to further improve the current knowledge on the effects of Hg on aquatic biota, the present study was designed to compare the cytotoxic effects of dietary MeHg on gills and liver of white seabream, 2O, heavy water; 99.8 atom %D; CAS-No. 7789-20-0) for NMR spectroscopy was obtained from Armar AG Chemicals . The 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt  was purchased from Sigma-Aldrich . The other chemicals required to conduct the metabolomics analysis, namely, methanol  and chloroform  were also purchased from Sigma-Aldrich . For the experimental exposure, MeHg chloride  was purchased from Sigma-Aldrich Chemical . Other routine chemicals used in this work were of analytical grade and acquired from local suppliers.Deuterium oxide (DDiplodus sargus), provided by the Aquaculture Research Station , were used in the experiment. Fish wellbeing deserved permanent attention in accordance with national and international guidelines for the protection of animal welfare. Fish were allowed to acclimatize to experimental conditions and routines for two weeks prior to MeHg exposure. Water temperature, salinity, and pH were monitored daily, varying as follows, respectively: 13.5 ± 0.3 °C, 35 ± 2, and 7–8. Juvenile white seabreams  under a 10 h light:14 h dark photoperiod. Seawater was renewed daily (around 80%), and fish were fed once a day, namely, 1–2 h before water renewal. In sampling days, fish were not fed in the 12 h preceding handling that started around 09:00 am. Fish were held in 300 L fiberglass tanks in an average density of 0.056 kg L−1 dry weight) used to expose fish to this Hg form were similar to those previously detected in natural food of D. sargus , as previously described [−1).Fish were fed with feed (3 mm pellets) produced by SPAROS Company  composed by 44% protein and 16% lipids. A solution of MeHg chloride (prepared in ethanol) was added during the process of pellet production, with a homogenous distribution of the toxicant throughout the batch. MeHg levels in contaminated pellets  purchased from Sigma-Aldrich, China, and buffered with NaHCO3. Fish were then weighed, measured, sacrificed by cervical transection, and properly bled from the cardinal vein (using heparinized Pasteur pipettes). Gills and liver were excised, and the two sets of samples stored at −80 °C until further processing for Hg determination and metabolomics.Fish were exposed to MeHg for 7 (E7) and 14 (E14) days. Then, to potentially allow recovery for 28 days, fish started to be fed with uncontaminated pellets  . Thus, tFirstly, gills and liver samples were lyophilized, homogenized, and then analyzed for total Hg (tHg) in an advanced mercury analyzer (AMA) , according to the U.S. EPA method 7473 . Briefly−1) and cold distilled water (0.85 mL g−1) in order to be homogenized using a TissueLyser LT bead mill , with the inclusion of a 3.2 mm stainless steel bead in each tube. Therefore, homogenization took place for 10 min at 50 vibrations/s. The homogenates, after addition of chloroform (4 mL g−1) and distilled water (2 mL g−1), were then vortexed for 60 s to be mixed and left on ice for 10 min to partition. Afterward, samples were centrifuged for 5 min at 2000 g at 4 °C, resulting in a triphasic mixture. The upper methanol layer (600 µL) with polar metabolites was then carefully removed and transferred into clean vials to be dried in a centrifugal vacuum concentrator (Eppendorf 5301). The resulting pellet was then kept at −80 °C. Prior to NMR analysis, the dried extracts were resuspended in 600 μL of a 0.1 M sodium phosphate buffer  with 1 mM DSS, used as internal reference, and then transferred to a 5 mm diameter NMR tube. DSS acts as an internal standard and provides a chemical shift reference (δ = 0.0 ppm) for the NMR spectra, while D2O provides a deuterium lock for the NMR spectrometer. Polar metabolites were extracted from liver and gill tissues of white seabreams (n = 6 per condition at each sampling time) by applying a “two-step” methanol/chloroform/water protocol, as reported in detail in previous works ,40. Brie1H NMR spectra, a PRESAT pulse sequence was applied for suppressing the resonance of residual water, with 6 kHz spectral width and a 2.0 s relaxation delay. A total of 128 transients was collected into 16,384 data points requiring a 10 min acquisition time for each sample under investigation. All data sets were zero filled to 32,768 data points, and exponential line-broadenings of 0.5 Hz were used before application of the Fourier transformation. A Chenomx Processor, a module of Chenomx NMR Suite  software, was then used for manually phasing, baseline-correction, and calibration (DSS at 0.0 ppm) of all 1H NMR spectra acquired. The Chenomx 500 MHz library database, another module included into the Chenomx NMR Suite software, was utilized for identifying the different peaks profiled within the acquired 1H NMR spectra from each seabream gill and liver in order to be assigned with reference to known chemical shifts and peak multiplicities. Furthermore, the use of Chenomx NMR Profiler, another module also included in the Chenomx NMR Suite software, allowed us to quantify the level of each individual metabolite detected in the 1H NMR spectra using the known concentration of the internal standard DSS, previously introduced in each sample prior to the NMR analysis [A Varian-500 NMR spectrometer working at a spectral frequency of 499.74 MHz at 298 K was used to analyze fish tissue extracts. To obtain one-dimensional (1-D) analysis ,49. Ther−1) measured in seabream gills and liver were presented as mean ± SD, and statistically significant differences (p < 0.05) between the control and exposed fish at all the experimental times were established by application of a two-way analysis of variance (ANOVA) followed by the post hoc Sidak’s multiple comparisons test. Metabolomics data were expressed in mM as mean ± SD. For each fish organ, metabolite changes were calculated via the ratio between the averages of exposed fish at each sampling time and control peak areas. The metabolic dataset was tested for normality using the Shapiro–Wilk distribution test, and after confirmation of normal distribution, data homogeneity was evaluated through the Levene test. Data were analyzed by one-way ANOVA using Dunnett’s multiple comparison test, which was aimed at finding significant differences between the control and exposed fish at each experimental time. Data were considered statistically significant at p < 0.05. GraphPad software  was used for all the statistical analyses. Bioaccumulation data on the levels of total Hg (μg g−1) and in the liver (10.2 μg g−1), with the latter organ depicting levels 2.7 times higher. It is worth noting that during the post-exposure period, tHg levels decreased for both organs but remained above the values recorded in the control fish.The levels of total Hg (tHg) measured at all experimental times in the gills and liver of white seabreams exposed to MeHg by diet, as well as those of control fish, are depicted in 1H NMR spectra of the metabolome of the gills and liver of juvenile white seabream D. sargus from the control group are shown in Typical 1-D Dietary exposure to MeHg provoked alterations in different classes of metabolites in fish gills at each experimental time . Indeed,Dietary exposure to MeHg provoked alterations in different classes of metabolites in fish liver at each experimental time . In regaD. sargus, with the aim of elucidating the cytotoxic effects of dietary MeHg and the potential recovery ability of the selected target organs during a successive post-exposure period, revealing differential impairments in various metabolic pathways.To date, the use of metabolomics has proven to be an extremely effective tool for investigations of the perturbed metabolic pathways in aquatic biota triggered by a variety of pollutants ,49,50,511H NMR-based metabolomics allowed for the simultaneous detection of 27 metabolites in the gills and 31 metabolites in the liver. Interestingly, some metabolites were in common among the two fish organs, though organ-specific metabolites were also observed. In detail, the presence of metabolites involved in neurotransmission  and osmoregulation (betaine) was observed solely in the gills, whereas some carbohydrates and molecules involved in energy metabolism , as well as ketone bodies , were predominantly recorded in the liver. The evidence of organ-specific metabolomic profiles, both in terms of presence of specific metabolites and in terms of their concentrations, as previously described in other aquatic organisms [The use of rganisms ,52, may rganisms ,43. Convrganisms ,53, besirganisms . The different physiological role of the two examined organs would also explain their dissimilar capacity to accumulate Hg. In fact, in the present study it was found that the liver, responsible for detoxification of pollutants, showed more than two times higher levels of total Hg than the gills following MeHg dietary exposure. In both organs, total Hg levels tended to decrease during the post-exposure period, though concentrations did not reach baseline levels after 28 days of depuration, as they remained statistically higher than the values recorded in the control, thereby attesting to the strong persistence of Hg within biological tissues ,55,56,57−1) provoked, at all the selected exposure times , an increase in the level of branched-chain amino acids , tyrosine, phenylalanine, and alanine, likely due to a MeHg-induced protein catabolism, followed by a noteworthy drop in their concentrations at the successive post-exposure period (PE28), perhaps associated with a possible occurrence of protein biosynthesis as a repair or cell renewal/turnover process. As is widely documented in the literature, BCAAs play a key function in the immune system by both regulating protein turnover and being precursors for the biosynthesis of new molecules , which are essential for cells such as lymphocytes to correctly perform their functions [It is well known that free amino acids are the most abundant components in the gills of fish ,41. In tunctions . Thus, tunctions ,60. TherMoreover, the higher levels of tyrosine and phenylalanine, coupled with the enhancement of acetylcholine detected at all the experimental times, may be associated with alterations in the neurotransmission system. As a matter of fact, tyrosine and phenylalanine are precursors of the neurotransmitter dopamine, which is implicated in the dopaminergic system , while aThe depression of neuronal activity could therefore result in an alteration of the normal functioning of the gills, also supported by the increased concentration of alanine as discussed above. The increased energy demand would seem to be partly counterbalanced by the enhanced levels of lactate, which may suggest a shift towards anaerobic energy metabolism . FurtherBesides changes in energy metabolism, in the gills of fish the osmoregulation system appeared also to be disrupted following exposure to MeHg via diet, as supported by the increased levels of taurine and glycerophosphocholine observed during all the exposure and post-exposure times. The MeHg effect on the concentration of osmolytes confirms the high sensitivity of the osmoregulatory system in aquatic organisms exposed to Hg species and other classes of pollutants ,67,68. HMoreover, among the changes observed in the metabolome of fish gills, it is interesting to note the increased levels of inosine, likely due to the breakdown of adenosine, which are a sign of cellular stress, commonly occurring during high energy demand . In addiFinally, it is interesting to note how certain metabolites, and in particular those involved in neurotransmission, osmoregulation, and cell membrane stability, appeared to be altered even during the post-exposure period (PE28). This may reflect a higher sensitivity of certain metabolic pathways than others to MeHg exposure and emphasize the persistence of certain harmful effects triggered by MeHg, even after a considerable time beyond the exposure phase ,72.The MeHg dietary exposure of white seabreams provoked alterations in some metabolic pathways also at the hepatic level that, with some exceptions, appeared differently from those revealed at the gills. Among amino acids, the changes observed in the levels of alanine were comparable with those recorded in the gills. In fact, the concentrations of alanine increased during all the exposure and post-exposure times, thus confirming the impact of MeHg in nitrogen metabolism ,64. Contrarily to the gills, changes in the amino acids glutamate and glutamine were observed in the liver. These metabolites, which are precursors of glutathione, were found to be reduced especially during the selected exposure times. These impairments, taken together with the drop recorded in the levels of glutathione, support the involvement of the antioxidant system to face the impact triggered by MeHg exposure, as already widely reported in the literature, since it is known that a decisive way to initiate Hg toxicity in organisms is through oxidative stress ,35,40,41Moreover, in the liver of seabreams dietarily exposed to MeHg, variations in the levels of glucose, ATP/ADP, creatine, and niacinamide were also noted and could be ascribed to disruptions in energy metabolism. In fact, the drop revealed in the glucose levels and the unchanged level of glycogen, despite the regular food administration to seabreams, suggest hepatic gluconeogenesis inhibition, contrarily to what was observed in the golden grey mullet collected in a highly Hg-polluted environment ,41. HoweContrary to the results observed in the gills, the levels of several metabolites detected at the hepatic level appeared to be altered even at PE28, indicating a higher sensitivity to MeHg of this organ than the gills, probably linked also to the higher Hg accumulation recorded in the liver. Therefore, these findings further support the occurrence of organ-specific responses elicited by fish in accordance with their sensitivity to Hg exposure, since it has been previously documented that MeHg is able to affect differently even parts of the same organ, as observed within the brain optic tectum of white seabreams . Diplodus sargus, as well as to reveal organ-specific cytotoxicity mechanisms induced by MeHg in the gills and liver. In brief, following exposure to MeHg via diet, an intense energy catabolism associated with disruption of neurotransmission and osmoregulation systems was observed in the gills, whereas impaired antioxidant and detoxification systems were revealed in the liver, coupled with changes in the energy metabolism. Although the post-exposure period of 28 days had allowed a partial recovery of both target organs, there was still evidences of oxidative stress and changes of the energy metabolism, revealing long-term effects of dietary MeHg in fish. Overall, organ-specific cytotoxicity mechanisms of dietary MeHg exposure were discerned, pointing out the vulnerability of fish health to this Hg form at highly impacted ecosystems and the need for increased surveillance in this direction, therefore emphasizing the urgency of the further development of new remediation strategies against this persistent environmental pollutant.In this work, the NMR-based metabolomic approach allowed us to shed light into the toxic metabolic pathways triggered by dietary MeHg exposure in juveniles of white seabream,