Lactobacillus plantarum is considered a potential probiotic supplementation for treating obesity. However, the underlying molecular mechanism is poorly understood. Our previous study displayed that L. plantarum FRT4 alleviated obesity in mice fed a high-fat diet (HFD) through ameliorating the HFD-induced gut microbiota dysbiosis. To explore the roles of FRT4 in obesity prevention, in this study, we investigated changes in serum metabolomic phenotype by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) and analyzed the pathway of HFD-fed Kunming female mice orally administered with FRT4 for eight weeks. Using orthogonal partial least squares discriminant analysis (OPLS-DA), metabolite patterns with significant changes were observed. 55 metabolites including phosphatidylcholine, lysophophatidylcholine, sphingomyelin, serotonin, indole-3-methyl aceta, indole-3-carbinol, indole-5,6-quino, 11,12-DHET, prostaglandin B2, leukotriene B4, and 3-hydroxybenzoic acid were identified as potential biomarkers associated with obesity, which were mainly involving in glycerophospholipid metabolism, tryptophan metabolism, and arachidonic acid metabolism. Perturbations of 14 biomarkers could be regulated by FRT4 intervention. These metabolites may serve as valuable biomarkers to understand the mechanisms by which intake of diets containing FRT4 contributes to the treatment or prevention of obesity. Thus, FRT4 can be a promising dietary supplement for the prevention of HFD-induced obesity. The availability of inexpensive, high-fat processed foods has contributed to the worldwide prevalence of obesity, which is one of the strongest risk factors for metabolic diseases, including type two diabetes mellitus T2DM), hypertension, and cardiovascular disease . The imbDM, hyperLactobacillus is a major component of intestinal flora and is commonly used as a probiotic. In obese individuals, the supplementation of different strains of Lactobacilli has been shown to reduce fat mass while reducing the occurrence and progression of T2DM and insulin resistance [Lactobacillus plantarum is one of the most well-studied species of Lactobacillus because of its versatility, allowing it to be distributed in a wide range of environments such as fermented foods and humans. Numerous studies have shown that L. plantarum has a variety of functional properties that exert beneficial effects on obesity. L. plantarum FRT10 had anti-obesity effects on obese mice, which was partly associated with activating PPARα/CPT1α pathway [L. plantarum NCU116 alleviated non-alcoholic fatty liver disease (NAFLD) through the regulation of the expression of genes associated with lipogenesis, lipolysis, and fatty acid oxidation [L. plantarum WCFS1 was found to increase the expression of immune response pathways [Increasing studies have shown that probiotic treatment can ameliorate the disorders of host metabolism by regulating gut microbiota . Lactobasistance . Lactoba pathway . L. planpathways  and epitpathways . Howeverpathways ,10. MetaL. plantarum FRT4 on intestinal flora and liver metabolomics. The results suggested that FRT4 could effectively prevent obesity via alleviating HFD-induced gut microbiota dysbiosis, the mechanism of which could be partly revealed by liver metabolomics (data not shown). However, the exact mechanism is still unclear. Systemic metabolic disorders in patients can be reflected by serum markers, which are more suitable for clinical application because of their advantages of being non-invasive and at a low cost. Therefore, based on our previous studies, in this study, we further studied the effects of the dietary intervention of FRT4 on serum changes of HFD-induced mice during eight weeks of feeding via metabolomics method based on UPLC-QTOF/MS. Then, in order to find more specific obesity-related biomarkers and metabolic pathways, OPLS-DA analysis was used to determine the number and the distribution of metabolites with significant differences among each group. The aim of this follow-up research was to study the effects of FRT4 on serum metabolomics profile and provide more evidence for the potential mechanism of FRT4 against obesity. Meanwhile, this study may provide new insights into the pathogenesis of obesity, and the candidate biomarkers may be used for the diagnosis of obesity and related diseases.We previously established an HFD-induced obesity model in mice to study the regulatory effects of L. plantarum FRT4 was isolated and identified from a kind of local yogurt in Xinjiang province and preserved in the Chinese General Microorganism Collection Center . It was cultured in MRS broth at 37 °C for 24 h. After centrifugation at 5000 rpm at 4 °C for 10 min, the bacterial cells were collected, washed with 0.9% saline buffer three times, and adjusted to 1 × 1010 colony-forming units (CFU)/mL.10 CFU/mL). All experiments were approved in accordance with the Laboratory Animal Ethical Committee and its Inspection procedures of the Institute of Feed Research of CAAS (AEC-CAAS-20090609).Mice  were supplied by Vital River Laboratory Animal Technology Co. Ltd. . After 1-week adaptation with the administration of a normal diet and water, the mice were randomly divided into two groups: control group  with a normal chow diet and HFD group (n = 18) fed a HFD based on the previous reports ,12. The After 8 weeks of gavage treatment, all of the mice were sacrificed to collect blood after fasting for 6 h. The serum was obtained by centrifugation of the blood samples at 3000 rpm, 4 °C for 10 min and then was stored at −80 °C for metabolomics analysis.4AC and NH4OH were supplied by Sigma Aldrich and Fisher chemicals, respectively.Acetonitrile and methanol were supplied by CNW technologies. NHv/v) were vortexed for 30 s, followed by ultrasonication in ice water bath for 10 min. After cooling down at −40 °C for 1 h, the mixtures were centrifuged at 12,000 rpm, 4 °C for 15 min. Supernatants of 400 μL were freeze dried, and the powders were solved in 150 μL methanol:acetonitrile  extract including isotope-labeled internal standard mixture, followed by vortex for 30 s and ultrasonication for 10 min. Then, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min, and supernatants of the same amount from the whole samples were mixed into QC samples for further analysis.EP tubes containing 100 μL serum sample and 400 μL extract  (A) and acetonitrile (B). The automatic injection temperature was maintained at 4 °C, and 3 μL was the injection volume.The UHPLC system  with a Waters ACQUITY UPLC BEH Amide column (1.7 μm × 2.1 mm × 100 mm) coupled to Q Exactive HFX mass spectrometer  was utilized to analyze serum metabolic profiling. The mobile phase consisted of 25 mM NHOn the basis of the information-dependent acquisition (IDA) mode, Xcalibur acquisition software (Thermo) was applied to acquire MS/MS spectra. The ESI source conditions were as follows: sheath gas flow rate, 30 Arb; Aux gas flow rate, 25 Arb; capillary temperature, 350 °C; full MS resolution, 60,000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; spray voltage, 3600 V in positive ion mode, and 3200 V in negative ion mode.The original data of MS were converted to the mzXML format by ProteoWizard and untargeted peak detection, peak extraction, peak alignment, and peak integration were processed by R package XCMS. Metabolites were identified using the in-house MS2 database (BiotreeDB). The cut-off value of the annotation was set to 0.3.2 and Q2. The permutation test of OPLS-DA was used to validate the model. The significantly differential metabolites were identified when variable importance for the projection (VIP) > 1 and p < 0.05 in the Student’s t-test.After preprocessing the original data, OPLS-DA was applied to process the metabolomics analysis using SIMCA software . The fitness and reliability of the OPLS-DA were verified by the parameter values Rp < 0.05) were searched from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database . Meanwhile, metabolites were retrieved from KEGG online database and corresponding metabolic pathways in KEGG were extracted. Compared with the model group, using differential abundance (DA) score was used to further screen the pathway affected by FRT4 intervention.Metabolites with significant differences  and the ESI- mode , indicating significant changes in serum induced by HFD. OPLS-DA score plots revealed that there was a significant difference between the FRT4 group and the model group  and negative ion (ESI-) modes. To reveal the differential metabolites among CT, HF and HF4H groups, the OPLS-DA model was used to determine the serum metabolic profile. As shown in el group C,D. The 2Y = 0.87, Q2 = −0.72 in the ESI+ mode, R2Y = 0.91, Q2 = −0.61 in the ESI- mode for HF versus CT group. As shown in 2Y = 0.97, Q2 = −0.27 in the ESI+ mode, R2Y = 0.98, Q2 = −0.23 in the ESI- mode for HF4H versus HF group, which displayed that the OPLS-DA models had good robustness and there was no overfitting problem.As shown in p < 0.05, significantly differential metabolites at the superclass level were classified into 13 categories, including organoheterocyclic compounds; phenylpropanoids and polyketides; organic nitrogen compounds; organic acids and derivatives; nucleosides, nucleotides, and analogs; organic oxygen compounds; alkaloids and derivatives; organic compounds; lipids and lipid-like molecules; benzenoids; nucleosides, nucleotides, and analogs; lignans, neolignans, and related compounds; and hydrocarbons. Finally, 55 differently abundant metabolites were identified (p < 0.05), while kynurenic acid, 3-methyldioxyindole, indole-3-carbinol, indole-5,6-quino, 11,12-DHET, prostaglandin B2, and leukotriene B4 were significantly decreased (p < 0.05). Notably, FRT4 intervention partly reversed the abnormal serum metabolites alterations caused by HFD.According to VIP > 1 and entified . CompareThe serum metabolites of HF group were significantly different from those in CT group. As shown in Additionally, 8 potential biomarkers associated with tryptophan metabolism were also identified, including serotonin, indole-3-methy aceta, indole-3-carbinol, indole-5,6-quino, indoleacetaldehyde, 5-methoxytrytophan, and N-methynicotinamide. The relative content of serotonin, indole-3-methy aceta, indoleacetaldehyde, and 5-methoxytrytophan increased significantly in HFD-fed mice compared to the CT group, while decreasing significantly after FRT4 intervention. In contrast, indole-3-carbinol, and indole-5,6-quino were significantly reduced, while FRT4 intervention showed recovery patterns for these metabolites. Moreover, FRT4 intervention significantly reduced the level of indoleacetaldehyde, indole, 3-indoleacetonitrile, and 5-hydroxyindoleacetic acid. The results displayed that tryptophan metabolism disorder was generated by HFD, whereas FRT4 treatment reversed this trend, which indicated that the effects of FRT4 on tryptophan metabolism might be one of the reasons for its obese efficacy 11,12-DHET, prostaglandin B2, leukotriene B4, 15-deoxy-d-12,14-PGJ2, 5-KETE, prostaglandin G2, and prostaglandin D2 involved in arachidonic acid metabolism were decreased 0.084, 0.36, 0.22, 0.071, 0.22, 0.0077, and 0.097-fold, respectively, while FRT4 treatment increased 11,12-DHET, prostaglandin B2, leukotriene B4, and 15-deoxy-d-12,14-PGJ2 2.65, 2.23, 2.17, and 44.78-fold, respectively. Besides, FRT4 treatment increased 8-isoprostaglandin F2a (2.01-fold) and lipoxin B4 (1.69-fold). These alterations were significantly prevented after FRT4 intervention, suggesting that the favorable effect of FRT4 on obesity might be associated with regulating arachidonic acid metabolism.Furthermore, three potential biomarkers related to phenylalanine metabolism were found in the HF group, including hippuric acid, 2-phenylacetamide, and L-tyrosine, which were significantly lower than those in CT group, while hippuric acid and 2-phenylacetamide were significantly reduced after FRT4 intervention. Finally, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid associated with phenylalanine, tyrosine, and tryptophan biosynthesis metabolism were significantly decreased compared to the CT group, while FRT4 significantly increased 3-hydroxybenzoic acid.p < 0.05 and pathway impact value, metabolic pathways with a significant difference in response to FRT4 treatment in obese mice were evaluated by KEGG annotation and pathway-based differential abundance (DA) analysis. As shown in On the basis of L. plantarum FRT4 could alleviate HFD-induced obesity in mice through modulating gut microbiota and liver metabolomics (data not shown). In this study, analysis of the serum metabolites profile by UHPLC-QTOF MS indicated that the mice fed HFD were abnormal in serum, mainly involved in glycerophospholipid metabolism, tryptophan metabolism, arachidonic acid metabolism, phenylalanine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis metabolism. In addition, FRT4 intervention had an inhibitory effect on obesity, which might be related to the recovery of some biomarkers.Our previous report has successfully established the obese model in mice with metabolic disorders in cecum contents and liver tissue after 8 weeks of continuous treatment of HFD . Supplemp < 0.05) after FRT4 intervention suggested that the administration of FRT4 suppressed PC degradation and protected the membrane. Phosphatidylethanolamine (PE) is a class of phospholipids that exist on the cell membrane of all living organisms and is the backbone of most biological membranes along with PC, phosphatidylserine (PS), and phosphatidylinositol (PI). In this study, PI(18:1(9Z)/18:1(9Z)) and PE(P-18:1(11Z)/18:4) were decreased after FRT4 intervention. Consistent with the stimulation of PCs, LysoPCs and LysoPEs, which were products or metabolites PCs and PEs, respectively, were significantly enhanced in the HF group as compared to the CT group. Altered levels of LysoPCs are identified to be related to diseases with the disorder of energy, such as obesity and hyperlipidemia [The disfunction of glycerophospholipid and fatty acid metabolism has been identified to be directly associated with hyperlipidemia. Glycerophospholipid levels in the HF group were higher than that in the CT group, suggesting that HFD caused lipid disorders. In accordance with our previous report, most glycerophospholipids in the liver of obese mice were significantly enhanced , and theipidemia . LysoPC, the most restricted essential amino acids  and precArachidonic acid (AA) and its derivatives play a critical role in the development of obesity, NAFLD, diabetes, cardiovascular disease, and other common diseases by linking nutrient metabolism with immunity and inflammation . 11,12-DAlthough we found these findings in this research, limitations to understanding the preventive effects of FRT4 on metabolic dysfunction remain. Therefore, further investigations are needed to characterize the metabolites, elucidate the specific functions of individual metabolites and the preventative effects of FRT4 on related metabolic pathways.The present study suggested that FRT4 intervention had a favorable inhibition effect on obesity, which might be related to the efficacy of FRT4 by restoring some of the identified biomarkers. 14 identified potential biomarkers in serum associated with the disturbance of the metabolism could be regulated by FRT4 in obese mice. FRT4 intervention showed partial recovery patterns for PCs, LysoPCs, SMs, PGs, and serotonin involving glycerophospholipid metabolism, tryptophan metabolism, and arachidonic acid metabolism, which were thought to be central factors in the prevention of obesity. Our results also predicted that the FRT4-treated mice could protect the cell membrane by remodeling glycerophospholipid metabolism and arachidonic acid metabolism. These metabolites could provide the basis for further understanding the pathogenesis of obesity and HFD-related diseases, increase the predictability of obesity risk, and evaluate the efficacy of anti-obesity drugs. These results support the concept that FRT4 had beneficial effects in HFD-induced obesity-associated metabolic disorders. These findings demonstrated the effects of FRT4 on serum metabolomics profile, which could provide new evidence for understanding the potential mechanism of FRT4 in preventing obesity.


Ovarian clear cell carcinoma (CCC) exhibits unique characteristics, including slow growth, glycogen accumulation in the cytoplasm, and poor prognosis for stress resistance. Several molecular targeting agents have failed to treat ovarian CCC. Recent reports have identified metabolic alterations through HNF1B, which is highly expressed in ovarian CCC. The Warburg effect, GSH synthesis, and mitochondrial regulation occur in CCC. The metabolic behaviors of ovarian CCC resemble the evolution of life to survive in stressful environments. Understanding the fundamental biology of ovarian CCC might help in the development of novel therapeutic strategies.AT-rich interacting domain-containing protein 1A and phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit alpha genes may contribute to the carcinogenesis of ovarian CCC, whereas hepatocyte nuclear factor-1-beta (HNF1B) plays crucial roles in sculpting the unique characteristics of ovarian CCC through metabolic alterations. HNF1B increases glutathione synthesis, activates anaerobic glycolysis called the Warburg effect, and suppresses mitochondria. These metabolic changes may be induced in stressful environments. Life has evolved to utilize and control energy; eukaryotes require mitochondria to transform oxygen reduction into useful energy. Because mitochondrial function is suppressed in ovarian CCC, these cancer cells probably acquired further metabolic evolution during the carcinogenic process in order to survive stressful environments.Clear cell carcinoma (CCC) of the ovary exhibits a unique morphology and clinically malignant behavior. The eosinophilic cytoplasm includes abundant glycogen. Although the growth is slow, the prognosis is poor owing to resistance to conventional chemotherapies. CCC often arises in endometriotic cysts and is accompanied by endometriosis. Based on these characteristics, three clinical questions are considered: why does ovarian cancer, especially CCC and endometrioid carcinoma, frequently occur in endometriotic cysts, why do distinct histological subtypes (CCC and endometrioid carcinoma) arise in the endometriotic cyst, and why does ovarian CCC possess unique characteristics? Mutations in  AT-rich interacting domain-containing protein 1A (ARID1A) and phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit alpha (PIK3CA) genes have been found in approximately half of the cases [hepatocyte nuclear factor-1-beta (HNF1B) gene, which is highly expressed in ovarian CCC [Ovarian cancer is the worst gynecologic malignancy and includes heterogeneous histological and biomolecular features ,2,3. Clehe cases ,12,13. Trian CCC ,15. In arian CCC ,12,13. Irian CCC ,17, and rian CCC . Thus, trian CCC . BecauseARID1A, PIK3CA, and KRAS have been identified in deep endometriosis, indicating that endometriosis is a neoplastic disease [HNF1B were found to be associated with endometriosis and modified the risk of ovarian cancer, as well as endometriosis [Endometriosis is one of the most common gynecological disorders, affecting approximately 15% of women in their reproductive years . Althoug disease . Single-etriosis ,15. ThesKRAS mutations and PIK3CA mutations, although these epithelia were histologically benign and normal [ARID1A loss-of-function mutation in an ovarian endometriotic cyst, further driver events may be needed for the malignant transformation of ovarian endometriotic cysts with ARID1A loss-of-function mutations [Why does ovarian cancer develop in endometriotic cysts? Suda et al. reported that endometriosis and uterine endometrium frequently exhibit cancer-associated somatic mutations, including d normal . Clonal d normal . The burd normal . In addiutations . These sutations ; therefoutations ,26. Dietutations ,28 and iutations ,30,31,32utations ,34. The utations . We confutations ; therefoThe second question we sought to address is why CCC and endometrioid carcinoma are more frequent in endometriosis-related ovarian cancer than high-grade serous carcinoma and mucinous carcinoma. The rate of co-existence of endometriosis is higher in CCC and endometrioid carcinoma than in high-grade serous carcinoma and mucinous carcinoma ,29. AnotHNF1B signaling. We assessed whether the signature is involved in the stressful environment of carcinogenesis in endometriotic cysts. When cells were cultured with stressful factors, such as the contents of endometriotic cysts and iron, the ovarian CCC signature was upregulated [Using expression microarrays of ovarian cancer, we identified a gene set composed of 320 genes which are specific to ovarian CCC and named them ovarian CCC signatures [egulated . Our preegulated , suggestegulated . In theiBased on the above, we hypothesized two models of pathogenesis of carcinogenesis in endometriosis-associated ovarian cancer . The firp16 gene mutations in a rat renal carcinogenesis model [ARID1A and PIK3CA mutations [HNF1B signaling [HNF1B signaling, which forms a network in the ovarian CCC signature, may be involved in ovarian CCC properties. HNF1B expression is associated with physiopathological cytoplasmic glycogen accumulation in ovarian CCC, endometrial CCC, and non-neoplastic endometrium in the mid-to-late secretory phase of the menstrual cycle and gestation [The third question is why CCC possesses unique characteristics, including glycogen accumulation in the cytoplasm, slow growth, and chemoresistance. The genomic alterations in endometriosis-related ovarian cancer are distinct from those induced by free iron. Free iron frequently induces is model ,39,40, wutations ,12,13. Tignaling . Oxidatiestation . A bioinestation ,42. As sHNF1 transcription. Both expression and DNA methylation analyses revealed that the HNF1B signal is activated through decreased methylation in ovarian CCC. In contrast, ERa signaling is activated in endometrioid carcinoma of the ovary. In our previous report, whole exome sequencing using ovarian CCC revealed genomic abnormalities in many signals, as well as genes belonging to the HNF1B signal in ovarian CCC [PIK3CA were found to occur in 54% and 26% of cases, respectively, and mutations and amplification of KRAS were identified in 10% and 18% of ovarian CCC cases, respectively [KRAS-PI3K signaling were identified in 82% of ovarian CCC cases; furthermore, 79% of CCC cases show copy number alterations in MYC-RB signaling, including MYC amplification and RB1 deletion in 81% and 31% of CCC cases, respectively [TP53 gene was deleted, and MDM2 and MDM4 were amplified in ovarian CCC. ARID1A mutations were identified in 62% of cases, and SMARCA2 and SMARCA4 were deleted in 21% and 41% of CCC cases, respectively [KRAS-PI3K signaling, MYC-RB signaling, and the suppression of the TP53 network and the SWI/SNF complex are associated with ovarian CCC [ARID1A and PIK3CA mutations [KRAS mutations are identified in 9–17% of cases, and TP53 mutations are observed in 5–15% of ovarian CCC cases [ZNF217 (Zinc finger protein 217) locus, is frequently amplified in ~36% of CCC cases [MET (chr7q31) and AKT2 (chr19q13.2) oncogenes have been identified in 31% and 24% of ovarian CCC, respectively, and deletions of the CDKN2A/2B (cyclin-dependent kinase inhibitor 2A/2B) tumor suppressor genes have been detected in 17% of cases [PI3K signaling, is high, and pMAPK, which reflects KRAS signaling, is also highly expressed, similar to the genomic characteristics. Based on the immunohistochemistry of nine subunits belonging to the SWI/SNF complex, 61% of the cases exhibited the loss of at least one SWI/SNF complex subunit; thus, genomic and epigenetic abnormalities occur in ovarian CCC, which differs from the other histological types.How is the expression of the ovarian CCC signature regulated? A comprehensive DNA methylation analysis showed that CCC had a profile distinct from that of other histological subtypes ,43. Thisrian CCC . Mutatioectively . Genomicectively . The TP5ectively ; moreoverian CCC . Other sutations ,46. KRASCC cases ,46,47,48CC cases . ChromosCC cases . Amplifiof cases ,51,52. Wof cases ,31. The HNF1B transcript signaling, as described above [HNF1B mutations cause maturity-onset diabetes in the young type five [From the distinct gene expression profile of ovarian CCC compared to other histologic subtypes ,49, HNF1ed above . HNF1B mype five ; therefoHNF1B induces glucose uptake into ovarian CCC cell lines through glucose transporter 1 (GLUT1) and activates glycolysis flux and lactate secretion . ComprehARID1A deficiency, which is identified in approximately half of ovarian CCC cases, suppresses glutathione synthesis. ARID1A-deficient cancer cells are specifically vulnerable to inhibitors of the antioxidant glutathione metabolic pathway and the glutamate–cysteine ligase synthetase catalytic subunit, a rate-limiting enzyme for glutathione synthesis [ARID1A deficiency.HNF1B also contributes to glutathione synthesis . An HNF1ynthesis . These rHNF1B suppresses cell proliferation  and reprWhy does the Warburg effect occur in ovarian CCC? Aerobic metabolism occurs in the mitochondria , which pHNF1B, resulting in CCC  oceans, other than possibly a few “whiffs”, atmospheric O2 was essentially nil until the great oxidation event (GOE) 2.3 billion years ago (2 production by oxygenic cyanobacteria and plants ~600 million years ago. Although oxygen production increased tremendously by the combined activity of cyanobacteria and plants, the oxygen was immediately reduced by the large amounts of H2S and Fe2+; subsequently, the oceans were oxidized, atmospheric oxygen increased to the present values, and led to an end to H2S as an energy source. In the Phanerozoic eon, the earth is supported by solar energy and an abundance of atmospheric oxygen. Mitochondria have evolved to give organisms the energy to exploit new, more complex morphologies interacting in increasingly complex ecosystems. Eukaryotic organisms with nuclei acquire mitochondria to use oxygen to efficiently produce energy; however, the abundance of oxygen poses a new threat to life. Mitochondria simultaneously emit oxidative stress which animals are forced to deal with. H2S-dependent animals either became extinct, retreated to isolated sulfide niches, or adapted. Mass extinctions were often associated with a fall in ambient O2 and an increase in H2S, perhaps providing a biological filter for descendants that retained some degree of tolerance to hypoxia and sulfide.Life has evolved for energy homeostasis. The earth was formed ~4.6 billion years ago, and is defined by four eons, including the Hadean, Archean, Proterozoic, and Phanerozoic eons . The Hadears ago . The ProDoes ovarian CCC use mitochondria for evolution or reversion? Charles Darwin hypothesized that all species of organisms arise and develop through the natural selection of inherited variations that increase an individual’s ability to compete, survive, and reproduce—called Darwinian theory . LamarckOur study, along with others, suggests two carcinogenic processes in endometriotic cysts. The first is carcinogenesis under persistent stressful environments, resulting in the carcinogenesis of ovarian CCC, because endometriotic cysts contain abundant oxidative stress through free iron. The second potential carcinogenesis process is primarily estrogen-dependent and gives rise to endometrioid carcinoma. In ovarian CCC cells, HNF1B plays crucial roles in establishing the unique characteristics of ovarian CCC, including morphology, slow growth, and resistance to conventional chemotherapies. Abundant glycogen accumulation in the cytoplasm is caused by the uptake of glucose, which increases anaerobic glycolysis activity by HNF1B. HNF1B suppresses the cell cycle, which is compatible with the clinical growth of ovarian CCC. HNF1B contributes to resistance against anti-cancer agents through glutathione synthesis and the Warburg effect. In the Warburg effect, mitochondrial function is suppressed to decrease ROS production. The purpose of life evolution is the efficient production of energy. Although eukaryotes require mitochondria to generate energy efficiently, ovarian CCC cells suppress mitochondrial function. A better understanding of the fundamental biology of ovarian CCC and innovative strategies targeting metabolism that overcomes evolution are necessary to develop useful therapies against ovarian CCC.


Chlorophylls, carotenoids, macro/micronutrients in leaf, and anthocyanin derivatives in grape peel were evaluated. Expression levels of 24 genes, including nitrogen, phosphorous, potassium, and anthocyanin pathways in leaf, peel, and pulp were validated by qPCR at L-29, L-33, and L-36 stages. Results indicated that Norway (21:7:12) and Peters 1 (30:10:10) increased carotenoids, chlorophylls, and anthocyanins in leaves, while Zhanlan (16:16:16) improved fruit biochemical attributes, and anthocyanin . However, a better grape yield was obtained by the application of Peters 1 (30:10:10). Potassium pathway genes were upregulated by Nutrivant (14:14:30), phosphorous pathway genes by Peters 2 (20:20:20), and nitrogen pathway genes by Peters 1 (30:10:10), while Nutrivant (14:14:30) upregulated anthocyanin pathway genes and simultaneously enhanced anthocyanin biosynthesis in berry peels. Results of two years’ study concluded that Peters 1 (30:10:10) was proved better to increase yield, while Zhanlan (14:14:30) was superior in improving anthocyanin biosynthesis.Fertilization, a fundamental aspect of a plant’s life, has been of great concern for agricultural specialists to minimize the yield gap between actual and potential yield. Around the globe, fertilizers with different NPK ratios are being used to attain a better yield of grape. To find the suitable commercially available fertilizer for quality grape production, a 2 years (2017–2018) study was conducted for the evaluation of 10 fertilizers with different NPK ratios. Commercial fertilizers included were Zhanlan (16:16:16), Garsoni (15:15:15), Acron (16:16:16), Norway (21:7:12), Peters 1 (30:10:10), Nutrivant (14:14:30), Peters 2 (20:20:20), UMAX (15:15:15), G2 (20:20:20), and Yara (15:15:15). The fertilizer application rate was 20 g plant Soil fertility decline is a major prevailing factor in reducing food production in tropical regions. Tumbling fertility is mainly attributed to the erosion of mineral via harvested crop, depletion of organic matters, and less addition of inorganic fertilizers . TherefoIncreasing the amount of N applied during the period of early growth promotes vegetative growth, which results in high yields. However, excessive N fertilizer applications impede differentiation of flower buds and yield related attributes ,10. IncrPotassium contributes a role in many functions in plants; it increases the photosynthesis rate, maintain turgor of stomata guard cell, balance osmotic concentrations, increase biomass production, and yields ,15,16,17VvNR (V. vinifera nitrate reductase), VvGDH (V. vinifera glutamate dehydrogenase), VvAS (V. vinifera asparagine synthetase), VvNIR (V. vinifera nitrite reductase), and VvGS (V. vinifera glutamine synthetase) [VvSORK (V. vinifera outward rectifying shaker-like K+ channel), VvSIRK (V. vinifera inward rectifying shaker-like K+ channel), VvKUP2 (V. vinifera K+ uptake and metabolism permeases 2), VvKUP1 (V. vinifera K+ uptake and metabolism permeases 1), VvPAP (V. vinifera purple acid phosphatase), VvPHO1 (V. vinifera phosphate (Pi) transporter 1), VvPHT2–1 (V. vinifera phosphate transporter 2–1), and VvPHT1–4 (V. vinifera phosphate transporter 1–4) [In the assimilation process of nitrogen, major enzymes are encoded by the grapevine nitrogen metabolism genes, including thetase) . Potassiter 1–4) ,19,24,25Balanced fertilizer is required for economical crops, which can be optimized by scientific methods. Therefore, agriculture production be under by the unscientific use of fertilizer. Residue of excess fertilizer disturbs the balance of soil-nutrients and prevents beneficial growth of plant. Economical crop can be achieved by application of required amount of correct fertilizer type ,27,28. BThe aim of this study was to evaluate the response of different commercial NPK fertilizers of different countries based on grape quality, and yield. Furthermore, the present study intended to illuminate gene expression-based practicability and workability of these fertilizers on grape that might be useful for farmers to choose from available NPK sources based on their locality and preferences. Furthermore, the study intended to help commercial industry to formulate new combinations of NPK for potential yield and quality grape.At stage L-38, TA%, TSS (Brix°), TSS/TA, and pH were measured from grape berries subjected to different fertilizer combinations. A significant interaction between fertilizer combinations and both years of study was found, so the data are presented separately for both years (2017 and 2018). Peters 1 and G2 in 2017 and Peters 1 in 2018 depicted the highest TSS/TA, as these fertilizers depicted 29%, 14%, and 28% increases, respectively, as compared to their respective controls. Peters 1 application showed the least TA% during both years of study, while its highest value was recorded by the application of Norway in 2017 .The application of Peters 1 in 2018 recorded the highest pH in fruits, followed by the same fertilizer in 2017 . Norway p < 0.5) contributing parameters in both years. Norway application in 2018 and Peters 1 application in 2017 and 2018 recorded significantly highest number of berries per cluster and weight per cluster (p < 0.5) highest weight of 100 berries in both years.Years and fertilizer combinations depicted significant interaction regarding the number of berries per cluster, 100 berries weight, weight per cluster, and fruit yield per plant. While the number of clusters per plant was non-significant regarding any treatment applied . Control cluster . Norway Peters 1 recorded the highest fruit yield (18.49 Kg per plant) during the first year of study as a ~4–5-fold increment was recorded compared to control followed by Norway. However, application of Norway resulted in the highest fruit yield during the second year along with Nutrivant, and it showed a 4-fold increment in fruit yield compared to control. Norway was the only fertilizer that resulted in higher and sustained grape yield during both years of study.Control treatment recorded minimum N, P, and K contents in grape leaves at all growth stages under study in 2017 a. SlightCalcium contents depicted an increment in 2018 as compared to 2017 regardless of growth stage measured. In 2017, Acron application exhibited a 23% decrease in Ca contents than the respective control at the L-36 stage b. The hiDifferent combinations of NPK fertilizers were applied at three different stages, significantly affecting the anthocyanin contents in grape berry peel. Anthocyanins were determined at L-38 stage. Twenty-five contents were detected in treated grape skin.O-(6″-acetyl-glucoside), cyanidin 3-O-(6″-caffeoyl-glucoside), cyanidin 3-O-(6″-p-coumaroyl-glucoside), cyanidin 3-O-glucoside, and cyanidin derivatives , delphinidin 3-O-(6″-p-coumaroyl-glucoside), delphinidin 3-O-feruloyl-glucoside, delphinidin 3-O-glucoside, delphinidin 3-O-glucosyl-glucoside, delphinidin derivatives was examined in grape berry peel. Control depicted minimum delphinidin contents while Norway, Peters 1, Peters 2, and G2 were statistically the same with control. The application of Zhanlan and Garsoni showed the highest increment in delphinidin contents with a ~4-fold increase than control (The response of delphinidin 3- control b.O-(6″-acetyl-glucoside), malvidin 3-O-(6″-caffeoyl-glucoside), malvidin 3-O-(6″-p-coumaroyl-glucoside), malvidin 3-O-glucoside, and malvidin derivatives contents were affected significantly by the application of different fertilizers combinations. Application of Norway and Peters 1 were not able to increase malvidin contents, and both these fertilizers were statistically at the same level as control. However, the approximately 4-fold increase was observed with the application of Zhanlan, Garsoni, and Acron as compared to control , pelargonidin 3-O-(6″-succinyl-glucoside), pelargonidin 3-O-(6″-succinyl-glucoside), pelargonid-in 3-O-glucoside, and pelargonidin derivatives were measured to evaluate the differential response of various fertilizers , peonidin 3-O-(6″-p-coumaroyl-glucoside), peonidin 3-O-(6″-p-coumaroyl-glucoside), peonidin 3-O-glucoside, and peonidin derivatives were measured at L-38 stage (Peonidin 3-38 stage b. IncremO-(6″-acetyl-glucoside), petunidin 3-O-(6″-p-coumaroyl-glucoside), petunidin 3-O-glucoside, and petunidin derivatives  and three different parts .Nitrogen pathway genes, including; NR, GS, GDH, AS, and NIR were studied for transcriptional analysis from grape leaves, peel, and pulp. NR relative expression depicted an increasing trend with progression in growth; however, this increase was more evident in leaves as compared to peel and pulp. An 11-18-fold increase in NR transcript level in grape leaves was observed by the application of Peters 1 at L-33 and L-36 as compared to control a. NutrivTranscript levels of PHT1-4, PHT2-1, PHO-1, and PAP were studied in grape leaves, fruit peel, and pulp at L-29, L-33, and L-36. Relative expressions of PHT1-4 and PHT2-1 depicted an increasing trend with the progression of growth stages as highest transcript levels were measured at L-36 . Peters Relative expressions of HKT1, SIRK, SORK, KUP1, and KUP2 were observed in grape leaves, fruit peel, and pulp at three different stages .Relative expressions of HKT1 remain the same at all growth stages, except for pulp. Nutrivant application resulted in maximum HKT1 relative expression among all fertilizers with a ~6-fold increase as compared to respective controls a. SIRK tPAL, CHS, F3′H, F3′5′H, DFR, LDOX, UFGT, OMT, and GST were studied for differential relative expressions in grape leaves, fruit peel, and pulp at L-29, L-33, and L-36.Relative expressions of PAL, CHS, F3′H, DFR, OMT, and GST were higher in fruit peel as compared to leaves . Peters In this study, an investigation of grapewine’s N, P, and K uptake and metabolism genes response to applied fertilizers of different NPK ratios was carried out. There are different types of N, P, and K fertilizers and these types have exclusive characteristics that is specific for different crops. Besides, yield and quality parameters of grapes can be optimized by the application of specific fertilizer combinations ,31,32.Nevertheless, it is challenging to forecast the precise nutritional requirements of grapes on various developmental stages. Internal and external factors influence these diagnostic techniques, and these are less sensitive to be employed as fertilization strategies. Though by exploring the transcript levels of NPK uptake and metabolism genes, the precise nutrient requirements of various developmental stages could be find out, and adequate fertilizer absorption time can be predicted, consequently permitting us to fertilize with the accurate nutrient type at the right time. At this point, the advanced effects can be included since berry ripening indicators such as; °Brix index, grape juice pH value, sugar to acidity ratio, along with the berry weight and fruit yield at harvest, were expressively affected by different fertilizers application. Consequently, the experiential effects of N on anthocyanin contents can be related to alteration in anthocyanin biosynthetic pathway regulation.NPK fertilizers having different NPK ratios from various companies were used in this study to evaluate their effects on anthocyanin contents, biochemical parameters, mineral content, related gene transcript levels, and yield contributing parameters of grapes. Among the ten fertilizers used, Acron, Norway, and Peters 1 had differential ratios of NPK. Acron and Norway had more nitrogen as compared to phosphorus and potassium, while Peters 1 had more potassium concentration. Plant nutrition diagnosis, soil testing, and formulation of fertilizers are the main focus of crop fertilization research ,35,36,37Azolla pinnata) [Chlorophyll and carotenoids were increased by fertilizers of the higher ratio of NPK. Nutrivant and Acron application recorded the highest and at par chlorophyll contents in 2018 at L-29 and L-33 stage. Chlorophylls  are the vital components of the plant’s photosynthesis. A drastic decrease in total chlorophyll content is obvious in plants with less N compared to other treatments. A probable clarification for the deterioration in chlorophyll during N famishment is that chlorophyll molecules structure has four N atoms. Thus, it becomes complicated for the cell’s organelles to manufacture these compounds in nitrogen source absenteeism . Mineralpinnata) . Anotherpinnata) .2−) and nitrate (NO3−) [Leaf nitrogen was highest in plants applied with Petrer1 due to the highest amount of nitrogen in this fertilizer. In both years of study, the maximum leaf N concentrations were detected in grapevines applied with the higher N application. This most possibly occurred as fertigation endorsed the urea dissolution by accelerating urease hydrolysis. This lead to ammonium carbonate formation, which decomposes quickly, forming hydroxyl, carbonate, and ammonium. Later on, through biological oxidation, this might be converted into nitrite (NOe (NO3−) , and mase (NO3−) . Increase (NO3−) ,41.Furthermore, in orange leaves an increase of N application also caused a decline in N concentration . The higBy the results of the study, it is clear that the berry yield by Norway and Nutrivant application is at the same level, and the same fertilizer recorded the highest fruit yield. Higher berry weight ultimately increased yield by highest N application along with P and K. Berry weight and yield tended to increase with increasing N ratio in combined fertilizer according to the previous findings ,52,53. IThe highest values of acidity and lowest TSS were found in grapes applied with Zhanlan having comparatively high NPK nitrogen. The possible explanation of these results is that the grapevines absorbed greater amounts of mineral N from the soil, that usually rouse shoot growth, causing the lower frequency of solar rays reaching into the grapevines. The more shading of the grape clusters inside the grapevine hampers canopy aeration from modifying temperature, that can lower sugar contents and upsurge the berry acidity . The higAnthocyanin belongs to flavonoid family of polyphenol phytochemicals. It gives bright red-orange to blue-violet color to numerous fruits, as well as vegetables also. Anthocyanin exists in the form of aglycone bind to different sugar moiety in the plants. Vital anthocyanin (stored in vacuoles) comprises of cyanidin, delphinidin, peonidin, pelargonidin, petunidin, and malvidin ,62. In tO-glucosyltransferase (UFGT) [Solanum lycopersicum leaves, nitrogen depletion caused an increase in transcript levels of PAL, CHS, and F3H [The structural genes of anthocyanin and potassium-pathway in the present study showed higher expression levels by the application of Nutrivant that has NPK in equal ratio. The Nutrivant depicted higher K contents in leaves, and yield contributing parameters were higher by the same fertilizer application. The same fertilizer showed increased anthocyanin contents as compared. The structural genes indoctrination of the enzymes accountable for each catalytic step have been categorized, and the knowledge of corresponding molecular regulation has significantly proceeded over past two decades ,71,72,73e (UFGT) . A previe (UFGT) . In Sola and F3H . Corresp and F3H .Different ratios of NPK play various roles in improving gape attributes. NPK fertilizer should be chosen on the basis of required parameters in grape. Although applying fertilizers based on gene expression data has been confirmed by the findings of our study and it can be regarded as a viable and applied fertilization approach, further appraisal of various fertilizer forms, mixtures, and doses rendering to transcript levels knowledge will permit us to accomplish a wider goal. Furthermore, that strategy will allow us to forecast the nutritional needs of plants. Additionally, fertilizers application with the optimal type at the most reasonable application rate will also be convenient. The present research can contribute to improving traditional fertilization practices, and it can serve as one of the important findings concerning the fertilizer application in vineyards. Despite the growth conditions were the same for all plants under study; however, the molecular makeup of plants can be affected by many other cultural practices and environmental factors.V. vinifera × V. labrusca) in 2017 and 2018. Experimental site was Grape Experimental Vine-Yard, Jiangsu Academy of Agricultural Sciences (31°62′ N/119°18′ E), Baima, Lishui District, Nanjing, China. Weather data of the experimental site was collected from Nanjing Institute of Meteorolgy, Nanjing, Jiangsu, China . Yield components include number of berries per cluster, total fruit weight per cluster (g), 100 berries weight (g) yield per plant (Kg), and number of clusters per plant. Weighed at least 10 clusters of grape samples for each product to calculate the average weight of the cluster, and used 100 berries per cluster to calculate the average weight of the berries.3) was added into tubes, and then placed for digestion for 2 h in above mentioned machine. After cooling samples at room temperature, the final volume of each sample was adjusted 10 mL with distilled water. Specific ion content was measured using an inductively coupled plasma optical emission spectrometer  . The blank samples were prepared and processed by adding 5 mL of concentrated nitric acid to an empty digestive vessel while standard solutions of each element were prepared in different concentrations [Grape leaf samples were collected to assess the amounts of plant nutrients at (E-L 29), (E-L 33), and (E-L 36) stages. Leaf samples were collected after 96 h of NPK application, for nutrient examination. Leaves were collected to the opposite side of a ring. The blade of the leaf was immediately removed from the petiole, dried under the shade then stored for 48 h in an oven at 65 °C. The samples dried were ground into a fine powder for analysis using a hand mill. Powdered leaves weighted 0.1gm were shifted into digestion tubes of ETHOS Touch control advanced microwave lab stations of Milestone microwave laboratories . After that 5mL of concentrated nitric acid , measured using a portable hand-held dialyzer digital Atago PAL-1 meter , and the titratable acidity (%) was titrated with 0.1 mol/L NaOH. pH was measured with Mettler Toledo FE20 Desktop pH Meter  .Grapes fruit skin samples were analyzed at the harvest stage (L-38) to determine anthocyanin components.Around 50 berries were picked from each pack, skins were peeled and powered with liquid nitrogen, then frozen for 24 h at −80 °C in FreeZone 4.5 Plus LABCONCO until weight was constant .v/v/v, 1 mol/L). The mixture was collected for 10 min under 100 Hz ultrasonic waves, and then shaken for 30 min at 25 °C in the dark at a rate of 150 rpm. Then, using a high-speed, refrigerated centrifuge, the sample was centrifuged at 10,000 rpm and 4 °C for 10 min, and the supernatant was collected. Then, we re-extracted the residue four times. Finally, both supernatants were dried with a rotary evaporator, then re-dissolved with mobile phase A in 10 mL. Prior to UPLC analysis, all extracts were filtered with a filter of 0.45 μm.Peel 0.50 g of powder was blended with 10 mL of solution HCl/methanol/water  was allowed for recalibration to the lock mass options. The ionization parameters were: the capillary voltage was 2.5 kV, the colliding energy was 40 eV, the source temperature was 120 °C and the temperature of the desolvation gas was 400 °C. Using MASslynx 4.1, the data acquisition and processing was carried out. Malvidin-3-O-glucoside  was used as a typical external standard, and it quantified other anthocyanin components.A 2μL solution aliquot with a flow rate of 0.4 mL/min was injected into the UPLC column (2.1/100 mm ACQUITY UPLC BEH C18 column containing 1.7 μm particles). Buffer A composed of 0.1 per cent formic acid in water while buffer B was 0.1 per cent formic acid in acetonitrile. The buffer B gradient was 5 percent of buffer B for 2 min, 5−95 per cent for 15 min, and 95 per cent for 2 min. Mass spectrometry was performed with MSe acquisition mode using an electrospray source, with a selected mass range of 50−1200 In this study, 24 genes , includiAccording to the research  total RN−ΔΔCt method and each control sample was selected as a reference.The volume of the reaction mixtures was 10 μL, including 5 μL of SYBR Green Supermix (Bio-Rad), 2 μL of diluted cDNA, and 0.2 μL of each primordial. Each pair of qRT-PCR primers had been validated with this pair of primers by cloning and sequencing the RT-PCR product. Each priming pair’s efficiency was quantified using serial dilution of a PCR product. All biological samples were tested in triplicates. Ubiquitin and EF1ÿ have been used as internal standards to normalize the expression. Expression levels were calculated on the basis of the 2p < 0.05 level for yield related attributes biochemical parameters, nutrient levels in leaves, and anthocyanin contents in berries. Gene transcript level study was comprised of three biological and technical replicates. Up and down regulation of transcript levels were determined by t-test (p < 0.5) and fold change. Graphs were drawn using Microsoft Excel 2019.Experiment was laid out using randomized complete block design with three replicates. All the fertilizers under study were randomized in three blocks on above mentioned growth stages. SPSS version 15.0  was used to construct ANOVA and statistical evaluation of result at In this research, we applied fertilizers of different N, P, and K ratios on three stages to evaluate impact of these fertilizers on grape in field for two years. We found different ratios have different impact on leaf, fruit parameters, and molecular mechanisms, i.e., high nitrogen increased yield might be because nitrogen metabolism genes were upregulated. If the grape yield is the ultimate goal to be achieved, then Norway must be considered because it performed better and sustained the grape yield on both years. However, decreased anthocyanin contents by the application of the highest nitrogen-containing Norway fertilizer is not a desirable character for grape quality. Though Zhanlan application increased anthocyanin profile of grapes, fruit yield cannot be compromised just for anthocyanin increase. Nevertheless, Nutrivant application not only increased the grape yield, but depicted a moderate increase in the anthocyanin profile as well. That depicts its ability to enhance yield and grape quality simultaneously. This article suggested that viticulturists select Nutrivant as potential fertilizer sources. Fertilizer industries may consider the result of this study to design more specified fertilizers for grape vines.


Polydatin, one of the natural active small molecules, was commonly applied in protecting and treating liver disorders in preclinical studies. Oxidative stress plays vital roles in liver injury caused by various factors, such as alcohol, viral infections, dietary components, drugs, and other chemical reagents. It is reported that oxidative stress might be one of the main reasons in the progressive development of alcohol liver diseases (ALDs), nonalcoholic liver diseases (NAFLDs), liver injury, fibrosis, hepatic failure (HF), and hepatocellular carcinoma (HCC). In this paper, we comprehensively summarized the pharmacological effects and potential molecular mechanisms of polydatin for protecting and treating liver disorders via regulation of oxidative stress. According to the previous studies, polydatin is a versatile natural compound and exerts significantly protective and curative effects on oxidative stress-associated liver diseases via various molecular mechanisms, including amelioration of liver function and insulin resistance, inhibition of proinflammatory cytokines, lipid accumulation, endoplasmic reticulum stress and autophagy, regulation of PI3K/Akt/mTOR, and activation of hepatic stellate cells (HSCs), as well as increase of antioxidant enzymes , glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), glutathione reductase (GR), and heme oxygenase-1 (HO-1)). In addition, polydatin acts as a free radical scavenger against reactive oxygen species (ROS) by its phenolic and ethylenic bond structure. However, further clinical investigations are still needed to explore the comprehensive molecular mechanisms and confirm the clinical treatment effect of polydatin in liver diseases related to regulation of oxidative stress. Increasing epidemic investigations have suggested that liver diseases remain one of the leading causes of deaths globally, and millions of people are suffering from acute or chronic liver disorders nowadays . CurrentOxidative stress is a state due to the imbalance of free radicals and antioxidative enzymes. The imbalance tends to be oxidized, which leads to inflammatory infiltration of neutrophils, increasing the secretion of proteases and large amounts of oxidative intermediate products. Oxidative stress is produced by the excessive free radicals in the body which is considered as one of the most important factors to aging and various diseases. Redox state constitutes a necessary background of multiple liver diseases . OxidatiO-β-D-glucopyranoside, PD), commonly isolated from the roots of Polygonum cuspidatum, can be also obtained from many dietary supplements like grapes, peanuts, cocoa products, hop flowers (Humulus lupulus), and other plants , alcohol liver disease (ALD), liver fibrosis, and HCC. The specific molecular mechanisms of protective and therapeutic effects of PD are concluded in αa (GADD45αa) in zebrafish larvae [In the past 30 years, alcohol consumption has a dramatically increased tendency due to the booming economy in China, leading to the highly incidence of alcohol liver disease (ALD) at 4.5% . Nowadayh larvae . Acetaldαa GADDαa in zebh larvae . Furtherh larvae , 47. Preh larvae .β (IL-1β), and tumor necrosis factor-α (TNF-α) through downregulating toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) p65 [There is increasing evidence that long-term excessive alcohol intaking can increase the release of inflammatory cytokines . PD trea-κB) p65 . Besides-κB) p65 , 46.To conclude, previous studies indicated that pretreatment of PD could alleviate liver diseases induced by alcohol. PD exerts its protective activity by resisting the oxidative stress induced by alcohol and restoring the antioxidant balance and the MMP/TIMP ratio of hepatic tissue.β-oxidation, leading to hepatic steatosis. Hepatic steatosis conversely exacerbates the degree of insulin resistance and accelerates the subsequent transition to steatohepatitis and fibrosis [α (PPAR-α) protein levels, decreased stearoyl-CoA desaturase-1 (SCD-1) and sterol regulatory element binding protein 1 (SREBP-1) protein levels, and reduced total cholesterol (TC) and TG levels in the fructose-fed liver of rats [Nowadays, nonalcoholic fatty liver disease (NAFLD) is emerging as one of the most common causes of chronic liver disease due to the increasing incidence of obesity, diabetes, and metabolic syndrome in the general population , 50. NAFfibrosis , 54. Supfibrosis . Besides of rats . These rin vivo and in vitro. In vivo, PD reduced the levels of malondialdehyde (MDA), ROS, and hydrogen peroxide (H2O2) and decreased thioredoxin-interacting protein (TXNIP) at concentrations of 7.5-30 mg/kg. In vitro, PD could reduce the levels of ROS and TXNIP and enhance miR-200a targeting Keap1/Nrf2 pathway in fructose-induced HepG2 and BRL-3A cells [In NAFLD pathogenesis, oxidative stress is considered as a vital factor . It is r3A cells . In path3A cells . Intrape3A cells .β were released in the liver of fructose-induced rats [+ cells and alleviate hepatocyte steatosis and decrease expressions of proinflammatory factors including S100A8, S100A9, and monocyte chemoattractant protein-1 (MCP-1) in the liver tissues of HFD mice [α, and CD68 macrophage activation related to the suppression of toll-like receptor (TLR) 4/NF-κB p65 signaling pathway [Apart from lipid accumulation and oxidative stress, inflammation also plays a crucial role in the development of NAFLD. Excessive fructose and HFD consumption cause NAFLD pathogenesis. PD could downregulate apoptosis-associated speck-like protein (ASC), and the NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein levels and IL-1ced rats . In anotHFD mice , 61. Bes pathway , 60.In vivo, oral administration of 100 mg/kg PD decreased hepatic lipid accumulation and alleviated inflammation and hepatocyte injury in MCD-induced db/db mice. In vitro, PD reduced palmitic acid-induced lipid accumulation in cultured hepatocytes. Both in vivo and in vitro, PD could restore lysosomal function and autophagic flux which was damaged by steatosis or NASH. In conclusion, PD inhibited PI3K/Akt/mTOR signaling pathway and increased the expression and activity of transcription factor EB (TFEB), a known master regulator of lysosomal function [Autophagy is a system which could regulate intracellular degradation. The development of NASH is considered to be related to impaired autophagic degradation of intracellular lipids. Autophagy regulates lipid metabolism and insulin resistance in the liver and protects hepatocytes from injury and cell death . In vivofunction .Currently, lots of the commonly used drugs, including analgesic, anticancer drugs, agent antiphlogistic, and antidepressant, might be hepatotoxicity for human being . Acetami4 can cause severe hepatocellular injury due to its highly toxic metabolite trichloromethyl free radical via the action of the cytochrome P450 system [4 (50 μL/kg) markedly induced liver injury in mice with increased serum levels of AST and ALT and upregulated IL-1β, TNF-α, iNOS, COX-2, and NF-κB in hepatic tissues. Besides, CCl4 increased the MDA and decreased the GSH, SOD, GST, CAT, and GPx in liver tissues. Interestingly, pretreatment with PD (25-100 mg/kg/day) for 5 days before CCl4 injection can improve the liver injury via upregulation of transforming growth factor-beta1 (TGF-β1) in the liver tissues [Sulfur mustard (SM), a chemical warfare agent applied in a series of military conflicts, possesses serious threat to civilians and military soldiers . Althoug0 system . Intrape tissues .Almost all organ systems including humans and animals could be affected by arsenic (As), as could cause several hazardous effects on animals and humans via inducing oxidative stress , 74. Preα, CHOP, and hepatocellular apoptosis in the cholestatic mice induced by alpha-naphthylisothiocyanate (ANIT) and bile duct ligation (BDL). The results suggest that PD may alleviate cholestatic liver damage by inhibition of oxidative stress, ER stress, and apoptosis [Cholestasis might be induced by intrahepatic and systemic retention of toxic hydrophobic bile salts. Cholestatic liver diseases can develop into periportal inflammation, liver fibrosis, cirrhosis, and even hepatic failure , 79. Thepoptosis .α, endothelial cell adhesion molecule-1 (ECAM-l), intercellular cell adhesion molecule-1 (ICAM-l), NF-κB, and myeloperoxidase (MPO) activities induced by LPS [Fulminant hepatic failure (FHF) is characterized by overwhelming hepatic injury with failure of hepatocyte function, resulting in a devastating clinical syndrome of hepatic encephalopathy, severe coagulopathy, jaundice, and hydroperitoneum , 83. Pred by LPS .α-smooth muscle actin (α-SMA) that synthesizes ECM proteins, such as type I collagen [α-SMA and suppress the increased collagen I and hydroxyproline, an amino acid contained in collagen in liver of mice [4. It is reported that PD could attenuate the proliferation and activation of HSCs via inhibiting SphK1 signaling pathway in CCl4-induced mice, contributing to the suppression of liver fibrosis [β1 signaling is one of the most critical profibrotic pathways [β1/Smad signaling, which was consistent with its protective effect on fructose-induced EMT and liver fibrosis. Inhibiting the nuclear translocation of ZEB1 by PD may be a new strategy to ameliorate EMT of liver fibrosis associated with high-fructose diet [It is reported that uncontrolled simple steatosis might develop to some serious liver diseases such as hepatitis, fibrosis, and cirrhosis , 85. Durcollagen . However of mice . Sphingo of mice , and Sphfibrosis . Using hpathways , 98. Zhaose diet .α, and TGF-β1 and mediators related to oxidative stress that induce quiescent HSCs to differentiate into activated myofibroblasts, the principle ECM-synthesizing cells which play as the vital executor in hepatic fibrogenesis. In return, the activated HSCs promote the recruitment of macrophages from the bone marrow to augment the already-large number of KCs, further aggravating the deterioration of inflammation and fibrogenesis [κB p65 signaling and proinflammatory cytokines secretion in macrophages and oxidative stress [However, liver fibrosis was a complex pathophysiological process. If therapeutic methods are only aimed at decreasing the activation of HSCs, it often leads to undesirable outcomes . Liver fogenesis , 96. Bote stress .β-catenin signaling in hepatocellular carcinoma. The invasion and migration of cancer cells are believed to promote the metastasis of cancer to a large extent. HCC cell invasion and migration invasion assay and wound healing assay were suppressed by treatment with PD [Cancers are the leading killers for human being in the world, especially for aged people over than 55 years old. Besides surgery, chemotherapy remains the best choice for treating various cancers. In recent years, increasing scientific evidences have suggested that natural agents are precious resources for finding more novel and safe candidate drugs for treating cancers, including hepatocellular carcinoma (HCC), lung cancer, and breast cancer , 101. Pa with PD . TherefoYi-du-Tiao-gan mixture, liver Corelle tablet, and Hu-gan-ning capsule, have been used in clinical practice for the treatment of liver disease in China. In these Chinese patent medicines, P. cuspidatum was one of the primary medicinal materials and PD was one of the main active ingredients [In clinical practice, there was no research published on PD single used for treating liver diseases. However, lots of Chinese patent medicines, such as redients –107. Onlredients . Besidesredients .50 of PD was (1000 ± 57.3) mg/kg injected intraperitoneally [50 of PD for human normal liver cells L02 is 263.05 μg/mL [PD has a favorable safety profile in animals (up to a dose of 200 mg/kg) and was well tolerated in humans . The safe evaluation was completed by the New Drugs Safety Evaluation and Research Center in the Chinese Academy of Medical Sciences, which demonstrated that no significant toxic effect existed after intravenous injection of PD for 30 days . Anothertoneally . Howevertoneally . Gavaged05 μg/mL .Collectively, according to the abovementioned effects and mechanisms of polydatin (PD) on liver diseases, it is highly suggested PD is an effective natural product for treating oxidative stress-associated liver diseases, including alcoholic liver diseases, nonalcoholic liver diseases, liver injury, liver fibrosis, and hepatocellular carcinoma. Experimental evidence indicated that PD exhibits curative effect against liver diseases through various signaling pathways, such as PI3K/Akt/Nrf2/HO-1 and Sirt1/Nrf2, PI3K/Akt/mTOR. Overall, this review highlights the potential application of PD as a potential agent against liver diseases.50 of PD, the study of reproductive, carcinogenic, and teratogenic toxicity was not involved. PD has a higher bioavailability than resveratrol, but its concentration in liver tissue was relatively low; intravenous injection of 20 mg/kg of PD, the maximum concentration in liver is 5.22 g ± 0.46 μg/kg; for oral administration of 50 mg/kg of PD, the maximum concentration in liver is 4.47 ± 2.51 μg/kg [In past decades, lots of researches have focused on the preclinical therapeutical effects of PD against various liver diseases. However, the detail molecular mechanisms were not thoroughly explored. Modern research methods such as genomics, metabolomics, proteomics, and metagenomics can be used to conduct more in-depth investigations on the corresponding mechanisms of PD on liver diseases, which provide strong support and theoretical basis for the subsequent clinical research and ultimately developed this natural compound to a new drug for liver disorders –116. Sec51 μg/kg , 118. Re51 μg/kg . It is n51 μg/kg . Therefo51 μg/kg . Finally


Metal nanoparticles are used in various branches of industry due to their physicochemical properties. However, with intensive use, most of the waste and by-products from industries and household items, and from weathering of products containing nanoparticles, end up in the waters. These pollutants pose a risk to aquatic organisms, one of which is a change in the expression of various genes. Most of the data that focus on metal nanoparticles and their effects on aquatic organisms are about copper and silver nanoparticles, which is due to their popularity in general industry, but information about other nanoparticulate metals can also be found. This review aims to evaluate gene expression patterns in aquatic organisms by metal nanoparticles, specifying details about the transcription changes of singular genes and, if possible, comparing the changes in the expression of the same genes in different organisms. To achieve this goal, available publications tackling this problem are studied and summarized. Nanometals were found to have a modulatory effect on gene expression in different aquatic organisms. Data show both up-regulation and down-regulation of genes. Nano silver, nano copper, and nano zinc show a regulatory effect on genes involved in inflammation and apoptosis, cell cycle regulation and ROS defense as well as in general stress response and have a negative effect on the expression of genes involved in development. Nano gold, nano titanium, nano zinc, and nano iron tend to elevate the transcripts of genes involved in response to ROS, but also pro-apoptotic genes and down-regulate DNA repair-involved genes and anti-apoptotic-involved genes. Nano selenium showed a rare effect that is protective against harmful effects of other nanoparticles, but also induced up-regulation of stress response genes. This review focuses only on the effects of metal nanoparticles on the expression of various genes of aquatic organisms from different taxonomic groups. The growing use of nanomaterials pose a threat to the aquatic environment as factory efflux and waste from products in common use such as textiles enriched with nano silver or paint-containing nanometals, enter the water directly . NanoparThere are many different materials that may be turned into nanoparticles. The classification based on the source material consists of carbon-based nanoparticles, such as fullerenes and nanotubes (NTs), ceramics NPs, metal NPs, and organic NPs, such as lipids, and all differ in size, shape, charge, surface modifications, etc. . To obtaThe toxic effect of NPs on living organisms is induced by reactive oxygen species (ROS), the generation of which can be stimulated by various mechanisms of action of NPs ,7. The pMost popular in various branches of industry are copper and silver nanoparticles due to their versatility and a wide range of interesting properties. For that reason, most of the related molecular studies tend to focus on the effects of those two types of metal nanoparticles ,11.The aim of this review is to study and compare changes in the gene expression of various genes from various aquatic organisms, which was achieved by a review of the available literature. Silver nanoparticles (AgNPs) found a place in general industry  mostly due to their antibacterial properties . They arOryzias latipes larvae and Danio rerio embryogenesis [Silver nanoparticles are known to be toxic to some aquatic plants and crustaceans ,15. It wogenesis ,17,18,19Strongylocentrotus droebachiensis, and in adult Danio rerio, nano silver triggers an immune response [In sea urchin, response ,21. Aparzgc:175127 (cellular component), sort1b (involved in lipid metabolism), idh3g (carbohydrate metabolism), thrab , lgals3l (lectin), caspb (proteolysis), zgc:114104 , pvalb5 . Overexpression of some of these genes, such as caspb and lgals3l, is involved in inflammation and apoptosis. Genes down-regulated by AgNCs in zebrafish embryos included per1b (response to light stimulus), nt5c3 (pyrimidine nucleoside metabolism), mif4gdb (regulation of translation), cbx7a (chromatin modification), mep1a.2 (proteolysis), slc45a3 (transmembrane transport), myf5 (muscle organ development) and irg1l (propionate metabolism). A study on the zebrafish embryos treated with silver nanocolloids (AgNCs) and silver nanotubes (AgNTs) caused both up-regulation and down-regulation of various genes . AgNCs cpepd (peptidase), sema3fb (semaphoring 3fb), crestin (crestin), zgc:103670, map2k2a (mitogen-activated protein kinase 2a), zgc:175127, anxa1b (annexin A), mylpfb (myosin light chain), fgf11a (fibroblast growth factor 11a), and cdh15 (cadherin 15), from which three are involved in calcium binding . The down-regulated gene from the AgNTs group involved those that take part in DNA-dependent transcriptional regulation, cell proliferation, and differentiation, i.e., tfdp2, fosb, nr1d1, jdp2, fox1a, nfil13-6, and fos) [Onorhynchus mykiss exposed to silver nanoparticles Ag N10, one gene was found to be overexpressed, namely cyp1a2, involved in general metabolism [The AgNTs-treated group mostly showed up-regulation of different and fewer genes, the top ten being and fos) . In Onortabolism .D. rerio embryos exposed to 4nm AgNPs showed significantly up-regulated hypoxia-inducible factor 4  genes, which implies that after exposure, a state of hypoxia occurred in the embryos. Another up-regulated gene was that encoding peroxisomal membrane protein 2 (Pxmp2), whose product participates in a continual flow of small solutes [D. rerio embryos to up-regulate the transcription of the transforming factor β (TGF-β) involved in cellular pathways, angiogenesis, apoptosis, and cell cycle, as well as signaling superfamily members pitx2, acvr2b, and smad1, crucial in forming various body structures; however, inhibin β B (inhβb), involved in oocyte differentiation, growth, and hormone activity, was down-regulated [Bax1, Noxa, andp21.  solutes . Anotheregulated . Anotheregulated  also shoD. rerio [Sublethal doses of AgNPs of 10 nm and 35 nm increased levels of MT2  in D. rerio . FurtherD. rerio .In zebrafish embryos 24 h post-fertilization treated with AgNPs, there was significant down-regulation of cytochrome 1a gene that is involved in circadian rhythm. Other differently transcribed genes included the down-regulated after 24 hpf (hours post fertilization) and up-regulated after 48 hpf proteasome subunit β type-1, which takes part in protein catabolism, and up-regulated ribosomal protein S6 modification-like protein B .Oncorhynchus mykiss treated with silver nanoparticles at 0.1 mg L−1 and 0.25 mg L−1 revealed increased transcription levels of MT, GST (glutathione transferase), and SOD (superoxide dismutase), which are involved in the antioxidative defense system, in the gills compared to a control group in a dose-dependent manner [t manner . While st manner . Chironomus riparius, exposure to 1mg L−1 AgNPs disrupts expression of the ribosomal protein gene L15 (CrL15), affecting the ribosomal assembly and at the same time induces up-regulation of the gonadotrophin-releasing hormone gene (CrGnRH1), which may lead to the over-activation of the gonadotrophin-mediated pathways and to reproductive failure, and Balbiani repeat gene 2.2 (CrBR2.2). Another study on C. riparius reported differently expressed CrGST genes after treatment with AgNPs for various times [C. riparius, AgNPs induced overexpression of Hsp70, a stress marker, but 0.2 mg L−1 after 24 and 48 h of exposure lowered the levels of CrRPL15  transcript levels, like in the previously mentioned study [In the fourth-instar larvae of us times ,29. In ted study . −1 and 25 µg L−1 silver nanoparticles, the liver of male medaka (Oryzias latipes) showed increased expression of S-glutathione transferase gene for 1 µg L−1 concentration and Hsp70, choriogenin, and vitellogenin genes for 25 µg L−1 concentration, whereas both concentrations of silver NPs induced higher expression of transferrin than in the control group [Mytilus galloprovincialis had been incubated for two weeks in water containing 10 µg L−1 AgNPs, it was found that CYP4YI (cytochrome) and cathepsin (protease) genes showed increased transcript levels, whereas GST and caspase genes (protease involved in apoptosis) exhibited down-regulation [After 28 days of exposure to 1 µg Lol group . After mgulation . S. droebachiensis immune cells, increased transcription of Hsp70 and Hsp60 was observed after keeping them for 48 h in an environment containing 100 µg L−1 AgNPs [Similarly, in −1 AgNPs .O. latipes). Coatings included polyvinylpyrrolidone (PVP), gum arabic (GA), and citrate (Cit), either solely citrate, or conjugated with either tannic acid or glutathione.Kwok et al.  tested dAll types of NPs and coatings caused up-regulation of CYP1a (stress response). Oxidative stress genes were up-regulated only by PVP coating and GA coating. NKA transcripts, however, were elevated by all types of AgNPs but those coated with Cit or Cit-Tannic acid. Chlamydomonas reinhardtii, which turned out to be up-regulated during the treatment. These genes included CYC6 , FDX5 , CTR2 , and CRD1 (copper response defect 1). In the study by Simon et al. [C. reinhardtii, AgNPs induced up-regulation of 141 genes and down-regulation of 86 genes. Wang et al.  tested tn et al.  on C. reCopper nanoparticles (CuNPs) can be found in paints, optical and medical instruments, water pipes, conductive materials, and antibacterial coatings. Their release to the environment is caused, among other things, by factories releasing them as waste efflux, by aerosols, and by exploitation of the products and their weathering .O. mykiss) [D. rerio [Epinephelus coioides after CuNPs treatment [Solea senegalensis [O. mykiss shows [Nano copper is a known toxicant towards aquatic organisms. It was noted to cause histopathological changes in rainbow trout ( mykiss)  and gillD. rerio , and injreatment  and in tgalensis . Toxic egalensis . It can ss shows  and causss shows ,41,42. CEpinephelus coioides subjected to a treatment with copper nanoparticles and copper sulphate, it was shown that after the treatment with either form of CuNPs some genes involved in lipid transport and metabolism were up-regulated, such as those encoding apolipoprotein Eb, carnitine O-palmitoyltransferase 1 and acetyl-CoA acetyltransferase, complement component 3, complement component 8 subunit 2β, and component 9 gene. Some of the down-regulated genes were the cytochrome P450 gene, and sorbitol dehydrogenase or α/β hydrolase domain-containing protein 14B [E. coioides after treatment with CuNPs, the anti-oxidative-related genes , GPx4 (glutathione peroxidase)) were down-regulated, and the up-regulated genes included p53, p38β, TNF-α [Oreochromis niloticus, 50 mg L−1 caused elevation of the transcripts for SOD, CAT, and GP, but TNF- α also showed up-regulation. Additional up-regulated genes consisted of IL-1β, IL-12, Hsp70, and caspase3 [In the study on tein 14B . In the β, TNF-α . On the Elodea nuttalii was subjected to a treatment with 1.4–2 mg L−1 CuNPs, it was discovered that the expression of the copper transporter COPT1 was down-regulated, which led to limiting the uptake of excess Cu [When aquatic macrophyte xcess Cu .−1) in adult zebrafish induced up-regulation of HIF-1 (response to hypoxia), HSP70 , and CTR  [Sublethal levels of nanocopper (1.5 mg Leceptor) .−1 of CuNPs administered for seven days to Cyprinus carpio induced up-regulation of diphosphomevalonate decarboxylase and selenide/water dikinase-1, as well as down-regulation of ferritin heavy chain, rho guanine nucleotide exchange factor 17-like, and cytoglobin-1, respectively involved in the synthesis of isopentyl dipshosphate, transport of phosphorus-containing groups, ferrooxydation, and stimulation of formation of Rho-GTP [Doses of 20 and 100 µg L Rho-GTP .. mykiss subjected to a treatment with CuNPs showed increased hepatic and intestinal levels of metallothionein , with much higher up-regulation in liver [Investigation showed that Oin liver . −1 of CuNPs to mussels M. galloprovincialis, 103 genes in the digestive glands and 119 genes in the gills showed different expression levels with a tendency of up-regulation of the expression in the gills and down-regulation of the expression in the digestive glans. Down-regulated genes included zinc-finger BED domain-containing protein 1 and caspase 1/7-1, whereas some of the up-regulated genes were S-glutathione transferase (defense against ROS), ATP synthase F0 subunit 6 (ATP synthesis), cathepsin L (protease), heat shock cognate 71, and precollagen-D [After 15 days of exposure of 10 µg Lllagen-D .Many other nanoparticulate metals are currently used in industry. Zinc nanoparticles (ZnNPs) are used in sunscreens because of their ability to absorb UV light, and they also show antibacterial properties. They are also extensively tested for their application in cancer therapy . Apart fTitanium nanoparticles (TiNPs) are suitable for use in pharmaceuticals, coatings, inks, and food products due to their preservative properties. They are also used in clothing and anti-fogging coating .Gold nanoparticles (AuNPs) found use in the medical industry in the targeted delivery of drugs, optical bioimaging, diagnostics . OutsideIron nanoparticles (FeNPs) can be used in water remediation, in dye removal, as antibacterial agents, and in medical field .Though very versatile and very useful, these nanoparticles also pose a threat to aquatic organisms. M. galloprovincialis was treated simultaneously with cadmium and TiNPs. Cadmium alone induced up-regulation of abcb1 gene (ATP Binding Cassette Subfamily B Member 1); however, when TiNPs were involved, the transcript level was lower [Danio rerio embryos, however, TiNPs negatively altered the expression of genes for such proteins as type I cytokeratin (regulation of kinases activity), cytochrome P450 and family 51 (involved in cholesterol synthesis), zona pellucida glycoprotein 3a.2 (component of zona pellucida), serum/glucocorticoid regulated kinase 1 (involved in cellular stress response), and prostaglandin D2 synthase (involved in smooth muscle contractions) and at the same time up-regulated the expression of, e.g., carboxyl ester lipase , as well as activin receptor 1ib (kinase) [Danio rerio embryos [C. reinhardtii, TiNPs caused up-regulation of 96 genes, and 80 genes were down-regulated [A rare case of nanoparticle metal having a positive effect on an organism was discovered when as lower . In Dani(kinase) . TiNPs m embryos . In the egulated .Daphnia magna exposed to sublethal concentrations of ZnNPs nanoparticles, genes for multicystatin (protease inhibition) were up-regulated in the concentration of both 2.2 mg L −1 of ZnNPs and 9 mg L−1, whereas in the same concentrations, the ferritin (iron binding), C1q domain protein (activation of complement system), and nucleoside transporter genes became down-regulated [In egulated .c-jun after 96 hpf, whereas MxA and TNF showed down-regulation of expression. The same zinc nanoparticles influenced zebrafish eleuthero embryos differently when for catalase and Cu/Zn SOD genes, which became less expressed after TNF as well, while other genes showed similar expression as in other embryos [bax, puma, and apaf1 genes in the zebrafish embryos treated with ZnNPs, and at the same time, the anti-apoptotic bcl-2 gene was under-expressed. Exposure of ZnNPs to zebrafish embryos caused up-regulation of catalase and Cu/Zn SOD after 48 hpf, but after 96 hpf, these genes were down-regulated; other up-regulated genes included metallothionein 2 after both 48 and 96 hpf, and  embryos . Zhao et embryos  found elogfrl2  and cyb5d1 , and reduction of intelectin 2 (development and innate immunity) was reported [In another study on developing zebrafish embryos treated with ZnNPs, it was found that 358 genes were up-regulated and 112 down-regulated. In the up-regulated group, there were such genes as reported .C. carpio, 100 µg L−1 ZnNPs suppressed the expression of steroidogenic enzyme genes  and transcription factors  [In the testis of p, wnt5) .C. reinhardtii, when subjected to a treatment with ZnNPs, were characterized by 156 up-regulated and 29 down-regulated genes [Green algae, ed genes . O. niloticus increased expression of TNF, IL-1β genes, and SOD. O. niloticus fed with SeNPs had down-regulated Hsp70 gene, a biomarker of shock by various stimulants to the organism [Dawood et al. ,66 perfoorganism ,66,67.sod1, sod2, cox1 , mt2, gaad (DNA repair), and ache (neurotransmission) genes in adult Danio rerio brain, but in the gills as well as in muscles, sod2, cox1, gaad and additionally rad51 (DNA repair) genes showed decreased expression, and, also in the muscles, down-regulation of hsp70 was noted [In the developing eyes of the zebrafish embryos treated with sublethal doses of AuNPs functionalized with a cationic ligand N,N,N-trimethylammoniummethanethiol, significant repression of transcription factors pax6a, pax6b, otx2, and rx1 occurs, as well as of pigmentation transcription factor sox10 . Spiked as noted .Sparus aurata) was exposed to 0.5 and 50 µg L−1 AuNPs. In the 0.5 µg −1 AuNPs group elevation of the expression of genes involved in response to xenobiotics, such as selenium binding protein 1, was elevated. Other groups of genes that were up-regulated consisted of genes encoding proteins that show oxidoreductase activity, such as L2HGDH, SULT1A3, and CYP2N, which involved in immune response, including NLRP3, ST6GAL-1, and integrin β 1 binding protein 3, as well as DNA repair/apoptosis regulation genes, such as perforin 1 or MSH6. Higher transcripts of transcription, DNA processing and translational regulation genes were also found, i.e., MEF2A, EIF4E, and RPL13a. Other genes included, among others, those encoding ETNK1 (lipid metabolism), mitotin (cell adhesion), and talin 1 (cytoskeleton organization).Teles et al.  performe−1 AuNPs, down-regulation of certain genes was also discovered. Those included, among others, glucose dehydrogenase (carbohydrate metabolism), VPS4B (cell cycle regulation), c-ski (repressor of TGFb), and ccdc59 (transcription factor).In the 0.5 µg L−1 AuNPs, were characterized by up-regulation of genes involved in response to xenobiotics (ABCF2), oxidoreductase activity, such as electron transport chains , and immune response (CCDC86). Genes involved in stress response also showed up-regulation, such as CHMP4C or HSC70, as well as genes involved in lipid/protein metabolism, i.e., farnesyl pyrophosphate synthetase, PITRM1, TMEM147, and PSMC3, as well as genes involved in transcription, DNA processing and translational regulation, such as exosome complex exonuclease RRP45, TBL3, WBP11.The second group, fish exposed to 50 µL LDown-regulated genes in the second group of fish involved chemokine CK-1 (immune response), NGFR, programmed cell death 7, MAP2K7 , elastase-like serine protease, ACTN1 (cytoskeleton protein), bloodthirsty (erythrocyte differentiation), and ankyrin repeat-containing protein (transcription factor). S. aurata. A 100 mg L−1 measure of SiNPs triggered up-regulation of stress-induced Hsp70 gene and cytokines genes, including TNF- α, IL-1 β, IL-12, and IL-8 in the gills and liver. In both organs, SOD gene responding to elevated ROS levels was found to be up-regulated, as well as CASP3 (caspase), which plays a role in apoptosis [SiNPs were also tested for changes in gene expression on poptosis .3ONPs, magnetite) were found to change the expression of 5571 genes, which included 59% down-regulated genes and 41% up-regulated genes in the gills of adult zebrafish. More differently expressed genes—5571—were observed in the liver. In both liver and gills, transcript levels of genes involved in iron metabolism were elevated, including such proteins as ferroportin-1, alas2, TfR1b, and TfR2. The liver was also characterized by elevated levels of pro-apoptotic genes transcripts  and up-regulation of tsc22d3 (leucine zipper transcription factor), a marker of inflammation. The expression of genes related to DNA repair was suppressed in the liver [FeNPs nanoparticles (Fehe liver .abcb4 (stress response), sgk1 (protein kinase), and tsc22d3 (anti-inflammatory response), whereas fen1 (DNA repair) and sirt7 (epigenetic regulation) were down-regulated [In the gills, magnetite nanoparticles induced up-regulation of such genes as egulated .2O3 (maghemite), administered to adult zebrafish, caused 953 genes in the liver to be expressed differently. Maghemite elevated the transcripts of antioxidant genes  in the liver. Genes related to ion binding, translation, and ribosome biogenesis were up-regulated as well [Another form of iron nanoparticles, γ-Fe as well .D. rerio cells subjected to a treatment with carboxymethyl cellulose stabilized iron sulfide nanoparticles revealed 3200 down-regulated genes , including cmc4  and pimr214 (Ser/Thr kinase activity), and 2593 up-regulated genes , including such genes as hist1h4l (histone H4 gene), hist1h4a (histone H4 gene), ighv-5 (V region of the variable domain of immunoglobulin heavy chains), and dut (deoxyuridine triphosphatase). Differentially up-regulated KEGG pathways included complement and coagulation cascades, serine, and threonine metabolism; PPAR signaling pathway; tryptophan metabolism; and fat digestion and absorption. Down-regulated KEGG pathways included cell cycle, DNA replication, pyrimidine metabolism, RNA transport and degradation, purine metabolism, and ubiquitin-mediated proteolysis [RNA-seq of teolysis . C. reinhartdii, were exposed to uncoated and coated either with polyacrylic acid (PAA) or citrate (Ci) CeNPs. The uncoated CeNPs group produced 688 DEGs, PAA-coated 315, and Ci-coated only 23. All types of CeNPs induced up-regulation of the genes involved in the xenobiotic resistance system. Uncoated and PAA-coated CeNPs caused over-expression of FAP16 gene, whose protein product induces detachment of the flagella and at the same time cause under-expression of the POC7 gene, which is related to flagella assembly. In the study performed by Morel et al. , green aThe growing interest in nanoparticulate metals in general industry inevitably leads to increasing environmental pollution by nanoparticles and ions released by them, which includes water pollution. This has led to a need to investigate the toxic effects of metal nanoparticles on aquatic organisms, and it was established that they have a gene-expression-modulatory effect.AgNPs were discovered to trigger overexpression of genes involved in inflammation and apoptosis and under-expression of genes whose protein products are involved in body development. Genes involved in calcium-binding might show overexpression, whereas those that participate in DNA-dependent transcriptional regulation, cell proliferation, and differentiation might be expressed, which might lead to delayed development, and genes participating in oocyte differentiation might also be repressed. Expression of genes for oxyradical scavengers and enzymes with antioxidative properties in organisms subjected to treatment with AgNPs is not uniform; some organisms present over-expression of those genes, and some show repressed expression. mRNA levels of genes involved in protein catabolism, those encoding transcriptional factors and involved in ribosomal assembly might be under-expressed which might lead to unbalanced levels of different proteins .CuNPs were also discovered to have a modulatory effect on gene expression of aquatic organisms. As with AgNPs, anti-oxidative related genes and genes involved in general stress response might be either over- or under-expressed, and metallothionein mRNA levels are found to be elevated. CuNPs were also involved in elevating the mRNA levels of genes that participate in lipid transport and metabolism, as well as cell cycle regulation and induction of apoptosis . Other nanoparticulate metals that were proven to have a modulatory effect on gene expression were ZnNPs, SiNPs, AuNPs, TiNPs, SeNPs, and CeNPs .ZnNPs had an up-regulating effect on genes involved in protease activity inhibition and response to oxidative stress. They also may elevate the transcripts levels of genes involved in binding of heavy metals and those that have pro-apoptotic activity. On the other hand, ZnNPs may suppress transcription of genes that play a significant role in immune responses.SiNPs may induce expression of immune-response genes as well as general-stress-response genes and oxidative-stress-response genes. Transcripts of genes with pro-apoptotic activity may also be elevated. FeNPs trigger up-regulation of such groups of genes as genes involved in iron metabolism, stress response genes, anti-inflammatory genes, pro-apoptotic genes, and antioxidants and ROS scavengers. DNA repair genes may, however, be under-expressed. AuNPs may induce over-expression of genes involved in oxidoreductive activity, immune response, apoptosis regulation, lipid metabolism, cell adhesion, cytoskeleton organization, response to xenobiotics, and electron transport chain, whereas cell cycle regulation genes and transcription factor may be under-expressed. There is also inconsistency in some cases in changes of expression of some genes induced by AuNPs in different organs. In the brain, genes involved in response to ROS and DNA repair may be up-regulated, but in the gills, they might be down-regulated.TiNPs disrupt the expression of genes that are involved in the regulation of kinase activity, cholesterol synthesis, and zona pellucida formation.SeNPs may protect cells from the toxic effects of other metals but can also induce cytokines’ gene expression, as well as expression of various genes involved in general stress response.CeNPs may induce detachment of flagella and inhibit flagellation, which is a marker of stress in flagellated organisms. Understanding the impact of nanoparticles on aquatic organisms and gene expression will help to assess the risk following their release into the water and could help minimize it. It is important to know exactly how extensive the toxic effects of metal nanoparticles on aquatic organisms are, so placing importance on the prevention of water pollution is justified. Knowing which genes specifically can be targeted by nanoparticles might be important as biomarkers in the evaluation of the pollution levels by molecular studies. It is limited, however, just to assess the changes in the expression of the genes and not of the synthesis of the proteins, which may be controlled on translational levels.