Lung cancer remains the leading cause of cancer-related death and is associated with a poor prognosis. Lung cancer is divided into 2 main types: the major in incidence is non-small cell lung cancer (NSCLC) and the minor is small cell lung cancer (SCLC). Although NSCLC progression depends on driver mutations, it is also affected by the extracellular matrix (ECM) interactions that activate their corresponding signaling molecules in concert with integrins and matrix metalloproteinases (MMPs). These signaling molecules include cytoplasmic kinases, small GTPases, adapter proteins, and receptor tyrosine kinases (RTKs), particularly the epidermal growth factor receptor (EGFR). In NSCLC, the interplay between ECM and EGFR regulates ECM stiffness, angiogenesis, survival, adhesion, migration, and metastasis. Furthermore, some tumor-promoting ECM components  enhance activation of EGFR and loss of PTEN. On the other hand, other tumor-suppressing glycoproteins and -proteoglycans can inhibit EGFR activation, suppressing cell invasion and migration. Therefore, deciphering the molecular mechanisms underlying EGFR and ECM interactions might provide a better understanding of disease pathobiology and aid in developing therapeutic strategies. This review critically discusses the crosstalk between EGFR and ECM affecting cell behavior of NSCLC, as well as the involvement of ECM components in developing resistance to EGFR inhibition. Lung caErbB1 gene mutations (The EGFR is a transmembrane glycoprotein receptor that belongs to the ErbB family of receptor tyrosine kinases (RTKs). There are four types of EGF receptors  that comprise a cysteine-rich extracellular ligand-binding domain (LBD), an α-helix transmembrane domain (single-pass), a C-terminal domain, and except HER3, a cytoplasmic tyrosine kinase (TK) domain . The EGFutations  that areutations . Tumor eIn the following sections, we highlight the different types of ECM proteins and their roles in mediating EGFR signaling to pinpoint their significance in NSCLC as biomarkers for diagnosis and prognosis and their potential as druggable targets.ECM is a significant part of all tissues’ microenvironment. It offers physical support for the neighboring cells, binds growth factors, and controls cell behavior under physiological and pathological conditions . ECM is 22.12.1.1Emerging data have indicated that the fibulin (FBLN) family comprising seven members (fibulin-1–7) of widely expressed ECM proteins is associated with lung cancer invasion and metastasis. FBLNs are ECM glycoproteins consisting of EGF-like domain repeats crucial for normal organogenesis and embryonic development . They aret al. used siRNA to knock down FBLN1C and FBLN1D expression in NSCLC Calu-1 cells to examine if FBLN1 isoforms could play a role in controlling EGFR signaling and function  and EGF-mediated EGFR activation. Conversely, overexpression of FBLN1D and FBLN1C inhibits EGFR activation, indicating a regulatory crosstalk between the two proteins.Fibulin-1 (FBLN1) expression levels are substantially downregulated in NSCLC . The rolfunction . WithoutIGF1R was discovered to be a direct target of FBLN3, which inhibits the action of IGF. Further, FBLN3 inhibits lung CSC and EMT by modulating the IGF1R/PI3K/AKT/GSK3 pathway, and that FBLN3 may be used as a CSC-centered therapeutic alternative ((MMP)-7 and MMP-2  were investigated . Moreoveernative . FBLN3 cnd MMP-2 . Again, nd MMP-2  that, innd MMP-2  and inhind MMP-2 , 35. Mornd MMP-2 .in vitro, and its amino-terminal domain serves as a ligand for cell surface integrins αvβ3, α9β1, and αvβ5 , a vascular integrin receptor ligand, is a distinct member of fibulins harboring the RGD (Arg-Gly-Asp) motif associated with endothelial cell adhesion . FBLN5 cand αvβ5 –41. FBLNand αvβ5 , and is and αvβ5 . FBLN5 w pathway .2.1.2Mucins (MUCs) are high M.wt glycoproteins synthesized by many epithelial tissues . They arCCNB1), cyclin-dependent kinase inhibitor 3 (CDKN3), cell division cycle protein , mitotic arrest deficient 2-like protein 1 (MAD2L1), protein regulator of cytokinesis 1 (PRC1), and ribonucleoside-diphosphate reductase subunit M2 (RRM2), which are involved in cell cycle and proliferation regulation and have been linked to poor outcomes in patients with lung adenocarcinoma , and necrosis induction via a ROS-dependent mechanism. Furthermore, in H1975 (EGFR L858R/T790M) mutant cells and A549 (K-Ras G12S) xenografts developed in nude mice after treatment with GO-203, tumor regressions were observed. These data suggest that MUC1-C is needed for PI3K-Akt pathway activation and survival in NSCLC cells  mutations and Hnf4α expression may participate in mucin expression profiles in these lung ADC subtypes  is present in multiple isoforms through alternative splicing, where 20 isoforms in humans have been discovered  expression than ADC, and these data were verified in external gene expression data sets. Overexpression of ITGB4 is also linked to venous invasion and a lower overall patient survival rate. The most highly 50 altered genes related to ITGB4 identified in SqCC were Lns, CD151, collagens, PI3K, and EGFR-associated pathway genes, other recognized signaling partners using cBioPortal. Finally, they show that ITGB4 is overexpressed in NSCLC and is an unfavorable prognostic factor (Laminins (Lns) are heterotrimeric extracellular glycoproteins found in all BMs. So far, more than 17 Ln isoforms have been identified with a cross-shaped and specific arrangement of α, β, and γ subunits . Ln-332 c factor . Overall2.1.5via activated thrombin considerably affects blood clotting, inflammatory response, wound healing, fibrinolysis and neoplasia. Increased fibrinogen activity considerably affects cancer cell growth, progression, and metastasis  Polymerase 1 (PARP1) and caspase 3 pathways. By regulating the PARP1/caspase 3 pathway, FGL1 promotes acquired resistance to gefitinib in the PC9/GR NSCLC cell line. As a result, FGL1 may be a possible therapeutic option for NSCLC patients who have developed resistance to gefitinib (Accumulating evidence indicates a correlation between fibrinogen and EGFR in lung cancer –81. A stefitinib .2.1.6Tenascin-C (TN-C) is a glycoprotein composed of 4 distinct domains interacting with matrix constituents, cell surface proteins, soluble factors, and pathogenic components. TN-C affects pulmonic blood vessel invasions by decreasing apoptosis and promoting cancer cell plasticity, thus, increasing lung metastasis  is a vital ECM protein known for its complex role in tumorigenesis  is a multifunctional glycoprotein found in blood and ECM. It binds collagen, glycosaminoglycans, the urokinase-receptor, and plasminogen and stabilizes plasminogen activation inhibitor-1 (PAI-1)’s inhibitory conformation. VTN can potentially control the ECM proteolytic degradation through its localization in the ECM and binding to PAI-1. VTN also binds to complement, heparin, and thrombin-antithrombin III complexes, suggesting an immune response role and clot formation control ( control . VTN is  control . EGF pro control . EGFR ac control .Nidogen (NID1 and NID2) are present in the BM and help maintain its stability by connecting COLIV and Ln networks in the ECM , a heparan sulfate proteoglycan (HSPG) family, consist of core proteins (60- to 70-kDa), heparan sulfate (HS) chains, and a glycosylphosphatidylinositol linkage . There a-smokers . GPC5 mRpression . Conflicr action . Therefor action . Althougr action , colon  family is a transmembrane protein that possesses HS chains on their extracellular domains  and consrecorded , 132.2.32.3.1HAS2 and HAS3 or CD44 suppresses the EGFR/AKT/ERK signaling pathway, cell proliferation, and survival  are the major ECM proteins  in the human body. They are arranged in a relaxed meshwork and possess elasticity to extreme tensile strength owing to their surrounding proteins like elastin and glycoproteins , which is mechanistically attributed to the PI3K pathway  fibrproteins –146. COL pathway .Notably, many studies addressed the significant effect of CAFs in tumorigenesis , 149. CAIn healthy tissues, the biosynthesis of COLs is highly controlled by a great counterbalance of many enzymes, including MMPs and their inhibitors and lysyl oxidases (LOX) , 158. In3Cas (Crk-Associated Substrate). To some extent, FAK activation results in the recruitment of other SH2-containing proteins, including PLC-γ, PI3K, and the adapter proteins Grb2 and Grb7, mediate ERK activation  expression, suggesting that β1 integrin maintains the endocytic machinery required for EGFR signaling  mice (NOG) mice  mice . Overall4Matrix degradation is a finely regulated process that occurs simultaneously with the formation of new ECM molecules. Tissue integrity is achieved through the actions of matrix-degrading enzymes such as matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), adamalysin group (ADAMs and ADAMTS), cathepsins, plasminogen activation system components, and glycolytic enzymes such as heparanase (HPSE) and hyaluronidases (HYALs) that cleave heparan sulfate (HS)/heparin chains on hyaluronan (HA) and proteoglycans (PGs) , 181. El4.1MMPs are a group of 24 proteinases, also known as matrix MMPs, matrixins, and zinc-dependent endopeptidases . TranscrG12D/+p53-/-) mouse model of lung cancer, overexpression of a soluble dominant-negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocks the invasion of lung cancer cells in collagen I matrix in vitro and reduces tumor incidence. MMP14 activity also triggers the proteolytic processing and activation of Heparin-Binding EGF-like growth factor (HB-EGF), which stimulates the EGFR signaling pathway and increases tumor proliferation and growth. These data pinpoint the potential for developing therapeutic strategies that target MMP14 in NSCLC, specifically targeting the MMP14-HB-EGF axis (in vitro and in vivo has been linked to tumor progression. Cox et al. linked the EGFR expression with MMP-9 upregulation in tumor cells in vitro in NSCLC patients. MMP-9 expression strongly correlated with EGFR expression and EGFR membranous expression, but not with cytoplasmic EGFR expression. MMP-9 and EGFR co-expression is associated with a poorer prognosis in NSCLC patients. Also, MMP-9 and EGFR are expressed in a large proportion of NSCLC tumors. The presence of these markers together indicates a poor prognosis. These findings suggest that the EGFR signaling pathway, via specific up-regulation of MMP-9, can play a key role in NSCLC invasion (When the ECM collagen becomes abundant, large amounts of MMPs are secreted in tumor tissues, and BM remodeling occurs , 194, leEGF axis . Increasinvasion .5et al. reported that COLI drives EGFR-TKI resistance through integrin-β (via increment of IL-6 mRNA and protein expression. IL-6 treatment increases α-actin and collagen expression, fibrosis markers, in lung fibroblast cells using a co-culture model. These findings indicate that IL-6 plays a role in EGFR-TKI-induced interstitial fibroblastic proliferation. Therefore, inhibiting IL-6 could be helpful to cancer patients receiving EGFR-TKI treatment to reduce the risk of side effects (via Akt and ERK1/2-independent pathway in NSCLC, leading to EGFR-TKI resistance. Combining EGFR-TKI and mTOR inhibitors may be a viable option for combating such resistance (via COLI uptake mediated by micropinocytosis (The biological features of the tumor microenvironment are affected by cancer cells, non-cancerous cells, and ECM . The inttegrin-β . Knockdotegrin-β . Interle effects . Further effects . Further effects . COLI is effects  and brea effects . The sensistance . ECM comsistance , 208. Raocytosis .in vitro and xenografted models, whereas NSCLC lines expressing vimentin and/or fibronectin showed resistance to the growth inhibitory effects of EGFR kinase inhibition (EMT is characterized by the downregulation of epithelial markers, especially E-cadherin, and upregulation of mesenchymal markers such as vimentin, N-cadherin, and fibronectin . EMT is hibition .FBLN1 isoforms regulate EGFR signaling and function in NSCLC. FBLN1 loss, using siRNA mediated knockdown of FBLN1C and FBLN1D, in NSCLC Calu-1 cells significantly increased EGF mediated EGFR activation, inhibited EGFR activation, promoted EGFR-dependent cell migration that inhibited upon Erlotinib treatment. Notably, FBLN1C and FBLN1D knockdown cells show a substantial increase in EGF-mediated EGFR activation, which promotes cell adhesion reduced by Erlotinib treatment. These data point out that FBLN1C/1D, as an ECM protein, can bind and regulate EGFR function and activation in NSCLC Calu-1 cells, highlighting tumor ECM role in affecting EGFR dependent lung cancers . In H197in vitro and in vivo and involved in the progression of lung ADC. Antagonizing integrin β3 improved the TKI sensitivity in vitro and in vivo, inhibiting anoikis resistance, proliferation, and EMT phenotype in lung cancer cells. Integrin β3 overexpression was also linked to the enhanced cancer stemness implicated in resistance development. Mechanistically, integrin β3 is induced by increased levels of TGFβ1 in acquired TKI-resistant lung cancer, which indicates the TGFβ1/integrin β3 axis as a potential target for combination therapy in EGFR-mutant lung cancer to overcome acquired resistance to EGFR TKIs (The activation of many signaling pathways imperils the clinical efficacy of EGFR-TKIs in EGFR-mutated NSCLC –218. TheGFR TKIs . FurtherGFR TKIs . The expGFR TKIs . Recentl6via crosstalk with EGFR, including COLs, MMP-9, MUC1, MUC5AC, Ln 5, and GPC5. Many ECM proteins can be used as therapeutic targets, such as COLs, PLOD2, FBLN3, MUC5AC, FN, FAG2, FG, GPC3, and HA by modulating their interaction with EGFR. ECM proteins can be tumor-suppressing or -promoting depending on their signaling context with EGFR and many signaling molecules. Despite the emerging data revealing the role of ECM components or/and EGFR in NSCLC, many gaps still exist in EGFR-ECM interactions. The correlation between EGFR and many ECM proteins, including COLI, COLIV, FBLN1, FBLN3, FBLN5 MUC1, MUC5, MUC6, and Ln5 was revealed, yet EGFR interactions with other types of COLs, MUC and Ln, FG, TN, Postn, VTN, NID, TSP, and versican in NSCLC still need further investigations. A future better understanding of the interactions between ECM components and EGFR-TKI might provide new insights for developing new therapeutic strategies for NSCLC patients.ECM components, along with integrin and MMPs, regulate many cellular processes relevant to lung cancer progression, including cell proliferation, adhesion, and migration through their direct or indirect interactions with EGFR. ECM proteins associated with poor NSCLC prognosis Conceptualization, SH and SI. Literature searches, SH and SI. Data curation, SI, SH, and AA. Investigation, SH, SI, and AA. Supervision, AA and SI. Figures design, SH. Writing—original draft, SH and SI. Writing—review and editing, SH, SI, and AA. This article is based primarily on the student MSc. thesis, SH. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


CYP27B1, CYP24A1, GC, and VDR genes were analyzed through real-time PCR. A total of 63 patients were enrolled. Calprotectin, hemoglobin, and C-reactive protein levels were influenced by SNPs in VDR, CYP27B1, and GC genes. After 3 months of therapy, clinical remission was predicted by smoke, systemic steroids, and VDR BsmI, whereas at 12 months by GC 1296AA/AC and VD supplementation. This study reports the association between VD pathway-related genetics and ADA treatment. Further studies are needed to confirm these promising data.Adalimumab (ADA) is a human anti-tumor necrosis factor (TNF-α) monoclonal antibody used in inflammatory bowel diseases, such as Crohn’s disease (CD). Vitamin-D (VD) is important for biological functions, such as the modulation of expression of genes encoding enzymes and transporters involved in drug metabolism and transport. ADA trough levels were associated with VD concentrations in patients with IBD, but no data are present in the literature concerning VD pathway-related gene single-nucleotide polymorphisms (SNPs) in affecting clinical outcomes. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission at 3 and 12 months in patients affected by CD treated with ADA. Patients affected by CD were included in this study. SNPs in  Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are multifactorial, lifetime, inflammatory diseases of the gastrointestinal tract, with an increasing prevalence worldwide . There iWorldwide, vitamin D (VD) deficiency represents a public health problem in all age groups, although studies are still lacking in most countries, particularly at -risk groups. The National Health and Nutrition Examination Survey (NHANES) shows that 79% of the elderly adult population have VD deficiency or insufficiency ,13. ThisMany studies displayed a correlation between VD reduced plasma levels and the onset of bone, neurological, cardiovascular diseases, diabetes, metabolic syndrome, infections, cognitive decline, and neurodegenerative diseases and neoplasms and autoimmune disorders ,18,19,20Ultraviolet light is responsible for VD synthesis in the skin; cholecalciferol is hydroxylated to calcifediol  in the liver through cytochrome P-450 (CYP) 27A1 and CYP2R1 and, in the kidney, calcitriol  is synthesized through CYP27B1. 1,25-VD is then transported in the bloodstream through the vitamin D binding protein (VDBP). Cholecalciferol is inactivated to calcitroic acid by the CYP24A1 enzyme, whose expression is partially affected by calcium and parathyroid blood concentrations and partially induced by 1,25-VD, which can also directly down-regulate the CYP27B1 gene. CYP3A4 and, to a lesser extent, CYP2B6 and CYP2C9 genes expression in human hepatocytes. In fact, they revealed that VDR recognizes and transactivates CYP3A4, CYP2B6, and CYP2C9 promoters binding to xenobiotic-responsive elements; in addition, it is targeted by the constitutive androstane receptor (CAR) and/or the pregnane X receptor (PXR). Moreover, the authors suggested that VDR, PXR, and CAR modulate several CYP genes’ expression by competitively interacting with the same battery of responsive elements [Through its receptor VDR, VD modulates the expression of genes encoding transporters and enzymes responsible for drug transport and metabolism, such as cytochromes (CYPs) . Drocourelements .Several drugs are metabolized by CYP3A4; this gene shows VD responsive elements (VDRE) and its expression is upregulated in presence of 1,25-VD. Lindh et al. suggested tacrolimus and sirolimus seasonal variability according to changes in VD levels, with lower drug concentrations in July to September than in January to March. Furthermore, VD influence on CYP3A4 expression results in altered metabolized drugs concentration as shown for immunosuppressants ,25,26.VD may interact with several drugs, potentially altering drug toxicity or efficacy, but drugs may also affect VD metabolism and status : The 25-VD plays an important role in IBD during anti-tumor necrosis factor (TNF) treatment: Higher rates of clinical response and clinical remission correlate with increased VD levels . Santos-An association between vitamin D pathway-related gene single-nucleotide polymorphisms (SNPs) and ADA clinical outcome, from the perspective of a putative use as a biomarker of the therapeutic response, has never been assessed. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission in patients affected by CD treated with ADA, for the first time in the literature.A total of 63 patients were enrolled in Turin and Pisa; their characteristics are reported in Subsequently, patients’ clinical characteristics were analyzed according to SNPs of genes related to VD metabolism, transport, and activity.CYP27B1-1260 TT was able to affect all three time points (CYP27B1 + 2838 CT/TT (p = 0.010), VDR ApaI CA/AA (p = 0.021), VDR Cdx2 AG/GG (p = 0.036), and GC 1296 CC (p = 0.034) had an influence; finally, a role was suggested for CYP27B1 + 2838 CT/TT (p = 0.023), VDR ApaI CA/AA (p = 0.021), and VDR TaqI TC/CC (p = 0.046) at 3 months of therapy. The levels of calprotectin were different according to the genetic variants ; particue points . At BL, CYP27B1-1260 TT was able to affect the three analyzed time points . Hemoglobin (Hb) levels were influenced by genetic polymorphisms ; also, ie points . CYP24A1VDR TaqI CC (p = 0.036) at BL, CYP27B1-1260 TT (p = 0.017) at 3 months, and, finally, VDR ApaI AA (p = 0.037) at 12 months.The C-reactive protein (CRP) was affected by VDR BsmI AA remained in the multivariate regression, whereas GC 1296 AA/AC and VD supplementation were retained in the final regression model at 12 months of therapy 27A1 and CYP2R1 hydroxylate cholecalciferol to calcifediol ; and in the kidney, CYP27B1 synthesizes calcitriol . 1,25-VD is then transported in the bloodstream through the vitamin D binding protein (VDBP) encoded by the  1,25-VD , which c7B1 gene ,35. VD l7B1 gene ,37.VD deficiency is highly prevalent in IBD, involving up to 80% of apparently well-nourished patients . HoweverIn this study, 63 patients affected by CD were enrolled and evaluated; particularly, predictive factors of biochemical biomarkers, such as calprotectin, Hb, and CRP, and clinical remission at three and twelve months of therapy were investigated.We focused on VD-related genetics, since no data are available in literature in this context. Polymorphisms in genes encoding its activation enzyme (CYP27B1), its inactivation enzyme (CYP24A1), its receptor (VDR), and its transporter (VDBP) were analyzed and related to clinical features.CYP27B1 -1260 TT was found to be associated with calprotectin and Hb at BL, three, and twelve months of therapy. In particular, this genotype patients showed higher calprotectin levels and lower Hb concentrations. These two findings could be related, since increased calprotectin exposure could be associated with a high grade of inflammation and blood loss, with consequently reduced Hb levels [CYP27B1 -1260 TT was associated with increased colorectal cancer risk; the CYP27B1 gene encodes a member of the CYP monooxygenases family (the 1α-hydroxylase enzyme), localized to the inner mitochondrial membrane of renal cells [CYP27B1 -1260 rs10877012 promoter polymorphism are inconsistent: Lange and colleagues suggested its influence on 1,25-VD serum concentrations, in contrast of what was observed by Kitanaka et al. [Interestingly, b levels ,40. Furtal cells . Availaba et al. ,43,44,45VDR BsmI AA genotype, in addition to smoke and use of systemic steroids, was a predictive factor at three months of therapy, whereas GC 1296 AA/AC was at twelve months. In particular, all the factors, with the exception of GC 1296 AC/CC, are negative predictors.For the first time, clinical remissions at three and twelve months after starting therapy were analyzed according to VD-related genetic variants. We suggested that the GC 1296 is unknown: It encodes the VDBP, which is a multifunctional glycoprotein produced by hepatic parenchymal cells and secreted into the circulation [The role of culation . Its funculation . Our recculation .49. It was supposed that VDR mRNA expression and protein levels could be lower in IBD affected subjects [VDR-mRNA expression, affecting its stability, confirming previous studies [VDR is the VD receptor that regulates the transcription of several genes; furthermore, it modulates several physiological systems, such as immune, calcium, and phosphorous homeostasis or apoptosis. Several VDR gene SNPs were able to affect certain features in different diseases, for example non-small-cell lung cancer or autoimmune hepatitissubjects . HoweverConcerning clinical remission at 12 months of therapy, VD supplementation remained in the final model as a negative predictive factor: It could be possible that these patients have lower VD levels, which could not be normalized, despite the supplementation. This finding is in line with the current literature, which displayed how the vast majority of patients with IBD have low levels of VD, regardless of their clinical or nutrition conditions ,50.The present study has several important limitations. Firstly, the number of patients was small; however, it is worth noting that this factor did not affect the significance of the analyses, thus suggesting that the putative associations of VD genetics and clinical outcome of ADA treatment in CD are conceivable. Secondly, data concerning VD exposures would have significantly improved our results, but unfortunately, these data were unknown. Finally, an endoscopic assessment at twelve months would improve the significance of the results, although fecal calprotectin is currently well recognized as a marker of endoscopic remission ,51.We performed a prospective, multicenter study at the Gastroenterology Unit of “Città della Salute e della Scienza di Torino”, Italy and the IBD Unit, Pisa University Hospital, Pisa, Italy. From January 2018 to January 2020, we recruited consecutive patients: (1) Affected by CD with indications to treatment with ADA; (2) naive to anti-TNF drugs or other biological drugs; (3) older than or equal to 18 years; (4) who agreed to sign the informed consent to participate in the study. We treated patients with moderate-to-severe disease activity or steroid-dependent disease with previous failure or intolerance to thiopurines with ADA. Exclusion criteria were: (1) Refusal to participate to the study; 2) pregnancy; 3) other concomitant auto-immune diseases . Clinical history, data on physical examination, recent biochemical examinations, and signed informed consent for the purpose of enrolment in the study were collected. All patients were treated with a total dose of 160 mg at day 1, 80 mg at day 15, and 40 mg of ADA by subcutaneous administration every two weeks. Before starting ADA therapy, 9 mL of venous blood was collected. The blood samples were associated with an anonymous numerical identification code and stored frozen at −80 °C. In a subgroup of patients, randomly identified, a second blood sample was collected after 3 months of treatment (T1).®, Trieste, Italy).Clinical characteristics were evaluated at BL , 3, and 12 months of therapy. At the same time-points, fecal calprotectin was assessed by ELISA . Clinical response to ADA therapy was defined as a decrease in the Harvey–Bradshaw index (HBI) score used to foster a systematic collection of clinical data related to Crohn’s disease: It considers five parameters, exclusively clinical . For each parameter, a specific score is assigned: Greater than or equal to 3 in the absence of corticosteroid therapy and with ongoing ADA therapy, clinical remission, and HBI <= 4, in agreement with the literature . The stuWhole blood was collected in EDTA tubes and genomic DNA was obtained from blood samples ; allelic discrimination was assessed through the real-time polymerase chain reaction system . The investigated gene SNPs were VDR (encoding VD receptor) rs7975232 (ApaI) C>A, rs731236 (TaqI) T>C, rs10735810 (FokI) T>C, rs11568820 (Cdx2) A>G, and rs1544410 (BsmI) G>A, CYP27B1  rs4646536 (+2838) C>T and rs10877012 (-1260) G>T, CYP24A1  production) rs2248359 (3999) T>C, rs927650 (22776) C>T, and rs2585428 (8620) A>G, and finally GC  rs7041 (1296) A>C.p-value < 0.05. Stepwise multivariate logistic regression analysis was then performed including variables with a p-value below 0.05 in univariate analysis to evaluate factors able to predict clinical remission at three and twelve months of therapy .All variables were tested for normality through the Shapiro–Wilk test. Normal variables were presented as the average and standard deviation, whereas non-normal variables were median values and the interquartile range (IQR) and categorical variables were numbers and percentages. Allele frequencies were analyzed for Hardy–Weinberg equilibrium. Kruskal–Wallis and Mann–Whitney tests were adopted for differences in continuous variables between genetic groups, considering a statistical significance with a two-sided Since no previous studies have analyzed the ability of VD genetics to predict clinical response to ADA in patients with CD, a prior calculation of the power of the study was not possible.In conclusion, this is the first study investigating the role of VD-associated genes in evaluating the clinical remission at three and twelve months of therapy with ADA in patients with CD. The novelty of our results is the most important strength of the present study. However, further studies are required in order to confirm these preliminary data and to evaluate their potential role in clinical management.


Autophagy is a widely conserved process in eukaryotes that is involved in a series of physiological and pathological events, including development, immunity, neurodegenerative disease, and tumorigenesis. It is regulated by nutrient deprivation, energy stress, and other unfavorable conditions through multiple pathways. In general, autophagy is synergistically governed at the RNA and protein levels. The upstream transcription factors trigger or inhibit the expression of autophagy- or lysosome-related genes to facilitate or reduce autophagy. Moreover, a significant number of non-coding RNAs  are reported to participate in autophagy regulation. Finally, post-transcriptional modifications, such as RNA methylation, play a key role in controlling autophagy occurrence. In this review, we summarize the progress on autophagy research regarding transcriptional regulation, which will provide the foundations and directions for future studies on this self-eating process. Macroautophagy, hereafter referred to as autophagy, is the main type of autophagy, which is characterized by the formation of autophagosomes. Autophagosome biogenesis involves a series of autophagy-related (Atg) proteins that accompany the different steps of the autophagic process. Autophagosome initiation is mediated by the activity of the ULK1/Atg1-ATG13/Atg13 protein kinase complex. Nucleation of the autophagosome requires the BECN1/Beclin 1/ATG6-PIK3C3/Vps34 ) complex. Elongation and maturation of the autophagosome involves two ubiquitin-like systems, i.e., Atg5–Atg12-Atg16 and LC3/Atg8–phosphatidylethanolamine (PE). Finally, the mature autophagosome fuses with the lysosome, leading to cargo degradation and completing the autophagic flux.Atg genes, WIPI1 (WD repeat protein interacting with phosphoinositides), p62/SQSTM1, and vacuolar (H+)-adenosine triphosphatases (V-ATPases), or by a variation of post-translational modifications of autophagy-associated proteins ..6A) modiATG5, ATG7, and LC3B through m6A modification of their mRNA, whereas β-elemene reverses gefitinib resistance in gefitinib-resistant PC9GR and HCC827GR derived from NSCLC cells by inhibiting METTL3-mediated autophagy [FOXO3 (in an 800 bp region of FOXO3 3′UTR containing the m6A modification site), which subsequently downregulates the expression of ULK1, ATG5, ATG7, ATG12, ATG16L1, and MAP1LC3B in human sorafenib-resistant hepatocellular carcinoma [TFEB mRNA and thus inhibits autophagy [METTL3 positively regulates autophagy by increasing the expression of utophagy . Moreoveutophagy .ULK1 expression by demethylating the adenine residues 3335, 3397, and 3784 at 3’ UTR of ULK1, thus promoting autophagy in Hela cells. In a mouse preadipose cell line, mRNAs of Atg5 and Atg7 are the targets of m6A reader protein YTHDF2, and FTO upregulates Atg5 and Atg7 expression in a YTHDF2-mediated manner to promote autophagy [ATG5 or ATG7 in turn attenuates the expression of FTO [ALKBH5, inducing its expression, whereas HsCG decreases METTL14 expression, leading to the activation of AMPK-ULK1 axis and autophagy occurrence [Demethylase FTO increases the autophagic flux in patients with chronic kidney disease ,85. In putophagy . There in of FTO . In Leydcurrence ,88 (FiguMiT/TFE factors from inhibition by mTORC1 promotes autophagy and lysosomal catabolism to maintain intracellular amino acids, playing the pro-survival role of cells in pancreatic ductal adenocarcinoma [Autophagy protects organisms against various pathologies, including pathogen infections, cancer, neurodegeneration, aging, and heart disease . Many starcinoma . Howeverarcinoma .Atg5 and Atg7, and thus triggers autophagy activation against infection with Zika virus [NF-κB is an important regulator of cellular immunity that is involved in the control of autophagy. NF-κB serves as a transcription factor with a dual effect on autophagy in different species. In the fly brain, NF-κB induces the expression of ka virus . Howeverka virus . FOXO3 cka virus . In summAtg genes [Atg gene expression to affect autophagy, whereas in a series of tumors circRNAs serve as sponges of autophagy-related miRNAs to regulate autophagy [Atg genes through competitive binding to microRNAs [An increasing number of non-coding RNAs are reported to regulate tg genes . The exptg genes . All micutophagy . LncRNAsutophagy . In addiicroRNAs . InformaicroRNAs . RecentlicroRNAs . The nov6A methylation modification plays a critical role in autophagy and drug resistance of tumors [6A modification is a novel process able to regulate autophagy. Whether other RNA modifications are involved in this scenario is still unknown and deserves further research.Modifications of histone proteins and DNA methylation are two common epigenetic regulatory mechanisms of gene expression . Recentlf tumors . Howeverf tumors . m6A mod


SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of  SDR superfamily has grown considerably since the initial family members were characterized in the late 1970′s [SDRs was performed in cyanobacteria [SDRs are divided into seven different categories termed “classical”, “extended”, “intermediate”, “divergent”, “complex”, “atypical”, and “undefined” according to their different cofactor binding sites, substrate binding sites, functions, and sequence structures [SDR superfamily genes is difficult because most family members share only 20–30% amino acid sequence similarity.Short-chain dehydrogenases/reductases (SDR) superfamily comprises NAD(P)(H)-dependent oxidoreductases that possess the conserved Rossmann-fold motif. They are a large and ancient gene family found in organisms as diverse as viruses and vertebrates .With thee 1970′s ,3,4,5. Gbacteria , nematodbacteria ,9, and pbacteria . GeneralOncorhynchus mykiss) and Atlantic salmon  underwent an additional round of genome duplication (4R) [SDR superfamily are still lacking. The available genome sequences of more and more species provided new resources to understand the evolution of SDR superfamily. Recently, genome sequences were released for elephant shark  [Latimeria menadoensis, an early sarcopterygian) [Lepisosteus productus, a non-teleost actinopterygian) [SDR superfamily in vertebrates provide insights into the evolution of this gene family after each round of WGDs.The diversification and the morphological innovations of vertebrates are attributed to large-scale gene or genome duplications at the origin of the group. These duplications are predicted to have occurred in two rounds of genome duplication, the “2R” hypothesis, or in one genome duplication plus many segmental duplications, although these hypotheses are still somewhat controversial . In addiion (4R) ,14. Therchthyan) , coelacaerygian) , and spoerygian) , which rEpinephelus coioides) [Paralichthys olivaceus), eight hsd17b family genes, including hsd17b3, -4, -7, -8, -10, -12a, -12b, and -14, were identified. Flounder hsd17b10, -12a, and -12b are highly expressed in the ovary, while hsd17b3 is dominantly expressed in the testis. In mammalian species, 11β-hydroxysteroid dehydrogenases (HSD11b1 and HSD11b2) catalyze the interconversion between active and inactive glucocorticoid [hsd11b1 proved to be a potent enzyme for the biosynthesis of 11-ketotestosterone (11-KT) from testosterone in testis [dhrs11 can act as an NADPH-dependent hsd17b and play an important role in the metabolism of 11-oxygenated-C19-steroids as well as estrogens, androgens, and androgen precursors [Rdh is another well-defined large subfamily of SDR which is involved in the first step of retinoic acid (RA) synthesis from vitamin A by catalyzing the oxidation of retinol to retinaldehyde [SDRs in different tissues and at different stages of gonadal development are largely unknown in vertebrates, especially in teleosts.Nowadays, identification and characterization of some subfamily of SDR superfamily are reported in several vertebrates. Genome wide identification and expression analysis of beta-hydroxysteroid dehydrogenase genes were reported in orange-spotted groupers (oioides) . Upreguloioides) . In humaoioides) ,21. In on testis . Previouecursors ,24. Rdh aldehyde . Howeveraldehyde . The oveOreochromis niloticus) is a commercially important farmed fish species in aquaculture worldwide. It is also a good model for study of sex determination and differentiation for the availability of genome sequences, mono-sex fry, and transcriptome data from different tissues [The Nile tilapia  had multiple copies located together in tandem arrays. These tandemly arrayed genes were on different chromosomes, except dhrs11 and dhrs13, which were located on LG14  in Nile tilapia went through tandem duplication, which resulted in 2, 13, 2, 2, 2, 3, 4, and 2 copies, distributed on LG19, LG14, LG14, LG4, LG5, LG10, LG13, and LG23, respectively.The 119  on LG14 . In totaSDR members of Nile tilapia, 114 genes could be designated into 49 subfamilies according to previous publications [SDR genes in Nile tilapia, spotted gar and human are listed in A phylogenetic tree was constructed using the conserved domain of amino acid sequences of all SDRs from Nile tilapia, spotted gar, and human. The SDRs were divided into five categories termed “classical”, “undefined”, “extended”, “complex”, and “atypical”. Most of the members belonged to the “classical” category. Only one member of the “complex” category was observed in human, spotted gar, and Nile tilapia, respectively. One member of “atypical” category was observed in human and spotted gar but none in Nile tilapia. “Classical”, “atypical”, and “complex” SDRs were clustered into different clades, while “extended” and “undefined” SDRs were clustered in one clade due to their high sequence similarity . “Intermications . CompareSDRs  were retained after the 3R-WGD events  were retained after the 2R-WGD , which retained two copies and had no tandem duplicates, were constructed  were tandem duplicated in different species, which resulted in multiple copies on the same chromosome  and downstream genes  were observed in Nile tilapia, zebrafish, and medaka. Similarly, conserved synteny of dhrs11-a and its upstream genes (tlcd1 and traf4a) and its downstream genes  were observed in Nile tilapia, zebrafish, and medaka but not in non-teleost vertebrates. Interestingly, 14 copies of dhrs11 genes were found in Nile tilapia, while there were only two copies in most teleosts and three copies in zebrafish  was found in Nile tilapia  were specifically expressed in the muscle, while gale was expressed only in the head kidney. Moreover, ten SDRs  were predominantly expressed in the liver. Importantly, hsd3b1 and kdsr were exclusively expressed in the testis, while hsd3b7, rdh12b, decr2, dhrs7b, sdr39u1, and spra were dominantly expressed in the ovary. Seven copies of the 13 dhrs11-a were highly expressed in both liver and kidney.Transcriptome data from eight adult tissues and gonads from four developmental stages of Nile tilapia allowed us to analyze the spatial and the temporal expression profiles of SDRs . There wSDRs were expressed in Nile tilapia gonads according to the threshold we set, while 56 SDRs were highly expressed . The expression of SDRs (RPKM) in Nile tilapia gonads at four developmental stages was listed hsd3b7, rdh10a, rdh12b, dhrs3, dhrs7b, dhrs9 sdr39u1, hpgd-b2, far1, spra, decr2) were found to peak at 90 dah, followed by 180, 30, and 5 dah. Expression levels of six testis-enriched genes  displayed similar expression profiles as the ovary-enriched genes but with higher expression in XY than in XX gonads  in gonads of Nile tilapia at 180 dah. Nine  of them were expressed dominantly in the cytoplasm of phase I and II oocytes in the ovary. On the contrary, hsd3b1 and hsd11b2 were expressed in the Leydig cells, and ak7-a was expressed in somatic cells of the testis. Expression of sdr39u1 was detected in both the spermatocytes of the testis and the cytoplasm of phase II oocytes in the ovary  shaped genome evolution in fish. In combination with phylogenetic and syntenic analyses, we concluded that SDR superfamily members increased evidently due to the 3R- and the 4R-WGD in fish. Although teleost fish experienced the 3R-WGD, the extended SDRs has not expanded much, indicating that a large number of the extended SDR subfamilies in teleost fishes were lost during evolution. Variation in the number of SDR superfamily in different representative species is consistent with multiple WGD events. Tandem duplications of dhrs11-a, hsd11b1b, cbr1-a, and dhrs7a were observed in seven cichlids analyzed, and tandem duplication of rdh10-b was only observed in four tilapiine species, indicating that they are lineage specific. In addition, tandem duplications of SDRs were also observed in other species, such as far, hsd11b1, and dhrs4 in humans and c-factor in spotted gar. These results demonstrated that tandem duplication is common in the SDR superfamily in vertebrates. The different number of SDRs observed in teleosts might be mainly attributed to different tandem duplication of different SDR genes among them. It is worth noting that some SDR genes have multiple copies located on different chromosomes in some invertebrates. These duplicates probably resulted from transposon-mediated replication. The origin of these copies remains to be elucidated.Studies on bacteria , nematodbacteria , plants bacteria , and humbacteria . Overallvolution . In thisor human . In contSDRs are expressed in multiple tissues, indicating that SDRs may play roles in various physiological and metabolic processes in Nile tilapia. In humans, DHRS7 is expressed in prostate, adrenal glands, liver, and intestine and participates in the reductive metabolism of both steroids and retinoids [DHRS7C) in mouse skeletal muscles induces enhanced glucose metabolism [dhrs7, dhrs7a-1, dhrs7a-2, dhrs7b, dhrs7c-a, and dhrs7c-b, were identified in Nile tilapia. Dhrs7b was expressed in ovary, indicating its possible role in steroid biosynthesis. Dhrs7c-a and dhrs7c-b, which originated from fish specific 3R-WGD, were highly expressed in muscle, suggesting their possible role in glucose metabolism. Previous reports demonstrated that human BLVRB has several biochemical functions, including biliverdin and riboflavin reductase (NADPH) activity. It catalyzes the reduction of biliverdin tetrapyrrole as an intermediary redox substrate in bilirubin generation in the heme degradation pathway [blvrb were detected in both heart and head kidney, suggesting their possible role in heme metabolism. Human DHRS11 exhibits enzymatic activities of both 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase, which efficiently catalyze the reduction of 11KA4, 11K-Adione, and 11KAST [dhrs11 in cichlids and the high expression of different copies in different tissues suggested their importance in multiple functions. High expression of dhrs11-a5, dhrs11-a6, and dhrs11-a10 in both kidney and liver indicated their essential roles in metabolism and homeostasis in Nile tilapia. In zebrafish, dhrs12 (named as 36K) was identified in myelin of the central nervous system to regulate membrane lipid composition and influence oligodendrocyte precursor cell differentiation and further myelination by altering the amount of transmembrane Notch ligands [dhrs12a gene was found to be expressed exclusively in Nile tilapia brain, indicating its critical role in brain functions.SDR superfamily encoded a large number of enzymes and displayed a broad spectrum of metabolic functions . Transcretinoids . Over-extabolism . In the  pathway . In Niled 11KAST ,35. The named as K was idehsd17b, hsd3b, and hsd11b in sex steroids biosynthesis were extensively investigated in several teleosts [hsd3b7, rdh10b-2, rdh12b, dhrs3, dhrs7b, dhrs9, sdr39u1, hpgd-b1, far1, spra, and decr2 were identified as ovary-enriched genes, while hsd3b1, hsd11b1-b2, hsd11b2, rdh14b, kdsr, and ak7-a were identified as testis-enriched genes. Sexually dimorphic expression of SDRs in Nile tilapia gonads indicated their essential roles in steroids and RA metabolism. However, the exact role of the newly identified ovary- and testis-enriched SDR genes remains to be elucidated. Further investigations of the functions of these SDRs in fish gonads will greatly promote the understanding of their roles in sex steroids and RA metabolism.It is well documented that sex steroids play essential roles in fish sex differentiation, gametogenesis, and reproduction ,38. Receteleosts ,42,43,44rdh12b, sdr39u1, decr2, and far1 were significantly up-regulated from 90 dah in females, indicating their indispensable roles in oogenesis. Previous studies showed that rdh12b recognizes both retinoids and lipid peroxidation products  as substrates and contributes to the reduction of all-trans-retinaldehyde [Decr2 gene encodes the peroxisomal 2,4-dienoyl-CoA reductase, which primarily converts 2,4-dienoyl-CoA to trans-3-enoyl-CoA. Biochemical studies and structural analysis suggest that human DECRs can catalyze the shortening of six-carbon-long substrates and shorten very long chain fatty acids [Far1 catalyzes the reduction of saturated and unsaturated C16 or C18 fatty acyl-CoA to fatty alcohols and plays an essential role in the production of ether lipids/plasmalogens in mammals [sdr39u1 in vertebrates is still largely unknown. Taken together, the abundant expression of SDRs in ovary indicated that anabolism of RA and fatty acid is critical for oogenesis in fish.Transcriptome analyses revealed that all of the ovary-enriched genes and the testis-enriched genes were highly expressed at 90 and 180 dah, indicating that they might be important for oogenesis and spermatogenesis, respectively. aldehyde . Decr2 gty acids ,47. Far1 mammals . These t mammals . In contHSD17b3 and HSD11b2 might be involved in the biosynthesis of 11-KT in human [RDH14 [rdh14 in testis of Nile tilapia further emphasizes the essentiality of retinol for testicular differentiation and functions. In humans, 11HSD1, which is ubiquitously expressed in most tissues, catalyzes cortisone to cortisol, while 11HSD2, expressed in the kidney and other classical target tissues for aldosterone, is responsible for the inactivation of cortisol to cortisone [11hsdb1-a1, -a2 and 11hsdb2 was found in testis, suggesting their possible roles in 11-KT production.In fish, both RA and 11-KT proved to play essential roles in testicular differentiation and spermatogenesis. Previous reports showed that both in human . RDH14 [n [RDH14  is a ubiortisone . In fishortisone . In thisNile tilapia fish used in this study were reared in recirculating freshwater tanks at 26 °C and under natural photoperiod. All female (XX) progenies were obtained by crossing the normal female (XX) with the sex-reversed XX pseudomales. All male (XY) progenies were obtained by crossing the normal female (XX) with YY supermales. Animal experiments were performed following the regulations of the Guide for Care and approved by the Institutional Animal Care and Use Committee of Southwest University.SDR sequences of human (Homo sapiens) and zebrafish (Danio rerio) were used as the query sequences to blast the genome sequences by tblastn (E = 2 × 10−5)) to identify the SDR genes in each genome analyzed. The identified SDRs were used to search against the NCBI  by blastx to reduce redundant matches. In addition, SDR sequences of the rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), Nile tilapia (Oreochromis niloticus), zebra mbuna (Maylandia zebra), Flier cichlid (Archocentrus centrarchus), Eastern happy , large yellow croaker (Larimichthys crocea), medaka (Oryzias latipes), fugu (Takifugu rubripes), channel catfish , spotted gar (Lepisosteus oculatus), coelacanth , elephant shark , tropical clawed frog , chicken , python (Python bivittatus), lamprey (Lampetra japonicavase), tunicate , black tiger shrimp (Penaeus monodon), domestic silkworm (Bombyx mori), fruit fly (Drosophila melanogaster), nematode (Caenorhabditis elegans), sponge (Amphimedon queenslandica), and paramecium (Paramecium tetraurelia) were collected from NCBI and Ensembl  databases. SDR sequences of blue tilapia (Oreochromis aureus), Mozambique tilapia (Oreochromis mossambicus), and blackchin tilapia (Sarotherodon melanotheron) were collected from unpublished genome database generated by our lab. Relatively high-quality genome sequences in these species allowed us to isolate more SDR members to reflect the true evolutionary history of the superfamily. These 29 species provided a broad evolutionary coverage of different animal groups to test the SDR superfamily variation. In order to distinguish genes derived from different rounds of WGD or tandem duplication, we developed a new system of nomenclature by adding numbers or letters as suffixes of the gene name. The gene derived before 3R-WGD was represented by the letters a, b after the gene name. The gene derived from 3R-WGD was represented by a hyphen followed by a, b, etc. The gene produced by tandem duplication was represented by a number after the hyphen or a letter after the hyphen.SDRs in Nile tilapia, spotted gar, and humans, and the maximum likelihood (ML) method was used for phylogenetic analyses of specific SDRs in many different species by MEGA 6.0 software  [SDRs from representative species in vertebrates were used in these trees, including those from several species of Cichlidae . The neighbor-joining (NJ) method was used for phylogenetic analyses of large numbers of pe, USA) . A bootsSDR genes. Bidirectional hierarchical clustering analyses were performed using the heatmap package [The transcriptomes of eight tissues from adult Nile tilapia, including brain, heart, liver, ovary, testis, kidney, muscle, and head kidney, were downloaded from NCBI database (Accession codes: PRJNA78915 and SRR1916191). RPKM was used to normalize the expression profile of  package . The tra package .SDRs were selected for validation of their gonadal expression at 180 dah by in situ hybridization (ISH). Among them, 4 SDRs were selected for further validation of expression profile by qPCR. To perform qPCR, gonads dissected from XX and XY Nile tilapia at 5, 30, 90, and 180 dah (about 6–200 fish for each sample depending on the fish size) were collected. The total RNA was isolated from each sample and reverse-transcribed using MMLV reverse transcriptase  according to the manufacturer’s protocol. Subsequently, qPCR examination was carried out according to the manufacturer’s protocol of SYBR Green I Master Mix . Nile tilapia eef1a1a was used as an internal control to normalize the expression of these 4 genes. The relative abundance of mRNA transcripts was evaluated using the formula R = 2−ΔΔCt. Data were expressed as mean ± SD for triplicates. Statistical analyses were carried out using Student’s t-test of the SPSS package, version 18.0.The 14 SDR family members in the developing gonads, ISH was performed using ovaries and testes from Nile tilapia at 180 dah. Fixation, embedding, and sectioning of dissected gonads and ISH were performed as described previously. Probes of sense and antisense digoxigenin (DIG)-labeled RNA strands were transcribed in vitro from linearized pGEM-Teasy-decr2/dhrs3/dhrs7b/dhrs11-a6/far1/hpgd-b1/ak7-a/ak7-b/hsd3b1/hsd3b7/hsd11b2/rdh10b-2/rdh12b/sdr39u1 DNA using an RNA labeling kit .To ascertain cellular localization of the SDR members assigned into 49 subfamilies and four categories were identified by comprehensive analyses of genome and transcriptome data in Nile tilapia. Phylogenetic and syntenic analyses demonstrated that tandem duplications in combination with multiple WGDs contributed to the expansion of SDR superfamily in vertebrates. In general, the variation of SDR numbers reveals the evolution process of SDR superfamily. Most SDRs were expressed in different tissues, including developing gonads. Some SDRs displayed tissue specificity or gender dimorphism. This study provided a new perspective for the evolution of the SDR superfamily and laid a solid foundation for revealing the role of the SDR genes in bony fishes and even vertebrates, especially in oogenesis and spermatogenesis.In this study, 119


Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration. Osteoarthritis (OA) is a degenerative joint disorder that affects more than 300 million people worldwide, often resulting in diminished quality of life and disability ,2. AlthoThe knee joint is a complex structure composed of several tissues including articular cartilage, synovial membrane, joint capsule, menisci, subchondral bone, infrapatellar and suprapatellar fat pads, and tensile connective tissues including tendons and ligaments . DegenerSeveral studies have transcriptionally profiled articular cartilage/chondrocytes using human tissue samples obtained during joint replacement surgeries, but generally these results represent late-stage OA cartilage only ,7,8,9, aIsolating chondrocytes from the articular cartilage is technically challenging due to the cells being embedded within a dense, fibrous ECM . In the Ten-week-old male C57Bl/6J (BL6) mice  were subjected to anterior cruciate ligament injury using a single non-invasive tibial compressive overload, as previously described ,13,18,19n ≥ 3/group) mice and processed for histological evaluation as previously described . C. C−; Tern (Cdh5)  whereas and Mcam ,31,32,33nd Htra1 D [7]. A nd Htra1 . Osteoblnd Htra1 , than tond Htra1 .Sox9 [Nfatc2, Runx3, Bhlhe40, Bhlhe41, Lef1, Tsc22d1, and Sox5) showed significant enrichment in all chondrocyte clusters, suggesting that these transcription factors play major roles in regulating chondrocyte differentiation or homeostasis  and glycoproteins  , Smpd3 (an enzyme involved in sphingolipid metabolism) [Papss2 and Chst11 (enzymes involved in the glycosaminoglycan metabolic process) [Next, we compared the gene expression profiles of chondrocytes to all other cell type clusters  to identify genes enriched in healthy chondrocytes compared to other connective tissue-forming cells in the joint . The masSox9  and seveeostasis . Previou Col27a1 . Chondrod Smoc2) . Severalabolism) , and Papprocess) ,36 also OA is a complex disease which affects multiple tissue types in the joint. Several human and animal model-based bulk gene expression studies have identified thousands of genes dysregulated in OA joints. However, for many of these genes the tissue/cell types that express these genes are not known. To identify OA-associated genes that are primarily expressed by chondrocytes we obtained a list of potential OA targets from OAtarget, a recently developed knowledgebase of genes associated with OA, and determined their expression levels in various cell types in the mouse joint . A totalCol2a1, Acan, Cnmd, Chad, Scrg1, Wwp2, cartilage intermediate layer protein (Cilp), Smpd3, osteoprotegerin (OPG/Tnfrsf11b), Wnt inhibitory factor 1 (Wif1), Matrilin 3 (Matn3), Col9a1-3, and Hapln1 , Bmp2, Ibsp, and cartilage intermediate layer protein 2 (Cilp2) (Tnfrsf11b), Wif1, and Cilp , Mgp, and Apoe  . This cland Cilp B. Clusteand Apoe . To defiSox9, Col2a1, and Acan .All cells from clusters expressing chondrocyte markers  were extracted and re-examined in greater detail. This included 1625 cells expressing high levels of and Acan A, accounand Acan . Clusternd Scrg1  and was  markers . Clusterdrocytes ,39,40, as [high’ . A subseertrophy  , Cilp2, and Prg4, and was annotated ‘Cytl1high’ , and Col1a1, Col3a1, and Abi3bp, in addition to Npy and osteopontin (OPN)/secreted phosphoprotein 1 (Spp1) , Efemp1, Spon1, Mgp, and Fxyd3, and was annotated ‘Chil1high’  . Cluster cluster ,42 , Dcn, and Chad showed enrichment in the Cytl1high, Tnfaip6high, and S100a4high clusters, and Lum, Srgn, Ogn, and Omd showed enrichment in the Chil1high cluster   . Several cluster . Interes cluster . The Chiand Apoe , but Il1drocytes . Additiofor Il11 . These rPrrx1, Bmp2, and Nifb were enriched in the Cytl1high, Tnfaip6high, and S100a4high clusters  with antibodies targeting four chondrocyte subtype markers marking distinct populations. Cytl1, which was robustly expressed in Cytl1clusters C was prind divCs , showed nd divCs D. Matn3  cluster C, was pr cluster E. Spp1,  cluster , was exp cluster F. Robust cluster F. These Ji et al. recently profiled human osteoarthritic chondrocytes using scRNA-seq and identified seven major chondrocyte subtypes labeled proliferative chondrocytes (proCs), pre-hypertrophic chondrocytes (pre-HTCs), hypertrophic chondrocytes (HTCs), fibrocartilage chondrocytes (FCs), effector chondrocytes (ECs), regulatory chondrocytes (RegCs), and homeostatic chondrocytes (HomCs) . Here, wCytl1 was described as a gene highly expressed in ECs [Cytl1 was robustly expressed in clusters 3, 4, and 5 as well  showed enrichment for Chad, Wif1, and Phlda1  with the mouse Tnfaip6high and S100a4high clusters  expressing cell-cycle genes like the mouse divCs [d in ECs , and we  as well A–C. Simid Phlda1 . Clusterclusters  but did nd Pth1r ,39,40  and 7-day post-injury joints (7DP1). Knee injury was induced by a tibial compression injury model in which the mice develop severe post-traumatic osteoarthritis (PTOA) by 6 weeks post-injury A 12,13,,45,46,47d Abi3bp .Col9a1-a3, Sox9, Prelp, Il17b, and Cilp were significantly downregulated, whereas Mmp3, Mmp13, Slpi, and Col10a1 were significantly upregulated in response to injury  gene expression in OA chondrocytes [Bhlhe40, Bhlhe41, or Tsc22d1 play a significant role in maintaining cartilage homeostasis. We also identified several potential OA targets enriched in chondrocytes compared to other cell types including Scrg1, Smpd3, Ppa1, Tsc22d1, and Msmo1, suggesting that these genes play a role in maintaining articular cartilage integrity  exhibited broad expression patterns across many chondrocyte subtypes in the mouse  showed enrichment for the Wnt signaling pathway in the human chondrocyte data as well [Wnt signaling is a major regulator of chondrogenic differentiation. Inhibition of Wnt signaling has been shown to enhance chondrogenesis , and ourof Wnt5b . Interes as well , suggesthigh, Tnfaip6high, and S100a4high clusters. These clusters showed significant enrichment for Bmp2 17 superfamily of cytokines [We also identified several injury-induced early molecular changes in chondrocytes, including up-regulation of ba in OA ,58,59, aba in OA ,61,62, aba in OA ,64, streba in OA . Il17b, ytokines , was dowThe joint is a complex structure composed of articular cartilage, subchondral bone, synovium, synovial fluid, joint capsule, menisci, infrapatellar and suprapatellar fat pads, nerves, vasculature, and tensile tissues including tendons and ligaments. It also includes resident immune cells that become activated during injury and tissue repair . TraditiWhile we were able to identify nine subpopulations of chondrocytes, the possibility still exists that more subpopulations are present in the healthy articular cartilage of rodents, as well as in humans. As tissue digests protocols improve, and viable cells are released from densely packed ECM and mineralized tissues like bones and cartilage, we will be able to sequence an exhausting number of cells, expanding our knowledge of rare and novel cell populations. The detailed transcriptional profiling of chondrocytes from injured and uninjured mouse joints allowed us to report here, for the first time, discriminative molecular markers for various chondrocyte subtypes in healthy joints as well as significant transcriptional changes at the single chondrocyte level, as a function of joint injury. These results provide insights into the potential roles some of these genes play in maintaining cartilage homeostasis. Furthermore, the genes we identified as differentially expressed in chondrocytes post injury may play a role in early OA development. Our findings expand our knowledge of cartilage biology and open new avenues for developing improved diagnostic and preventive strategies for mitigating the long-term damaging effects of joint trauma on the articular cartilage.


Bacillus tropicus strain UPM-CREST01, which was isolated from the bulk soil at a paddy cultivation area in Kampung Gajah, Perak, Malaysia. The final draft assembly of 5,252,705 bp, with a G+C content of 35.23%, was found to harbor 5,368 coding sequences, including several plant-growth-promoting genes.We characterized the draft genome of the potentially beneficial  Bacillus are thought to possess a variety of positive characteristics that benefit vegetation explicitly or implicitly via nutrient absorption or general growth enhancement via the synthesis of plant growth hormones, including resistance against infections and other abiotic stresses .Members of the genus stresses . Hence, y sector . Here, w600) before being subjected to genomic DNA extraction using the NucleoSpin tissue kit (Macherey-Nagel). The concentration of the genomic DNA was determined using an Invitrogen Qubit 4.0 fluorometer, and sequencing libraries were generated using the Nextera XT DNA library preparation kit (Illumina) with 1 ng of input DNA, according to the manufacturer’s guidelines. Upon paired-end sequencing (2 × 150 bp) with the Illumina NovaSeq platform, the reads were subjected to preprocessing using Trimmomatic v0.39   , a total9–JAJAQE010000000. The associated BioProject, SRA, and BioSample accession numbers are PRJNA763714, SRR15910132, and SAMN21447887, respectively.The whole-genome shotgun project has been deposited in NCBI under the accession number


Wide nanotechnology applications and the commercialization of consumer products containing engineered nanomaterials (ENMs) have increased the release of nanoparticles (NPs) to the environment. Titanium dioxide, aluminum oxide, zinc oxide, and silica NPs are widely implicated NPs in industrial, medicinal, and food products. Different types of pollutants usually co-exist in the environment. Heavy metals (HMs) are widely distributed pollutants that could potentially co-occur with NPs in the environment. Similar to what occurs with NPs, HMs accumulation in the environment results from anthropogenic activities, in addition to some natural sources. These pollutants remain in the environment for long periods and have an impact on several organisms through different routes of exposure in soil, water, and air. The impact on complex systems results from the interactions between NPs and HMs and the organisms. This review describes the outcomes of simultaneous exposure to the most commonly found ENMs and HMs, particularly on soil and aquatic organisms. Engineered nanomaterial (ENMs) have distinctive physical and chemical features, e.g., rapid diffusion, large surface areas, liquid or gas-phase reactivity, and small sizes . NumerouDuring the last decade, many studies on the toxicity of NPs have been conducted. The exposure of living organisms to NPs or NPs/pollutants adducts can induce adverse results in various physiological systems ,10. In m2NPs) and Cd can co-exist in different systems. This is due to TiO2NPs release from wastewater treatment plant effluents to freshwater bodies [+2 has been described as a concern [2NPs as nano-fertilizer and nano-pesticide [+2 is a soil primary contaminant [It is unlikely that no other toxic species co-exists with ENMs in a realistic environment. Thus, the emitted NPs can interact with pre-existing pollutants, resulting in bioaccumulation and/or toxicity. Thus, the outcomes of interactions between NPs and co-existing contaminants have become a rising issue of scientific investigation . For insr bodies ,26, and  concern . In paraesticide ,29 couldtaminant ,31,32,33, et al. [2, silica oxide (SiO2), silver (Ag), and CdTe/CdS core/shell quantum dots (QD) on the Cd+2 toxicity to the freshwater algae Chlamydomonas reinhardtii. Their results demonstrated that both SiO2NPs (400 mg L−1) and TiO2NPs (100 mg L−1) diminished the Cd+2 toxicity to C. reinhardtii. On the other hand, QD (0.5 mg L−1) augmented the Cd+2 toxicity to algae, while no significant change was detected in the toxicity of Cd+2 to algae with combined exposure to Ag NPs (0.2 mg L−1). Therefore, this review discusses the reported co-exposure outcomes between ENMs and HMs in the different in vivo and in vitro experimental models  larvae, carp (Cyprinus carpio), and Daphnia magna augment significantly due to TiO2 NPs [+2 and Cu+2 toxicity, in the presence of TiO2NPs, decreased in algae (Chlamydomonas reinhardtii and Microcystis aeruginosa) and amphipod (Gammarus fossarum) due to their reduced bioavailability [+5 toxicity to the freshwater flea (Ceriodaphnia dubia) may be worsened or eliminated by TiO2NPs, depending on the ratio of TiO2NPs to As+5 [2NPs and HMs, differences in model organisms, and physicochemical parameters of the co-exposition medium may be underlying these conflicting effects.Engates and Shipley  have sho, et al.  reported, et al.  and Deng, et al.  demonstrTiO2 NPs ,47,52,55lability ,71,72. I to As+5 . Species, et al. [2NP (5 and 15 nm) effect on the Cd+2, As+3, and Ni+2 bioaccumulation and toxicity in Caenorhabditis elegans during the sedimentation process in the aquatic environment. The authors verified that the HMs hastened the aggregation of TiO2NPs. The TiO2NPs quick aggregation and sedimentation altered the vertical distribution of HMs through adsorption and prolonged benthic species exposure. The main factors affecting the sedimentation rate were aggregate particle size and ion strength. TiO2NPs at non-toxic levels competently increased the HMs bioaccumulation and reproductive toxicity to C. elegans in a dose- and size-dependent manner; however, the TiO2NP effect on As+3 was lower than that on Cd+2 and Ni+2. These data demonstrate clearly that TiO2NPs potentiate the HMs toxicity to C. elegans due to their increased accumulation in the presence of TiO2NPs. Importantly, the interactions and fate of TiO2NPs and HMs that occur during sedimentation and the responses in organisms induced by co-exposure should be considered a necessary and integral part of the ecological system risk assessment  and humic acid (HA) on Cd+2 bioaccumulation in zebrafish were investigated by Hu, et al. [2NP (5–20 mg/L) in HA-containing water could change the effects of exposure of zebrafish to Cd+2 and other probable HMs. There is no clear mechanism underlying these combined effects.The single or joint effects of TiO, et al. . They fo, et al. [Daphnia magna and oligochaete Lumbriculus variegatus. Results showed that the total body burden and Cd+2 toxicity to L. variegatus were unaffected by TiO2NPs exposure, showing that Cd+2 adsorption to TiO2NPs did not disturb total bioavailability. In addition, in D. magna, no change in toxicity was observed despite facilitated Cd+2 uptake by TiO2NPs and increased total body burden of Cd+2.Hartmann, et al.  evaluate, et al. [2 NPs effect on Cd+2 bioavailability and toxicity to Chlamydomonas reinhardtii green algae. They reported that Cd+2 toxicity to green algae cells was reduced in the presence of TiO2NPs. In addition, no measurable TiO2NP amount was found to be associated with the algal cells. Hence, the authors demonstrated that TiO2NPs could decrease the free Cd+2 concentration, reducing its bioavailability and toxicity to C. reinhardtii. Furthermore, the electrostatic and potentially steric repulse between TiO2NPs and algal cells may interfere with their direct contact and prevent TiO2NPs from being internalized into the cells.Yang, et al.  investig+2 and Cd+2 toxicity were investigated after exposure of freshwater zooplankton, Daphnia magna, to 1 mg/L TiO2NPs for 2 days. The authors verified that D. magna pre-exposure to TiO2NPs resulted in a marked increase in Zn+2 and Cd+2 uptake from the dissolved phase. In addition, the metallothioneins and ROS measurements proved that the TiO2NPs provide potential adsorption binding sites for Zn+2 and Cd+2 within the D. magna gut.In the study of Tan and Wang , the abs, et al. [2NPs on Cd+2 (112 μg/L) biouptake and toxicity for Corbicula fluminea, a freshwater bivalve. The authors confirmed that Cd-uptake by C. fluminea were not affected by TiO2NP presence.Vale, et al.  evaluate, et al. [2NPs increased Cd+2 accumulation in the Tetrahymen thermophila ciliate. In addition, Tan, et al. [+2 and Zn+2 were heavily uptaken and retained in Daphnia magna when adsorbed to TiO2NPs.Yang, et al.  found th, et al.  showed t, et al. [+2 and TiO2NPs for 7 days on various genotoxicity indicators in the European sea bass, Dicentrarchus labrax. Their results verified that individual Cd+2 and TiO2NPs exposure reduced genome template stability. The chromosome alteration was due to TiO2NPs exposure alone, although the damage to DNA was ineffectual; the opposite was seen in Cd+2 exposed specimens. On the other hand, joint exposure inhibits chromosomal damage and partially recovers the genome template stability.Nigro, et al.  investig+2 and TiO2NPs in the cells of soil and aquatic organisms, in vitro studies have been performed using human and rodent cell lines. For instance, using human embryo kidney 293T (HEK293T) cells, Xia, et al. [+2 and TiO2NPs on the oxidative stress indicators, including the activities of catalase and superoxide dismutase enzymes and concentrations of ROS and malondialdehyde. The study findings verified that Cd+2 and TiO2NPs exerted synergistic effects on the cellular oxidative damage of HEK293T cells.Despite the lack of in vitro studies evaluating the outcomes of mutual exposure to Cd, et al.  evaluate2NPs have a high adsorption ability for As ions because of their large surface area and the presence of high-affinity hydroxyl surface groups [2NP and As, either as As+3 or As+5, resulted in both As and TiO2NP accumulation in vital organs [As is an extremely toxic pollutant, highly detected in groundwater . TiO2NPse groups . For insl organs ,47. High2NPs and As+5 interaction on Ceriodaphnia dubia and concluded that the decline in residual As+5 amounts decreases the toxic effect. In addition, the As+5 sorption onto the TiO2NPs surface adds to the toxicity once NPs enter the body. Additionally, Li, et al. [2NPs, As+5 adsorbed onto TiO2NPs could disassociate and lead to increased D. magna toxicity. In contrast, Yan, et al. [2NPs alleviated As+5 toxic effects in Artemia salina nauplii by increasing efflux and reducing As+5 amounts in the sensitive cellular fractions, including heat-sensitive proteins and organelles.Wang, et al.  assessed, et al.  revealed, et al.  reported, et al. [2NPs (1 mg/L) and As+3 (50 μg/L) co-exposure for 48 h on Laeonereis acuta, an estuarine polychaeta. Their findings revealed that TiO2NPs and As+3 mutual exposure increased ROS levels, decreased total antioxidant capacity, increased GR activity, and damaged macromolecules, including DNA, lipid, and protein. Moreover, the TiO2NPs and As+3 co-exposure affected the As metabolization capacity, leading to increased formation of a moderately toxic compound, known as dimethylated As. Nunes, et al.  evaluate, et al. [2NPs exposure on the bioaccumulation and methylation of As in two freshwater algae (Scenedesmus obliquus and Microcystis aeruginosa), reared in water contaminated with inorganic As. The transmission electron microscope examination showed that TiO2NPs entered exposed algae. The TiO2NPs within the algae significantly increased As+3 and As+5 accumulation in S. obliquus and M. aeruginosa, respectively. S. obliquus was more sensitive than M. aeruginosa to As connected with TiO2NPs, thus, it has higher As methylation. Luo, et al.  evaluate, et al. [2NPs on the trophic transfer of As+5 from Nannochloropsis maritima microalgae to Artemia salina nauplii shrimp. The authors found that TiO2NPs considerably facilitated As+5 sorption on a N. maritima 24 h exposure period. This sorption promoted As trophic transfer from the algae to A. salina. Nevertheless, after depuration for 48 h, the As+5 retention in A. salina fed As+5-TiO2NPs-contaminated algae was lower than that in A. salina fed As+5 -contaminated algae at equal exposure levels. This result demonstrates that the higher food chain transfer of As+5 in the presence of TiO2NPs can be elucidated by As+5 adsorption onto TiO2NPs in contaminated algae. Still, the As+5 bioavailability in A. salina is decreased with the NPs presence. Yang, et al.  explored, et al. [2NPs  and As+3 (50 μg/L) for 48 h on accumulation, metabolization, and toxicity of As+3 in the golden mussel Limnoperna fortunei. Results showed that both crystalline TiO2NPs forms affected the metabolization ability and enhanced more As+3 accumulation. In addition, TiO2NPs alone or in combination with As+3 induced oxidative stress in L. fortunei gills.Nunes, et al.  evaluate+2 is a frequently found metal ion in water and a vital micronutrient for aquatic organisms, but it may result in acute toxicity at high levels. Evidence shows that Cu+2 toxicity is associated with the interaction between adsorption of Cu+2 and co-substrates coordination [+2 raises concern about enhanced toxicity for Cu+2, even if at low levels.Cudination . The coe2NPs coexistence with Cu+2 ion potentiated the toxicity of Cu+2 to daphnids even at low concentrations [2NPs could compete with sulfhydryl groups by adsorbing or binding free Cu+2 ions, which cause metallothionein detoxification to be inhibited. Additionally, in Daphnia magna, Fan, et al. [+2 increased oxidative stress and physiological damage in the presence of TiO2NPs due to Cu+2 sorption. In addition, TiO2NPs may have inhibited Na+/K+-ATPase by hindering the transfer channel of Na+/K+.A previous study revealed that the TiOtrations . The met, et al.  reported, et al. [2NPs (2 mg /L) and Cu+2 (40 g /L) for 24 days on the amphipod Gammarus fossarum mortality and health. In the presence of TiO2NPs, Cu+2-inducing toxicity was mostly eliminated. It was proposed that the Cu+2 toxicity reduction is linked to Cu+2 elimination from the water column through TiO2NPs agglomeration and sedimentation and the metal ions absorbed [Rosenfeldt, et al.  investigabsorbed .+2 is a heavy metal that has been used for thousands of years in the manufacture of human utilities. These include wine, pigments, glass, recipients, and, more recently, antiknock fuel additives, batteries, and electronic components [+2 release to the environment [+2 is one of the major toxic pollutants [Pbmponents . Industrironment . As suchllutants ., et al. [2NPs (50 and 120 nm) and Pb+2 interaction in adult mice and verified that no synergistic interaction exists between TiO2NPs and PbAC in orally administered mice. Still, Pb+2 may increase the TiO2NPs acute toxicity to some extent. In human embryo hepatocytes, Du, et al. [2NPs  and Pb+2 (1 μg/ mL) in combination induced cytotoxicity and oxidative stress in the absence of photoactivation.Zhang, et al.  investig, et al.  confirme, et al. [2NPs (0.1 mg/L) and/or Pb+2  exposure for 6 days post-fertilization on nervous systems and thyroid function of zebrafish (Danio rerio) larvae. The results of their experiment suggested that TiO2NPs increase Pb bioconcentration, leading to the disturbance of the neuronal system and the thyroid function in zebrafish larvae. Additionally, in zebrafish larvae, Hu, et al. [2 NPs on Pb+2 bioconcentration, depuration, and neurotoxic impacts. The results indicate that TiO2NPs may act as a Pb+2 carrier and augment its bioconcentration, although free Pb+2 concentration decreases due to the NP-Pb complex formation, thus decreasing toxicity to larvae.Miao, et al.  examined, et al.  examined, et al. [Hoplias intermedius, to TiO2NPs (100 mg/L) and/or Pb+2 (0.033 mg/L) toxicity for 96 h. The authors verified that TiO2 NPs alone caused DNA damage in the cells of vital tissues, including the brain, gill, and kidney, but the muscle AChE activity reduced in the Pb-only exposed group. However, the metallothionein concentration was significantly increased in the TiO2NPs+Pb+2 co-exposed group.Vicari, et al.  evaluate2NPs and Pb+2 and their impacts on the copepods feeding behavior in a basic food chain, comprising the cyclopoids copepods (Eucyclop sp.) and freshwater alga Chlorella ellipsoides. The results showed that the TiO2NPs and Pb+2 mutual exposure impaired microalgae ingestion and filtration via cyclopoid copepods and increased antioxidant enzymes, lipid, and carbohydrate levels because of stress.Matouke and Mustapha  assessed, et al. [2NPs and Pb+2 interaction effect on the biochemical and genetic biomarkers in the freshwater fish Rhamdia quelen. The study results showed that TiO2NPs alone and co-exposure of TiO2NPs and Pb+2 can produce significant short exposure toxic effects. Oya-Silva, et al.  investigZinc oxide nanoparticles (ZnONPs) have been used extensively as sunscreens that can easily be released into the water . Because, et al. [+2 at tolerable doses to healthy overweight and healthy normal weight mice. Compared to normal mice, the ZnONPs enhanced Pb+2 deposition in all major organisms in the overweight mice. In the overweight mice, higher levels of hepatic ROS, proinflammatory cytokines, and liver damage were found. These findings emphasized the potential increased risk of co-exposure of NPs/HM in the sensitive overweight population.Jia, et al.  assessed, et al. [+2 toxicity using two oral-administered pregnant mouse models during peri-implantation or organogenesis. The authors verified that combined exposures to ZnONPs and Cd+2 resulted in a higher fetal deformity rate at the organogenesis stage rather than peri-implantation stage co-exposures. Moreover, after Cd+2 adsorption, the surface charge of ZnONPs was modified. The resulting nanoadducts caused the shedding of endothelial cells and damage to placental barriers. Furthermore, lower expression of tight junction proteins, including claudin-4, -8, and ZO1, was observed. These molecular and cellular events increased maternal-fetal transmission of both pollutants and worsened embryotoxicity.Teng, et al.  evaluate+2 10 mg/kg b.wt.) for 8 weeks on the thyroid gland of adult rats compared to their individual exposure. The results revealed that the mutual exposure of ZnONPs and Pb+2 resulted in greater thyroid dysfunction represented by reduced serum levels of triiodothyronine and tetra-iodothyronine but increased thyroid-stimulating hormone levels. In addition, the concentrations of Zn+2 and Pb+2 in the serum and thyroid were greater in the ZnONPs and Pb+2co-exposed rats.Khayal, et al.  assessed+2 is another known toxic air pollutant from anthropogenic activities. Feng, et al. [+2 could worsen cardiovascular toxicity through hypercoagulation, endothelial damage, and cardiac injury.SiNPs are widely utilized in biomolecular detection, medication delivery, imaging, diagnosis, photodynamic therapy, and gene therapy ,96. SiNP, et al.  evaluate, et al. [2 (1.5 mg/kg BW) in mice once a day for seven successive days significantly increased Cd+2 biodistribution density in the kidney and liver but did not alter the Si distribution in all examined organs. In addition, SiNPs and Cd+2 co-exposure resulted in greater severe oxidative stress in the renal and hepatic tissues.Guo, et al.  showed t2O3NPs are among the most common ENMs, with various industrial and biomedical applications, including cutting tools, packaging materials, refractory products, semiconductor materials, and cosmetic fillers [2O3NPs exposure was accompanied by increased ROS production, mitochondrial dysfunction, protein damage, and impaired cell morphology [2O3NPs may lead to harmful effects, including genetic and DNA damage [Al fillers . Its ele fillers . Despiterphology . AdditioA damage , apoptotA damage , inflammA damage , and carA damage .+5, Al2O3NPs products commonly remove As from drinking water [2O3NPs and As mixture. Hence, the possible effects of Al2O3NPs and As co-exposure on the ecosystem have gained researchers’ attention. For instance, Wang, et al. [Ceriodaphnia dubia to Al2O3NPs and inorganic As+5 resulted in an enhanced toxic effect, as As+5 was adsorbed on the Al2O3NPs surface.Due to their strong affinity to Asng water ,107,108., et al.  demonstr2NPs increased the Cd+2 toxicity to the freshwater green alga, Pseudokirchneriella subcapitata, but 300 nm TiO2NPs reduced the Cd+2 toxicity to the same algae species [Scenedesmus obliquus substantially reduced in the presence of TiO2NPs anatase [2NPs significantly increased the Zn+2 toxicity to Anabaena sp. [2NPs reduced Cd+2 toxicity to C. reinhardtii [+2 toxicity to P. subcapitata [+2 [NPs are released into the environment in large amount due to being extensively used and may affect the toxicity of other pollutants already found in the environment, such as HMs . Hence,  species . Additio anatase , while raena sp. . Moreovenhardtii  but incrcapitata . Of notecapitata ,114,115.capitata . The precapitata . These ccapitata . The frecapitata . In additata [+2 . Furthertata [+2 . Hence, The outcomes of joint HM and NP exposure may be affected by the duration of exposure. It is well known that, in reality, species can be exposed to contaminants not only throughout their entire life but also over many generations, which may have a greater impact on the population . Thus, d+2 and AL+3, and cause toxicity rather than the HMs that are adsorbed to the NPs [2NPs and As II co-exposure to worm Laeonereis acuta influenced the As metabolization capacity via increasing dimethylated As, a moderately toxic form, damaged lipids, and DNA [Several mechanisms could be underlying the synergistic toxic effects due to the co-exposure of NPs and HMs. The first mechanism involves the internalization of NPs within cells, releasing toxic concentrations of HMs . Initial the NPs . Through the NPs ,57,124.  the NPs . For ins and DNA . The thi and DNA . In this and DNA . The fou and DNA . By the  and DNA ,132., et al. [2NPs and SiO2NPs surfaces were responsible for their adsorbance to the algal cell surface. At the same time, a minute amount of NP-Cd complexes were adsorbed, and the rest was aggregated and sedimented in the surrounding environment. Subsequently, both the soluble Cd+2 concentration around algae and the Cd+2 internalization were reduced. Additionally, Dalai, et al. [+6 toxicity to Scenedesmus obliquus in the presence of TiO2NPs was mainly linked to the Cr+6 adsorption on the TiO2NPs surface, resulting in its aggregation and precipitation.On the other hand, the amelioration of toxicity of HMs at the co-exposure with NPs could be associated with the potent antioxidant activity of some NPs ,111. In , et al.  reportedOverall, the NP and HM co-effects are a complex problem. Various aspects still need to be covered, such as the environmental factors and species differences determining the positive or negative outcomes of NP and HM mutual exposure. Consequently, experimentally developing proper test designs for evaluating such mixture effects is considered a huge challenge. In addition, the ability to standardize test guidelines for the testing of these mixtures is another important challenge. The appropriate test design should consider several factors, such as the exposure media effect, NPs aggregation behavior in the test medium, the physical interactions between NPs and HMs, the physical interactions between NPs and organisms, the possible cell membrane disruption, and the potential free metal ions release. In addition, due to the growing number and variety of NPs, e.g., carbon-based NPs, other factors to be considered for the development of test designs include NP size, shape, and even surface functionalization . MoreoveAs production of ENMs increases rapidly, the potential NPs eco-toxicity impacts cause global concern. The reviewed studies’ collective findings underline that studying the potential interactions of NPs with existing environmental pollutants are vital in evaluating the possible NPs environmental risks. Identifying the expected hazards of co-exposure to ENMs and environmental contaminants could greatly help determine the safe strategies for combating such hazards. This could benefit the health of the different living organisms, environment sustainability, industrial companies, and international standardization organizations.