CCNB1/CDC42/MAPK7/CD44 oncogenic signatures. Accordingly, we used computational simulation and identify these signatures as regulators of the cell cycle in GBM, which leads to cancer development and metastasis. We also showed the antiproliferative and cytotoxic effects of SJ10 compound against a panel of NCI-60 cancer cell lines. This suggests the potential of the compounds to inhibit CCNB1/CDC42/MAPK7/CD44 in GBM.Glioblastoma multiforme (GBM) remains to be the most frequent malignant tumor of the central nervous system (CNS), which accounts for approximately 54% of all primary brain gliomas. Current treatment modalities for GBM include surgical resection, followed by radiotherapy and chemotherapy with temozolomide (TMZ). However, due to its genetic heterogeneity, GBM tumors always recur, due to treatment reasistance. The aim of this study was to identify molecular gene signatures, responsible for cancer initiation, progression, resistances and to treatment, metastasis, and also evaluate the potency of our novel compounds SJ10 as potential target for 1/S and S phases, which may potentially lead to resistance to radiation. Through computational simulations using bioinformatics, we identified several GBM oncogenes that are involved in regulating the cell cycle. Cyclin B1 (CCNB1) is one of the cell cycle-related genes that was found to be upregulated in GBM. Overexpression of CCNB1 was demonstrated to be associated with higher grades, proliferation, and metastasis of GBM. Additionally, increased expression levels of CCNB1 were reported to regulate activation of mitogen-activated protein kinase 7 (MAPK7) in the G2/M phase, which consequently modulates mitosis; additionally, in clinical settings, MAPK7 was demonstrated to promote resistance to temozolomide (TMZ) and poor patient survival. Therefore, MAPK7 is a potential novel drug target due to its dysregulation and association with TMZ resistance in GBM. Herein, we identified MAPK7/extracellular regulated kinase 5 (ERK5) genes as being overexpressed in GBM tumors compared to normal tissues. Moreover, our analysis revealed increased levels of the cell division control protein homolog (CDC42), a protein which is also involved in regulating the cell cycle through the G1 phase in GBM tissues. This therefore suggests crosstalk among CCNB1/CDC42/MAPK7/cluster of differentiation 44 (CD44) oncogenic signatures in GBM through the cell cycle. We further evaluated a newly synthesized small molecule, SJ10, as a potential target agent of the CCNB1/CDC42/MAPK7/CD44 genes through target prediction tools and found that SJ10 was indeed a target compound for the above-mentioned genes; in addition, it displayed inhibitory activities against these oncogenes as observed from molecular docking analysis.Current anticancer treatments are inefficient against glioblastoma multiforme (GBM), which remains one of the most aggressive and lethal cancers. Evidence has shown the presence of glioblastoma stem cells (GSCs), which are chemoradioresistant and associated with high invasive capabilities in normal brain tissues. Moreover, accumulating studies have indicated that radiotherapy contributes to abnormalities in cell cycle checkpoints, including the G Glioblastoma multiforme (GBM) is one of the most frequent malignant tumors of the central nervous system (CNS) ,2, whichCD44-expressing cells were shown to escape exogenous DNA damage from radiation-induced double-stranded breaks (DSBs); it ultimately promoted tumor recurrence and resistance to radiation [1/S and S phases, which may potentially lead to resistance to radiation [CCNB1) is one of the cell cycle-related genes that was reported to be a potential biomarker in GBM [CCNB1 was demonstrated to be associated with higher grades, proliferation, and metastasis of GBM [CCNB1 was shown to regulate cell mitosis at the G2/M phase through interacting with cyclin-dependent kinase 1 (CDK1) [CCNB1/CDK1 may be potential diagnostic and prognostic markers of GBM [One of the stem cell markers that is commonly expressed in various cancer types, including GBM is cluster of differentiation 44 (CD44), a surface adhesion receptor which promotes cancer progression and metastasis ; its expadiation . Moreoveadiation ,18. Cyclr in GBM ,20. Overs of GBM . Additio1 (CDK1) . Thus, Cs of GBM ,24,25.MAPK7) is a member of MAPKs, which regulate signaling transduction cascades [MAPK7 were identified in GMB tumors compared to adjacent normal brain tissues. In clinical settings, MAPK7 promotes resistance to TMZ and poor patient survival; however, its role in GBM still remains to be further investigated [MAPK7 in the G2/M phase, which consequently modulates mitosis [MAPK7 is a potential novel drug target due to its dysregulation and association with TMZ resistance in GBM [CDC42) is a protein which is also involved in regulating the cell cycle through the G1 phase. Overexpression of CDC42 in GBM was demonstrated to promote tumor cell invasion and migration; additionally, CDC42 was associated with low survival rates and drug resistance in GBM patients [CCNB1/CDC42/MAPK7/CD44 oncogenic signatures in GBM through the cell cycle.Mitogen-activated protein kinase 7 -60 central nervous system (CNS) cell lines to determine responses of single-dose and dose-dependent treatments with SJ10 [Integrated bioinformatics analyses have been extensively applied in the early stages of drug discovery and development and have significantly accelerated the process, as well as reduced costs. Computational simulation approaches and molecular structural analyses of ligand-protein interactions have contributed to the identification and prediction of novel diagnostic and prognostic biomarkers in cancer research ,36. In treatment . Fortunareatment ,39, we sreatment . Therefoith SJ10 .http://www.ncbi.nlm.nih.gov/gds/, accessed on 4 August 2021), an international public repository for high-throughput microarray data [https://www.ncbi.nlm.nih.gov/geo/geo2r/, accessed on 4 August 2021), an interactive online platform to identify differentially expressed genes (DEGs) [p values (adj. p) to control the false discovery rate (FDR) and maintain the balance between the possibility of false-positives and the detection of significant genes. The fold-change (FC) threshold was set to 1.5, and adj. p < 0.05 was considered statistically significant. Venn diagrams were constructed using the Bioinformatics & Evolutionary Genomics (BEG) online tool .Gene expression profiles (GEPs) from GBM patient samples were downloaded from the Gene Expression Omnibus (GEO) database (ray data . (3) difs (DEGs) , which whttp://www.way2drug.com/passonline/predict.php, accessed on 17 August 2021). In addition, we explored the Swiss target prediction tool , a web-based algorithm that uses the principle of similarity to predict drug targets of bioactive small molecules [Potential SJ10 target genes were predicted using an open-source web tool based on the Prediction of Biological Activity Spectra (PASS)  , a web-based tool for the analysis of interactions between genes of interest and immune cells. The relative gene expression level is indicated as transcripts per million (TPM) and the expression value was normalized by log transformation. Moreover, we explored the Chinese Glioma Genome Atlas (CGGA)  to analyze gene expression correlations between the two datasets into positive and negative correlations, with positive Pearson correlation coefficients and p < 0.05 considered statistically significant.Expression profiles of genes showing differential expression between GBM tumor and adjacent non-tumor tissues in The Cancer Genome Atlas (TCGA) database were analyzed with TIMER  to compare expression levels between tumor samples and normal samples. The HPA database represents the protein expression in 44 major human tissues and some cancer tissues by IHC [p value was determined using the Mann–Whitney U-test. Moreover, for further analysis, the predicted genes were validated by an independent bioinformatics tool, the CGGA [To validate expression levels of identified DEGs in GBM, we explored the Human Protein Atlas (HPA) database for immunohistochemistry (IHC) , a web tool developed to analyze interactions of PPIs, such as physical and functional associations [p < 0.05 considered significant. For further analysis, we used Network Analyst , a web-based visual analytics platform for comprehensive gene and protein expression profiling [To assess PPIs, we used the search tool for the Retrieval of Interacting Genes/Proteins database , an online portal used for analysis of brain tumor expression [CCNB1, CDC42, MAPK7, and CD44 oncogenes, in order to evaluate overall survival (OS).To predict prognostic outcomes in GBM, radiomics signatures were constructed using the GlioVis database , and further explored the GlioVis database for the KM analysis. The ROC curve was based on true positive (sensitivity) and false positive (specificity) rates in GBM patients. We evaluated whether the test measurement had a specific condition. We assessed the area under the curve (AUC), and an AUC of 0.5 indicated no discrimination, while an AUC of 1.0 indicated discrimination of the curve that includes all possible decision thresholds from a diagnostic test result, which were patients who experienced disease onset and individuals who did not.To evaluate and validate the diagnostic and prognostic significance of http://www.swissadme.ch/index.php, accessed on 2 September 2021), and molecular in silico (molsoft) tools , to evaluate the PKs, drug likeness, medicinal chemistry friendliness, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of SJ10 [Identifying novel and potential drug candidates in the early stage of drug discovery and development is crucial, as it reduces time and costs; herein, we applied the drug-likeness concept based on specific criteria ,54. We e of SJ10 ,56. We a of SJ10 . Moreove of SJ10 . The Abb of SJ10 .https://dtp.cancer.gov/, accessed on 11 September 2021). The compound was tested at an initial dose of 10 μM. Results showed that SJ10 exhibited antiproliferative activities against CNS cell lines.SJ10 was submitted to the National Cancer Institute (NCI)-Development Therapeutics Program (DTP) to be screened for potential antiproliferative and cytotoxic effects against a panel of NCI-60 CNS cell lines, in agreement with the outlined protocol of the NCI -approved standard inhibitors of CDC42 and MAPK of CASIN and BAY-885, respectively. Accordingly, the 3D structure of SJ10 was assembled with the Avogadro molecular visualization tool [https://pymol.org/2/, accessed on 6 October 2021). In addition, the crystal structures of CCNB1 (PDB:2B9R), CDC42 (PDB: 2ODB), MAPK7 (PDB:4H3Q), and CD44 (PDB:1UUH) were downloaded from the Protein Data Bank (PDB). For further processing, we converted all PDB files to PDBQT file format using autodock software  and, finally, performed docking. To visualize and interpret the docking results, we applied BIOVIA discovery studio software for analysis [Receptor-ligand interactions were predicted using a molecular docking analysis, a technique used to predict the predominant binding ability of a ligand with a protein’s three-dimensional (3D) structure . To asseion tool , the 3D analysis .CCNB1/CDC42/MAPK7/CD44 expressions in GBM cancer types. The statistical significance of DEGs was evaluated using the Wilcoxon test. * p < 0.05 was accepted as being statistically significant.Pearson’s correlations were used to assess correlations of p-value in the volcano plot was used to indicate threshold indicators for adjusted p-values, which was further used to show all the genes that are statistically differentially-expressed with an adjusted p value threshold of 0.05 considered significant.Gene expression profiles (GEPs) from GBM samples and normal brain samples tallied from different studies were extracted from the microarray dataset. The analytical results showed that 100, 256, and 30 GBM samples and normal samples were, respectively, obtained from the GSE4290, GSE68846, and GSE30563 datasets. Further analysis with a Venn diagram demonstrated 87 overlapping upregulated genes from the three datasets A and 50 We explored the SwissADME algorithm developed by the Swiss Institute of Bioinformatics and molecule in silico (molsoft) to evaluate the PKs, drug likeness, medicinal chemistry friendliness, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of SJ10 ,56. We aCCNB1/CDC42/MAPK7/CD44 in pan cancers, including GBM tumor tissues compared to normal tissues from TCGA) RNA levels of om TCGA) A–D. Relapatients E–H, withCCNB1/CDC42/MAPK7/CD44 gene signatures in GBM, we explored the HPA database for IHC to compare gene expression levels between GBM tumor tissues and normal samples. CCNB1 displayed medium staining, with strong intensity and quantity (25%)   A, while ty (25%) B, and MAty (25%) C,D in GB tissues E–H. In a (ANOVA) I–L, withCCNB1/CDC42/MAPK7/CD44 genes in GBM, we explored HPA IF staining, using the U251-MG GBM cell line. The following antibodies were used for staining: CCNB1 (HPA030741), CDC42 (CAB004360), MAPK7 (CAB018561), and CD44 (CAB000112). Staining results of the U251-MG cell line exhibited the location of genes, with antibodies shown in green, nuclei in blue, and microtubules in red. CCNB1 was localized in the cytosol, CDC42 was detected in microtubules, while the localization of MAPK7 was in the nucleoplasm and CD44 was found in plasma membranes , a web tool developed to analyze interactions of PPIs, such as physical and functional associations. The clustering analysis had nine nodes and 15 edges, with an average local clustering coefficient of 0.917 and a PPI enrichment p value of 0.0293. Moreover, the interaction score confidence was set to > 0.4, and considered most significant. Active interactions were based on text mining, experiments, databases, co-expressions, neighborhood, gene fusion and co-occurrence , a web-based visual analytics platform for comprehensive gene and protein expression profiling [1/CDC42/MAPK7/CD44 oncogenes in the same network cluster, and results were viewed from the network topology in a force atlas layout analyzed from the Igraph R package  were 2.83, 6.62, 3.9, and 3.48, respectively  and false (selective) positive rates of responses in GBM patients. AUC scores of CCNB1, CDC42, MAPK7, and CD44 were 0.534, 0.515, 0.541, and 0.538, respectively /50% inhibitory concentration (IC50), and results ranged 1.14~2.15 μM in the CNS cell lines, with SNB-75 more sensitiv at 1.14 μM, followed by U251 at 1.59 μM, SF-268 at 1.64 μM, SNB-19 at 1.67 μM, SF-539 at 1.69 μM, and SF-295 at2.15 μM, showing a smaller response to SJ10  percentage showed that SNB-19 cells were more sensitive, with GI of 67.25%, followed by SF-539 at 36.91%, SF-268 at 34.33%, SF-295 at 23.75%, and SNB-75 at 22.34%, as shown in  to SJ10 B,C.CDC42 and MAPK7, CASIN and BAY-885 respectively. The interaction of CDC42 in complex with CASIN exhibited binding energy of  and MAPK7 in complex with BAY-885 displayed binding energy of , these results exhibited a much lower binding affinities as compared to SJ10, this suggesting the potential inhibitory effects of SJ10 in GBM expression CCNB1, CDC42, MAPK7, and CD44 oncogenic signatures , CDC42 , MAPK7 , and CD44 . When compared to standard inhibitors of CASIN (CID: 2882155) for CDC42 and BAY-885 (CID: 134128280) for MAPK7, they showed lower binding energies of −7.4 and −7.3 kcal/mol, respectively. For a further analysis, we used Pymol and Discovery Studio to visualize the analytical results. The SJ10/CCNB1 complex displayed interactions by conventional hydrogen (H) bonds with ALA128 (2.07 Å) and ARG68 (2.73 Å). The interactions were stabilized by van der Waals interactions , pi-sigma (GLY132), and pi-alkyl  displayed in their binding pockets. The SJ10/CDC42 complex displayed van der Waals interactions , pi-sigma (ILE21), and pi-alkyl  in their binding pockets, while SJ10/MAPK7 exhibited conventional hydrogen bond with SER153 (2.23 Å) and was further stabilized by van der Waals interactions , carbon hydrogen bond (GLU33), pi-sigma (THR190), pi-pi stacked (TYR113), and pi-alkyl (PRO152 and LYS151) in their binding pockets. Interactions between the SJ10/CD44 complex displayed van der Waals interactions , carbon hydrogen bond (CYS77), pi-pi T-shaped (TYR42) and pi-alkyl (ILE91) in their binding pockets . For furgnatures , Table 2Despite improvements in standard therapies, including surgical resection, radiation, and chemotherapy with TMZ, patients with GBM still exhibit poor clinical outcomes, with a median survival of only about 15 months , mainly CCNB1, CDC42, MAPK7, and CD44 oncogenic signatures as target genes for SJ10. Moreover, we explored the SwissAMDE and molsoft algorithms to evaluate the PK, drug-likeness, medicinal chemical friendliness, and ADMET properties of SJ10 [2), solubility (Log S (ESOL) = −4.7), flexibility (rotation = 4), saturation (Fraction Csp3 = 0.2), and pKa (=0.5) of the SJ10 compound. In addition, the SJ10 compound demonstrated highly probable GIA absorption, a bioavailability score (55%), and good synthetic accessibility (2.89). The compound reached the BBB with a score of 4.98, and further displayed a drug-like model score of (−0.68) (SJ10 (NSC7772862) is a small molecule and a derivative of a quinolone and piperazine derivative and was recently synthesized in our laboratory. Through application of the Swisstarget and PASS prediction tools , we pred of SJ10 ,56. The  (−0.68) .CCNB1/CDC42/MAPK7/CD44 oncogenes in pan cancers, including GBM tumor tissues compared to normal tissues from TCGA, using the TIMER bioinformatics tool. These results were further validated using the HPA and GlioVis database analyses, which showed similar outputs displaying overexpression of CCNB1/CDC42/MAPK7/CD44 gene signatures in WHO grade II, III, and IV GBM tumors using an ANOVA. For further analysis, we used the STRING online web tool and showed that CCNB1/CDC42/MAPK7/CD44 actively interacted with each other in the same clustering network, based on text mining, experiments, databases, co-expressions, neighborhood, gene fusion, and co-occurrence and also exhibited enrichment of GO involving BPs and  were 2.83, 6.62, 3.9, and 3.48, respectively , CDC42 , MAPK7 , and CD44  compared to the standard inhibitors of CASIN (CID: 2882155) for CDC42 and BAY-885 (CID: 134128280) for MAPK7, which showed lower respective binding energies of −7.4 and −7.3 kcal/mol . Therefore, the above-mentioned results suggest that SJ10 exhibits drug-like characteristics, with anticancer activities and is a potential oral drug candidate. Further in vitro and in vivo studies are both currently in progress in our laboratory.The potential anticancer activities of SJ10 were evaluated against NCI human CNS cell lines. Accordingly, an initial dose of 10 μM exhibited antiproliferative activities against the CNS cell lines, as shown in CCNB1/CDC42/MAPK7/CD44 oncogenic signatures are potential biomarkers of GBM therapeutic-resistant tumors, and potential drug targets of our novel small molecule, SJ10. We further showed that SJ10 exhibits anticancer activities against a panel of NCI human CNS cancer cell lines when administered at an initial dose of 10 μM and also in a dose-dependent manner. We evaluated receptor-ligand interactions using a docking analysis and identified unique and higher binding energies of SJ10 in complex with the CCNB1, CDC42, MAPK7, and CD44 oncogenes, compared to their interactions with two FDA-approved inhibitors. Further in vitro and in vivo studies are both currently in progress in our laboratory.In summary, our obtained results showed that


In addition, they exhibited anticancer properties by a diverse biological mechanism including induction of apoptosis or inhibition of cell migration and invasive potential. Finally, phlorotannins 1–7 and bromophenols 12–29 represent the most researched phenolic compounds, of which the former are recognized as protective agents against UVB or gamma radiation-induced skin damages. Finally, phenolic metabolites were assorted into six main classes: phlorotannins, bromophenols, flavonoids, coumarins, terpenophenolics, quinones and hydroquinones. The derivatives that could not be attributed to any of the above-mentioned classes were grouped in a separate class named miscellaneous compounds.Since the middle of the last century, marine organisms have been identified as producers of chemically and biologically diverse secondary metabolites which have exerted various biological activities including anticancer, anti-inflammatory, antioxidant, antimicrobial, antifouling and others. This review primarily focuses on the marine phenolic compounds and their derivatives with potent anticancer activity, isolated and/or modified in the last decade. Reports on the elucidation of their structures as well as biosynthetic studies and total synthesis are also covered. Presented phenolic compounds inhibited cancer cells proliferation or migration, at sub-micromolar or nanomolar concentrations (lamellarins D ( The malignant diseases have represented one of the greatest challenges to the modern society and health care systems, and have induced the extensive research and clinical investigations by both the pharmaceutical industry and the scientific community . AccordiNotwithstanding the fact that a great progress has been made in the treatment of malignancies arising from the development of combination therapy and immunotherapy as a form of personalized medicine, traditional chemotherapeutics are still the most widely used. However, due to insufficient selectivity towards cancer cells, together with the developed resistance, they continue to cause severe side effects, weakening the immune system of the organism ,9,10. Co®, 1969), nelarabine , trabectedin , fludarabine phosphate , eribulin mesylate , brentuximab vedotin , midostaurin , plitidepsin , polatuzumab vedotin  and enfortumab vedotin  .,47.46,47p-coumarid acid by cinnamate 4-hydroxlyase (C4H) chromene-2,7-dione structural feature, characteristic for both aspergiolides A (40) and B (41), might be used for the synthesis of new anticancer derivatives. Moreover, an in silico analysis indicated that 41 is a potential EGFR-TK inhibitor displaying low binding free energy in active site containing MET-766, THR-790 and THR-854 amino acid residues anthracene derivative. More precisely, 69 is known for its unique C-bounded angolosamine unit, with a dimethylamino group at the C-5 of the skeleton. Mayamycin (69) exhibited potent activity with determined IC50 values of 0.2, 0.3, 0.2, 0.16, 0.29, 0.13, 0.15 and 0.33 μM against eight cancer cell lines, HepG2, HT-29, GXF251L, LXF529L, MAXF401NL, MEXF462NL, PAXF1657L and RXF486L, respectively. As published by the authors, 69 was also cytotoxic toward a mouse fibroblast cell line (NIH-3T3) [69, N-acetyl-N-demethylmayamycin (70), was obtained in 2016 from another marine Streptomyces strain, 182SMLY, by Liang et al. [70 resulted in 50% of proliferation inhibition at the concentrations of 0.7, 1.4, 3.9 and 0.5 μM, against U251, U87-MG, SHG-44 and C6 glioma cell lines, respectively, as determined by sulforhodamine B assay. Furthermore, it was shown that 70 can induce apoptosis in U251 cells [The culture broth of the NIH-3T3) . An analg et al. . The aut51 cells . Synthet51 cells ,213.Streptomyces sp. Strain, M7_15, of which monacyclinone F (71) showed the highest activity against rhabdomycosarcoma cancer cells (SJCRH30) displaying an EC50 value of 0.73 μM. The authors concluded that the structural characteristic arising from the presence of two epoxide rings, aminodeoxysugar and ketone moiety could be of great importance for biological activity [In 2015, six new angucyclinone derivatives were extracted from the sponge-derived activity .72) and lupinacidin A (73), were isolated from the marine-derived Streptomyces spinoverrucosus by Hu and colleagues [50 values of 5.0 and 12.2 μM for 72 and 8.8 and 3.1 μM for 73, respectively. Recently, Sottorff and his team isolated both 72 and 73 from the sea anemone (Gyractis sesere) from Easter Island, but further confirmed the Actinobacteria of the genus Verrucosispora as the exact producer of those compounds [Anthraquinones with alkyl substituents, galvaquinone B (lleagues . They scompounds .Phormidium ectocarpi to isolate hierrdin B (74), a methylated hydrouinone with a long aliphatic chain which showed antiplasmodial activity [74 from the marine picocyanobacterium Cyanobium sp. LEGE 06113, and determined its selective, but weak, activity towards HT-29 cells with an IC50 value of 100.2 µM [74). The authors pointed out that 74 targets mitochondrial activity by increasing the mRNA expression of VDAC1, a key protein involved in mitochondria-mediated apoptosis. That observation was accompanied with the inhibition of cell cycle progression induced by 74 [74, norhierridin B (75), was synthesized with improved inhibitory activity against several cancer cell lines by activating the p53 pathway. The IC50 values of 0.61, 0.77, 0.68, 2.0, 0.61 and 3.2 µM against MDA-MB-231, SKBR3, MDA-MB-468, A375, Huh-7 and HCT116 were measured, respectively. Therefore, the authors suggested that the presence of two hydroxy groups in the quinone skeleton is of great importance for the improvement of anticancer activity [In 1998, Papendorf and his colleagues used the marine cyanobacterium activity . However100.2 µM . The samed by 74 . In the activity .Didymellaceae was used to isolate several phenol derivatives including diorcinol L (76) , a compound somewhat similar to diorcinol L (76), was very recently isolated from the Red Sea-derived fungus, Aspergillus falconensis [78 inhibited 50% growth of a mouse lymphoma cell line (L5178Y), while MDA-MB-231 cell migration was inhibited at 70 µM. The authors also performed docking studies which revealed inhibition activity of 78 against the CDK-2, TOP-2 and MMP-13 proteins.The ethyl-acetate extract of the sponge-derived fungus of the genus l L (76) . It exhiectively . Interes.0 µg/mL . On the conensis . At a co79) is a phenolic derivative with a unique spiro skeleton that was isolated from the marine fungus Penicillium citrinum. It showed a promising and moderate activity against various solid tumor types in vitro, with A-375 cells being the most sensitive. At the concentrations of 30.88, 12.78 and 7.06 µM, a treatment with 79 for 24 h, 48 h and 72 h, respectively, resulted in 50% growth inhibition of the abovementioned cell line. Furthermore, Liu and co-workers investigated the mechanism of action in detail regarding apoptotic and metastatic activity. The authors revealed that penicitrinine A (79) induced apoptosis of A-375 cells by decreasing and increasing the expression of the Bcl-2 and Bax proteins, respectively. Additionally, 79 induced cell migration suppression by downregulating MMP-9 and upregulating TIMP-1 levels [Penicitrinine A (1 levels .80), was isolated from the marine sponge of the genus Pseudoceratina [80 is manifested in the regulation of the expression of proteins involved in angiogenesis, DNA replication, apoptosis, proliferation and invasion. Therefore, it has been revealed that psammaplin A (80) inhibits aminopeptidase N (APN), mycothiol-S-conjugate amidase (MCA), topoisomerase II, farnesyl protein transferase, histone deacetylases (HDACs) and leucine aminopeptidase [80 and its analogues, which were found as more potent inhibitors of HDACs [80 [An unusual bromotyrosine metabolite containing oxime and disulfide moieties, psammaplin A . The authors demonstrated selective cytotoxic effects of 81 towards PANC-1 cells  which were adapted to certain nutrient conditions associated with the assumption of the mechanism of action via the inhibition of the Akt signalling pathway [81 displayed potent cytotoxicity against both sensitive and multidrug resistant cells with determined IC50 values being: 6.8, 6.4, 5.3, 4.9, 5.1 and 4.9 µM against S1, S1-MI-80, H460, H460/MX20, MCF-7 and MCF-7/ADR, respectively. In addition, 81 was found to induce cell death through c-Jun/Src/STAT3 signalling by inhibiting the proteasome-dependent degradation of c-Jun [81), however, isolated from the terrestrial source. It was revealed that 81 exhibited antitumor activity in both normal (HUVEC) and MCF-7 cancer cells through the Akt/mTOR/p70S6K pathway resulting in the inhibition of eNOS and ERK phosphorylation together with MMP degradation as key pro-angiogenesis factors [Tang et al. used the culture extract of the marine-derived  pathway . Furtherof c-Jun . Another factors .81, secalonic acid F (82), has been isolated from the marine-derived fungal strains, Penicillium sp. F11 and Aspergillus aculeatus [82 was more potent towards HepG2 cells than the positive control (5-FU), exhibiting IC50 values of: 45.5, 8.7 and 7.7 µM after 24, 48 and 72 h treatment, respectively. Those observations were, in relation to mitochondrial-mediated apoptosis, more precisely the activation of caspases-3 and -9. The authors also performed in vivo studies that demonstrated lower tumor weights after treatment with 82 [. A new biological evaluation of secalonic acid F (82) resulted in the identification of a potential target, MARCH1, for the treatment of hepatocellular carcinoma, in which the downregulation suppressed the migration and invasion of HepG2 and Hep3B cancer cells [82 against leukemia and multiple myeloma cells with regard to normal cells mediated through apoptosis and necrosis, as well as tubulin disassembly [On the other hand, an isomer of culeatus ,232. A sculeatus . Further with 82 . A new ber cells . Finallyassembly .Due to novel strategies and methods of the sampling of organisms from harsh marine environments and the constant progress in technologies and methodologies of extraction, purification, identification and characterization of natural products, the complex and diverse marine flora and fauna have been more and more accessible and recognized as a rich source of secondary metabolites with often unique chemical structures specific only for the marine environment. Although sometimes confusing as the same compound can be categorized to more than one class, marine natural products have been traditionally classified as peptides, alkaloids, macrocylclic polyethers, terpenoids and steroids and phenolics. Especially the latter have been widely studied in terrestrial organisms, mainly plants, while systematic investigations of marine phenolic compounds connecting their isolations, chemical characterizations and biological evaluations are still very scarce. The bioproduction of phenolics in marine species is triggered by both biotic and abiotic stress, with the purpose to quickly respond and to adapt to the varying ecosystem conditions. For that reason, the difference in the phenolic content could differ depending on geographical distribution, time and season of collection, as well as on the applied laboratory methodology of extraction and purification. Furthermore, despite the known main biosynthetic pathways for the biosynthesis of phenolic compounds, i.e., shikimic acid, the malonate-acetate and mevalonate-acetate, the complete biosynthesis of phenolic compounds is often specific to one or several marine species and it may imply postbiosynthetic modifications by symbiotic organisms.Natural products of a marine origin have displayed a broad range of pharmaceutically important biological effects. Especially, marine-derived phenolic metabolites and their derivatives or analogues have attracted great attention due to antioxidant, anti-inflammatory, antimicrobial and anticancer activities resulting from their abilities to inhibit reactive radicals through the single electron and hydrogen atom transfer reactions, or to interact and to bond to target proteins and other biologically important molecules.37), M (38), K (39), aspergiolide B (41), fradimycin B (62), makulavamine J (66), mayamycin (69), N-acetyl-N-demethylmayamycin (70) or norhierridin B (75)). Furthermore, they exhibited anticancer properties by the induction of apoptosis or the inhibition of cell migration and invasive potential. Finally, phlorotannins 1–7 and bromophenols 12–29 represent groups of the most studied phenolic compounds, of which the former are recognized as protective agents against UVB or gamma radiation-induced skin damages.In this review are summarized anticancer potentials of recently isolated highly active marine phenolic compounds as determined through in vitro, in vivo or in silico assays. The most promising cytotoxic marine-derived phenolics and their biologial activites are depicted in In order to obtain clinical evidence about the anticancer effects of marine phenolic compounds, these promising results should be further evaluated in in vivo studies in cancer patients. It is also of great significance to develop new bioprospecting programs and to promote further technological advancements in sampling from harsh marine environments. The improvements in the isolation and characterization of natural products present in traces and novel chemical synthetic strategies to overcome the scarcity of phenolic secondary metabolites from the marine matrices as the main limiting factor have to be also achieved. Taking this into consideration, it is expected that research in the field of marine phenolic compounds will become increasingly important and will more and more contribute to the future arsenal of anticancer agents.


Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make  Moreover, antibiotics such as landomycin E, which can induce a rapid generation of H2O2, have been applied to treat bacterial infections [In nature, bacteria live under various environmental stresses such as oxidative stress, which is one of the most common challenges for bacteria living in aerobic conditions. Reactive oxygen species (ROSs), reactive nitrogen species (RNSs), and reactive chlorine species (RCSs) are three major sources of oxidative stress . In bactry chain . Althougry chain , a high ry chain ,5. Diffeproteins . An excein cells . For insy system . Oxidatil growth . Differel growth . Due to fections ,12.2O2 damages, bacteria have evolved various defense pathways. Besides enzymatic scavengers such as catalase, superoxide dismutase, and peroxidase [2O2 damages [kat family , sodA (superoxide dismutase), and ahpCF , as well as other proteins involved in amino acid biosynthesis and cell wall synthesis [metB, metK, and metN), while downregulating intracellular iron levels [To protect themselves from Hroxidase , the str damages ,14. As aynthesis ,16. For ynthesis ,18. HOClynthesis . For insn levels . Interesn levels . TherefoE. coli as the representative bacterium, while H2O2 and HOCl were selected as oxidizing agents. The ASKA collection, which is a complete set of E. coli strains for overexpressing individual E. coli K-12 genes, was screened to identify oxidizing agent resistance genes. Different from most screening studies with the ASKA library which used either pooled strains or plasmids [E. coli K-12 MG1655 strain to avoid potential impacts of the host strain of the ASKA library, the AG1 strain, which has been engineered for high transformation efficiency. In our list of genes, there are several genes which were known for their functions or association with oxidative-stress responses. Further studies are ongoing to explore their specific mechanisms.Previous studies on oxidative stress responses have mostly focused on analyzing gene expression and regulation in cells after exposure to oxidizing agents. Some genes associated with oxidative stress responses might not be expressed under experimental conditions. Thus, we aimed to obtain a comprehensive list of genes whose expression can make cells resistant to oxidizing agents. In this study, we used plasmids ,22, we t2O2 and NaOCl solution, LB medium, M9 medium, chloramphenicol, β-D-1-thiogalactopyranoside (IPTG) were purchased from VWR International . The ASKA library and Keio collection were originally from the Coli Genetic Stock Center at Yale University. The no-insert control of pCA24N was from our previous study [All the chemicals, reagents, bacterial growth media such as Hus study . The pla2O2 or NaOCl. Each plate had three biological replicates. The 96-well plates were sealed with oxygen-permeable membranes . Cell growth was monitored by reading the absorbance at 600 nm with microplate readers at 37 °C, continuously. MIC determination was performed by using varying concentrations of oxidizing agents with 1 mM increments from 1 to 20 mM, or 0.25 mM increments from 0 to 1 mM in LB or M9 minimal medium. The lowest concentration at which bacteria could not grow was recorded as the corresponding MICs.Individual plates of the ASKA library were replicated by inoculating 5 µL stock culture into 200 µL fresh LB medium with 50 µg/mL chloramphenicol in each well of the new plates and incubating them at 37 °C overnight. The absorbance at 600 nm of each well was read by a microplate reader. On the next day, the overnight culture in each well was diluted to OD600 nm = 0.15 with a total volume of 200 µL fresh LB medium, 50 µg/mL chloramphenicol, 0.1 mM IPTG, and corresponding concentrations of HE. coli Database by typing individual gene names into the database [The subcellular localization of proteins was obtained from the EcoCyc database . The idedatabase  and analdatabase . Proteindatabase , in whic2O2 for the no-insert control strain of the ASKA library (the AG1 strain with pCA24N empty vector). MIC was 2 mM. Then, we used a 2-fold MIC (4 mM H2O2) to screen the complete ASKA library for H2O2 resistance genes. Each strain was tested three times. Only those growing in all three replicates were recorded as candidates for further analyses. There were 217 candidate genes after the screening. We listed them into categories based on their biological functions , ttdB (tartrate dehydratase), dmlA , ybiW (formate acetyltransferase), sucC (succinyl-CoA ligase), sucA (2-oxoglutarate dehydrogenase), leuA , and prpE (propionyl-CoA ligase); (2) flagellar biosynthesis and mobility including flhD, flgE, flgK, fliS, motB, cheW, and cheA; (3) protein synthesis including thrA (threonine synthesis), hisB (histidine synthesis), ilvA (threonine synthesis), guaA (GMP synthase), rho (transcription termination), yidC (ATP synthase), and rpmG ; (4) oxidative stress responses including sodB (superoxide dismutase), katE , btuE (Thioredoxin/glutathione peroxidase), katG , tpx (thiol peroxidase), and galU (UTP-glucose-1-phosphate uridylyltransferase).Bioinformatical analyses of these candidate genes were performed . About t2O2, we used a 3-fold MIC for the wild-type strain (6 mM H2O2) to screen those 217 candidate genes. Only 20 strains could grow, including appY, citG, damX, ilvA, katE, katG, kefC, leuA, metN, prpE, rho, sapC, yajQ, ybhC, ydhL, yhjJ, yncC, yncG, yqhA, and yrbB, which were selected for further validation. A summary of the current knowledge about these genes is shown in katE, katG, and yncG, which are oxidative stress response genes, most genes play roles in either membrane transport or gene expression and regulation. More details are described in the Discussion section.To identify genes which mediate stronger resistance to H2O2, there were fewer resistance genes for HOCl. Only 114 strains could grow in 2 mM NaOCl. We divided them into categories based on their biological functions (Similar screenings were performed with HOCl (in the form of NaOCl) as the oxidizing agent. The MIC was 1 mM for the no-insert control strain. Then, we used a 2-fold MIC (2 mM NaOCl) to screen the whole ASKA library. Compared to Hunctions .2O2 results, a higher number of inner membrane-associated proteins appeared to be coded by HOCl-resistance genes. Similar to H2O2-resistance genes, the functions of these candidate genes also cover a wide range of biological processes and focus on stress responses, membrane functions, metabolism, and gene expression. A little higher proportion of genes involved in membrane functions were identified for HOCl-resistance. The protein–protein interaction network only showed two clusters: (1) the respiration chain including hydrogenase  and ubiquinol oxidase (cbdA); (2) flagellar biosynthesis and mobility including fliP, fliS, motB, cheW, and tap, which was found also in H2O2-resistance genes.Bioinformatical analyses were performed on these candidate genes . CompareelaA, exbD, frmB, hyaC, hyaE, ilvA, marC, motB, prpE, rnc, rsuA, sanA, sucC, tap, ybhR, ycbB, yccJ, ycdK, yedQ, yfdY, yhdJ, yoaE, and ytfI. They were selected for further validation. A summary of the current knowledge about these genes is presented in 2O2 results, membrane transport and gene expression are also two important functions of HOCl-resistance genes. Furthermore, cellular redox balances and DNA damage responses are two unique processes of HOCl-resistance genes. More details are described in the Discussion section.To identify genes which mediate stronger resistance to HOCl, we used a 3-fold MIC for the wide-type strain (3 mM NaOCl) to screen the identified 114 candidate genes. Only 23 strains could grow, which expressed 2O2 reported in E. coli physiology studies. The LB medium is a rich medium containing amino acids that react with oxidizing agents to potentially affect cellular responses. Therefore, we used both the LB medium and the M9 minimal medium (0.2% glucose) to determine MICs in the validation experiments. MICs of H2O2 and NaOCl for the no-insert control strain (MG1655 with pCA24N empty vector) were 2 mM for H2O2 and 2 mM for NaOCl. Then, we determined the MICs of H2O2 or NaOCl for each resistance gene overexpressed in MG1655 cells  of the MIC for the control strain. On the other hand, overexpression of only one gene (yoaE) required a slightly higher MIC (8 mM NaOCl) for HOCl resistance.The host strain for the ASKA library is AG1, which was engineered for high transformation efficiency. Therefore, the AG1 strain may have potential inferences for the screening. To validate the effects of selected candidate genes which made cells resistant to concentrations that were 3-fold the MICs for wild-type cells in the first screening, we overexpressed each of them  made E. coli cells more sensitive to H2O2 or NaOCl compared to the wild-type strain, confirming that resistance genes identified from the ASKA library could help cells survive in the presence of oxidizing agents. Compared to gene overexpression testing, in which all the candidates showed highly significant effects (∆katE). This observation indicates that there are other defense mechanisms which can compensate for the inactivated resistance genes.To further confirm the resistance induced by the selected genes in MG1655 overexpression tests, we determined the MICs of H13 cells . As show effects , gene inE. coli cells whose expression can induce resistance to H2O2 or HOCl, this study utilized the ASKA library and further validated candidate genes in common E. coli K-12 strains. In total, ~4200 ORFs from the ASKA library were tested individually. Besides some well-known genes such as katG and katE for oxidative stress responses, this study identified a number of genes (105 genes for H2O2 and 63 genes for HOCl) which had not been shown to associate with oxidative stress responses before. On the other hand, some well-known response genes such as oxyR for H2O2 responses and hypT for HOCl responses were not identified in our study. To confirm this result, we determined the MICs of MG1655 cells expressing oxyR or hypT individually using the same protocol as that for candidate genes. The results showed that the engineered cells had the same MICs as those of WT MG1655 cells. One possible reason is that these response proteins are activated upon oxidation by oxidizing agents [Aiming to identify genes in the whole genome of g agents  and, whe2O2 resistance and 114 candidate genes for HOCl resistance from our genome-wide screening. Only 27 genes were identified in both sets of candidate genes, including agar, agaS, cheW, exbD, fliS, gloA, hyaA, ilvA, leuA, motB, nrfC, prpE, sbmC, slyB, sucC, ubiD, upp, yagM, ybhC, ydeQ, ydfB, ydfD, ydhL, yhaJ, yodA, yoeE, and yrbB. A summary of the current knowledge of these genes is presented in 2O2 or HOCl, indicating distinct mechanisms in oxidative stress responses. Further studies will be implemented to explore the mechanisms of the genes associated with oxidative stress responses.We identified 217 candidate genes for H2O2 resistance  than the one overexpressing katE (12 mM), the ΔkatE strain (MIC 0.5 mM) apeared more sensitive to H2O2 than the ΔkatG strain (MIC 2 mM). One possible explanation for this paradox is that a threshold concentration of H2O2 is required for katG expression [katG gene is regulated by the OxyR regulon [katE gene is permanently induced in aerobic environment [katE can quickly protect cells when katG is inactivated.Unsurprisingly, pression . Moreove regulon , while tironment . Thus, krho encodes the transcription termination factor Rho, which is responsible for the termination of over half of the transcripts [E. coli [2O2 resistance (MIC 12 mM) when overexpressed but is not very sensitive to H2O2 when inactivated. This could be explained by the previous finding that the activity of Rho in bacteria could be altered under stressful conditions [rho could compensate the negative effects brought by a dysfunctional Rho under stress conditions.The gene nscripts  and is r[E. coli . It medinditions . Overexp2O2 stress [appY and mcbR allowed E. coli cells to grow in 10 mM H2O2. AppY was found to function as a transcriptional activator of energy metabolism genes under stressful conditions such as anaerobiosis and phosphate starvation [E. coli strain was more sensitive to H2O2 than the wild-type strain [2O2 responses in avian pathogenic E. coli by downregulating the expression of the stress response genes yciF and yciE [The transcriptional regulator OxyR has been known as the major regulon for responses to H2 stress . In thisarvation . It was e strain , which iand yciE .yncG, a gene for a putative glutathione S-transferase, was also shown to induce obvious H2O2 resistance in our tests. Although one previous study demonstrated that YncG does not exhibit GSH activity when expressed in cell-free systems [In addition to the genes discussed above,  systems , YncG ma systems .ycbB encodes the L,D-transpeptidase with a role in protecting outer membranes during cell envelope stress [yedQ gene encodes a probable inner membrane protein with predicted diguanylate cyclase activity [sanA is implicated in strengthening membrane permeability in stationary-phase stress responses [marC, ybhR, exbD, yfdY, motB, yoaE, hyaC, and tap), which encode proteins in membrane systems. Among them, yfdY was indicated as a participant in biofilm formation, which is a defense mechanism against HOCl in E. coli [Twenty-three genes were identified to mediate stronger HOCl resistance . Among te stress ; The yedactivity . Expressesponses . It is responses . Besides E. coli .sucC in the citric acid cycle; prpE in propionate metabolism) [ilvA in isoleucine biosynthesis) [yhdJ for methylation of genomic DNA; rsuA for pseudouridylation of 16S rRNA) [rnc) [In addition to genes coding for membrane-associated proteins, we also identified genes involved in other biological processes such as metabolism (abolism) , amino anthesis) , DNA and6S rRNA) ,61, and A) [rnc) . One pos2O2, MIC determination tests showed no significant differences among the 23 candidate genes for HOCl. Only yoaE, which encodes a putative transport protein, induced slightly stronger HOCl resistance than other genes. Although there is no previous report indicating the role of YoaE in E. coli stress responses, a recent study demonstrated that the expression of the yoaE gene in Salmonella enterica could be upregulated by CpxR, which plays an important role in repairing bacterial envelope damages [Different from the results for H damages .E. coli ASKA collection and identified 217 candidate genes for H2O2 resistance and 114 candidate genes for HOCl resistance. Among them, 105 genes for H2O2 and 63 genes for HOCl were not shown to associate with oxidative stress responses before. Further studies are necessary to validate the genes here identified, which appear as promising new candidates for oxidative stress studies. Furthermore, because the disinfecting mechanisms of many antibiotics are related to oxidative stress, this study is expected to facilitate antibiotic development.In this study, we performed genome-wide screening of the


Sapap3 knockout mouse (Sapap3-KO), the current reference model to study such repetitive behaviours, presents both associative as well as sensorimotor cortico-striatal dysfunctions. Previous findings point to deficits in both macro-, as well as micro-circuitry, both of which can be affected by neuronal structural changes. However, to date, structural connectivity has not been analysed. Hence, in the present study, we conducted a comprehensive structural characterisation of both associative and sensorimotor striatum as well as major cortical areas connecting onto these regions. Besides a thorough immunofluorescence study on oligodendrocytes, we applied AxonDeepSeg, an open source software, to automatically segment and characterise myelin thickness and axon area. We found that axon calibre, the main contributor to changes in conduction speed, is specifically reduced in the associative striatum of the Sapap3-KO mouse; myelination per se seems unaffected in associative and sensorimotor cortico-striatal circuits.Pathological repetitive behaviours are a common feature of various neuropsychiatric disorders, including compulsions in obsessive–compulsive disorder or tics in Gilles de la Tourette syndrome. Clinical research suggests that compulsive-like symptoms are related to associative cortico-striatal dysfunctions, and tic-like symptoms to sensorimotor cortico-striatal dysfunctions. The  Repetitive, ritualistic actions are a core feature of animal behaviour, including humans, which allow for efficient learning and automatisation of behaviour ,2,3. HowCortico-striatal circuits are the principal anatomical region that underlies the development of RB ,5. TheseSapap3-KO mouse [Sapap3), which is highly expressed in the cortex and striatum. The Sapap3-KO mouse model has been mainly reported and studied for its disproportionate and injurious levels of self-grooming, its increased anxiety, and its behavioural rescue with fluoxetine, a first-line treatment for OCD patients [Sapap3-KO mice but also in other mouse models of pathological RBs [Sapap3-KO mice have been observed and shown to specifically occur in cortico-striatal but not thalamostriatal pathways, which also heavily project to the striatum [Rodent RBs have been extensively studied in translational psychiatric approaches, with a particular focus on excessive self-grooming as a behavioural marker for compulsive behaviours ,25,26. SKO mouse ,28,29,30patients . Consistical RBs ,32. Laststriatum ,33. Morestriatum . In rodestriatum ,35.Efficient connectivity and conductivity heavily depend on neuronal structure and integrity. On a population level, cell density and ratio can affect the integration and modulation of information; for example, in the context of RBs, a reduced number of PV interneurons has been shown to fail the regulation of striatal activity ,28. On aSapap3-KO mouse model. Hereby, we covered cortico-striatal candidate regions that have previously been characterised as affected both in human patients with tics or compulsions as well as in according animal models. We selected the associative (AS) and the sensorimotor (SMS) regions of the striatum and their respective major cortical projection areas, i.e., lOFC and primary/secondary motor cortex (M1/M2). In these candidate regions, we morphologically characterised myelinated axons in the Sapap3-KO mouse model, measuring both axon area and myelin thickness as the determinant parameters of axon conductivity. To achieve this, we took advantage of the open source software AxonDeepSeg [Different neuroimaging studies have found structural connectivity deficits in OCD patients ,48. HowenDeepSeg , which aAnimals. Adult Sapap3-KO and wild-type, age-matched controls (Sapap3-WT)  of C57BL/6J background were generated in heterozygous breeding trios. Animals were housed at the animal facilities of the Paris Brain Institute in Tecniplast, ventilated polycarbonate cages under positive pressure with hardwood bedding and provided with ad libitum food and water. The temperature was maintained at 21–23 °C and the relative humidity at 55 ± 10% with a 12 h light/dark cycle . All experiments were approved by the French Ministry of Research under the agreement number (APAFIS) #1418-2015120217347265. Founders for the Sapap3-KO colony were kindly provided by Dr. Ann M Graybiel and Dr. G. Feng, MIT, Cambridge, USA. Genotyping was performed as in the original publication [lication .Selection of regions of interest. In order to determine the delimitations of the associative (AS) and sensorimotor striatum (SMS), we analysed anterograde projection patterns documented for the lateral orbitofrontal cortex (lOFC) and primary and secondary motor cortices (M1/M2) in the Allen Mouse Brain Connectivity Atlas [ty Atlas . Bregma ty Atlas . Bregma Electron microscopy. Animals (n = 4 per genotype) were deeply anaesthetized with pentobarbital (200 mg/kg) and transcardially perfused with fresh 5% glutaraldehyde (GA)/5 mM CaCl2. Brains were collected, post-fixed overnight in 5% GA at 4°, then rinsed and kept overnight in 0.12 M phosphate-buffered solution (PB) (pH = 7.4).Brains were cut in 200 µm slices on the vibratome. Slices comprising the regions of interest were selected, and their left-hemispheric part punched under a stereomicroscope using a biopsy punch with a plunger 1 mm in diameter . Punched samples were incubated with 4% osmium tetroxide in 100 mL sodium cacodylate buffer for one hour at RT and then carefully rinsed with sodium cacodylate buffer, three times. Next, samples were immersed in 5% uranyl acetate in 100 mL sodium cacodylate buffer for one hour and carefully rinsed in sodium cacodylate buffer, three times. The punches were then dehydrated in increasing concentrations of ethanol  for five minutes, and finally twice in 100% acetone for ten minutes. In order to prepare the samples for epoxy resin embedding, they were first immersed overnight, at 4 °C, in a solution of 50:50 acetone and epoxy resin. Next, samples were transferred into embedding moulds and immersed in pure resin for two hours. Finally, samples were left to polymerise at 60 °C for 48 h in a new dose of epoxy resin. Samples were then cut into 70 nm slices on an ultramicrotome, placed onto a Transmission Electron Microscope (TEM) grid and immersed in lead citrate for fifteen minutes. Samples were imaged at 100.0 k× for quantifications of axon calibre, myelin thickness and g-ratio, and at 6.2 k×, for the quantification of the density of myelinated axons in the Transmission Electron Microscope . In order to be specific on the quantification of local striatal cells, myelinated axons within the fibre bundles that cross the striatum, i.e., axons corresponding to the internal capsule, were excluded from analysis. Electron microscopy analysis.Myelin thickness and axon area/calibre measurements. We quantified approximately 150 myelinated axons per region and animal using AxonDeepSeg [Quantification of myelinated axons. For each animal, we analysed 50 images per region of interest  and manually assessed the number of myelinated axons in each image, using Fiji, ImageJ2 [nDeepSeg . In orde ImageJ2 ,53. In oImmunohistochemistry. Animals (n = 12 per genotype) were deeply anaesthetized with pentobarbital (200 mg/kg) and transcardially perfused with 2% PFA in 1X PBS. Fixed brains were dissected and post-fixed overnight in 2% PFA at 4 °C. Samples were immersed in 15% sucrose in 1X PBS for 24 h and subsequently immersed in 30% sucrose in 1X PBS for 48 h. Samples were then embedded in Tissue-Tek O.C.T. compound and frozen on dry ice. Brains were sliced coronally in 12 µm sections on a cryostat, transferred onto a coated glass slide , and stored at −80 °C. For immunohistochemical labelling, we performed heat-inducing antigen retrieval with an unmasking solution and, after cooling down at RT for twenty minutes, we rinsed the samples with 1X PBS three times for five minutes. Samples were then blocked in 4% BSA/0.1% Triton X-100 for 1 h. The primary antibodies against Oligodendrocyte transcription factor 2 (Olig2) mouse IgG2a, a nuclear marker for all cells of the oligodendroglial lineage, and against adenomatous polyposis coli clone CC1 (CC1) mouse IgG2b, a cytosolic marker specific for mature oligodendrocytes, were diluted in 4% BSA/0.1% Triton X-100 with a concentration of 1/500 and 1/100, respectively. The brain sections were incubated in this primary antibody solution overnight at 4°C. Samples were rinsed with 1X PBS and incubated in the dark with the secondary antibodies rat anti-Mouse IgG2a 488 and goat anti-Mouse IgG2b 555 Alexa in a 1/2000 dilution for 1 h at RT. The slides were rinsed under agitation in the dark, mounted in Mowiol mounting medium, coverslipped, and left overnight to dry at RT; hereby, the stained sections were protected from light. Fluorescent samples were imaged in a Zeiss Axio Observer 7 at 20× magnification. The ROIs were chosen visually by an experienced user and corresponded to one field of view (665.60 µm × 665.60 µm). We imaged both right and left hemispheres of two slices per region and animal, separated by 72 µm, summing up to a total of four images per region for each mouse.Immunohistochemistry analysis. For the quantification of oligodendrocyte density, we used Fiji, ImageJ2 [ ImageJ2 ,53, and Data analysis and statistics. All data and statistical analysis were performed using Matlab R2017b. Given the small sample size, we exclusively performed non-parametric Mann–Whitney-U tests. We furthermore used two open source software packages available on GitHub for the visualisation of our data: raincloud plots [ud plots  as well ud plots . Clusterud plots . Sapap3-KO mouse model of tic- and compulsive-like behaviours, we characterised axon calibre and myelin thickness, two parameters that crucially contribute to axonal conductivity . However, there was no difference in axon area in the sensorimotor striatum    or the sensorimotor striatum  as a cell marker. For the pool of mature oligodendrocytes, i.e., those cells which actively perform myelination, we applied the criterion of a positive double-immunolabelling for both Olig2 as well as adenomatous polyposis coli clone CC1 (CC1)  , and the other cluster contained 15% and 18% of axons (Cluster 2), respectively . However, axons of Sapap3-KO mice assigned to Cluster 2 were significantly smaller than those of their wild-type, age-matched controls   b.Sapap3-KO mice is specific to a small subpopulation of axons characterised by larger axons, here assigned to Cluster 2. Thus, reduced axon calibre is not a general feature of all myelinated axons in the associative striatum.These results suggest that the observed reduction in axon calibre in the associative striatum of Sapap3-KO mice? The latter possibility would be reflected in a decreased overall number of myelinated axons in the associative striatum of Sapap3-KO mice. Hence, we quantified and averaged the density of myelinated axons in 50 transmission electron microscopy images per animal in the associative striatum, again comparing Sapap3-KO to age-matched, wild-type controls. Attending to the fact that the striatum is traversed by innumerous fibres of the internal capsule, which largely project outside of the striatum, we visually excluded the bundles of the internal capsule  and primary motor/M2 cortical projections to the dorsolateral striatum (DLS) [Axon calibre is the main regulator of conduction speed ,60. In iysiology ,31,33,64ysiology ,19,65,66patients ,21,22,23se model . In a mose model ,17,18,19se model . Indeed,um (DLS) ,68. One Currently, a knowledge gap on myelination of specific cell types, including in the striatum, hampers the deduction of the cell identity from the size of the axon calibre of cluster number 2. Indeed, the present-day state of knowledge does not allow for the conclusion as to whether the observed structural striatal deviation arises from afferents such as cortico-striatal or thalamostriatal connections or whether the reduced axon calibre represents an inherent striatal microcircuitry dysfunction. For example, to date, there is no direct evidence whether cortico-striatal pathways are actually myelinated ,70,71 orIn the first scenario, reduced axon calibre in the associative striatum could be explained as a macrocircuitry dysfunction of cortico-striatal loops. Concretely, the observed reduction in axon calibre in a small cluster of striatal axons might arise from a population of associative cortico-striatal projection neurons. Given that axonal calibre is positively related to synaptic strength ,39, the Sapap3-KO mouse [Sapap3-KO mice [In a second scenario, Cluster 2 neurons might correspond to PV-positive interneurons. These interneurons are crucially implicated in regulating striatal activity through a powerful mechanism of fast-forward inhibition ,85, and KO mouse . ConsequKO mouse . A reducKO mouse  and withKO mouse ,64. AlteKO mouse ,83 as weKO mouse ,86, incl-KO mice ,64. PV i-KO mice ,87.In a third scenario, striatal interneurons expressing choline acetyltransferase (ChAT) also need to be considered as a candidate for Cluster 2 neurons. ChAT-positive interneurons, though sparse, highly contribute to the modulation of MSNs . ChAT-poSapap3-KO mouse, here assessed for the first time using a structural, deep learning-based approach. This study thus contributes a novel and complementary aspect to the discussion of deviations in macro- and microcircuitry in the context of RBs in clinical as well as fundamental studies. Future structural neuroanatomy studies are required to define the affected striatal cell type within the pool of currently prominent candidates, such as cortico-striatal projections or PV- or ChAT-positive striatal interneurons.In summary, our study corroborates previous findings of deficits specifically within the associative striatum of the Sapap3-KO mouse. This axon caliber reduction was not found in cortical areas projecting onto the associative striatum and neither in sensorimotor cortico-striatal regions. The difference in axon calibre arises from a smaller cluster of cells, the identity of which will need to be determined in neuroanatomic follow-up studies. Potential cell-type specific candidates for this cluster, in the context of pathological RB, include cortico-striatal projections, PV- or ChAT-positive striatal interneurons. Our results further corroborate, from a structural point of view, neurophysiological deficits previously observed in associative cortico-striatal circuits of the Sapap3-KO mouse, which are known to be implicated in compulsive-like behaviours. In our study, we found that axon calibre in the associative striatum is reduced in the


The importance of innate immune cells to sense and respond to their physical environment is becoming increasingly recognized. Innate immune cells (e.g. macrophages and neutrophils) are able to receive mechanical signals through several mechanisms. In this review, we discuss the role of mechanosensitive ion channels, such as Piezo1 and transient receptor potential vanilloid 4 (TRPV4), and cell adhesion molecules, such as integrins, selectins, and cadherins in biology and human disease. Furthermore, we explain that these mechanical stimuli activate intracellular signaling pathways, such as MAPK , YAP/TAZ, EDN1, NF-kB, and HIF-1α, to induce protein conformation changes and modulate gene expression to drive cellular function. Understanding the mechanisms by which immune cells interpret mechanosensitive information presents potential targets to treat human disease. Important areas of future study in this area include autoimmune, allergic, infectious, and malignant conditions. Path (1 kPa) .in vitro environment, macrophages can develop into the different classically described macrophage phenotypes, pro-inflammatory (“M1-like”) or pro-healing (“M2-like”), however direct extrapolation of the in vitro macrophage phenotypes to in vivo function is problematic , and cell adhesion molecules (CAMs), such as integrins, selectins, and cadherins, have been shown to play a role in mechanotransduction in innate immune cells , 14. In 2+, magnesium Mg2+, potassium K+, and sodium Na2+) along their electrochemical gradients. Ca2+ plays a critical role in innate immune cell signaling. For example, in macrophages, Ca2+ has been shown to be an important second messenger in Tumor Necrosis Factor-α (TNF-α) secretion and phagocytosis  or indirectly  . In addimembrane . The mecmembrane . In the membrane . During id cells . There iilaments . Tractioa Piezo1 .2+ influx leading to activator protein-1 (AP-1) activation, production of endothelin-1 (EDN1), and stabilization of hypoxia inducible factor 1α (HIF1α) to modulate important genes that lead to the production of pro-inflammatory mediators, such as IL-6, TNF-α, chemokine ligand 2 (CXCL2), and prostaglandin E2 , the Piezo1 signal integrates with that of chemical pro-inflammatory signals to regulate macrophage function. In response to cyclical hydrostatic pressure in murine bone marrow derived macrophages (BMDM), Piezo1 mediates Caandin E2 . Stifferandin E2 . BMDM cufunction .2+ influx via interaction with Piezo1 in response to LPS (LysM) have increased signal transducer and activator of transcription 6 (STAT6) activation, and decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation, leading to a pro-healing phenotype after stimulation with IFN-γ/LPS . The CLP model, CD11b+ myeloid cells from Piezo1ΔLysM mice had decreased pro-inflammatory cytokines, enhanced peritoneal bacterial clearance, and less sepsis-related death than their Piezo1fl/fl littermates  has been shown to mediate Cae to LPS . MacrophFN-γ/LPS . It has vo model . Howevertermates . These c2+ influx via Piezo1  family of ion channels has many diverse roles, with TRPV4 being the most studied mechanically-gated channel for its role in innate immune cell function . TRPV4 i signals , 38. TRP signals . The TRP signals . The exa signals . Similar+ influx . Additio+ influx , 43. Con+ influx . Indirec+ influx . These d+ influx . Activatfunction .2+ is necessary for proper phagosome maturation. M. tuberculosis can inhibit TRPV4 expression in macrophages, reducing intracellular Ca2+ resulting in dysfunctional delivery of mycobacteria to phago-lysosomal components and impaired acidification of phagosomes, which are necessary for effective infection control   . Interesype mice . Improveype mice . M. tubeia TRPV4 . Furtherg matrix . This isin vitro and in vivo models  had lower total YAP protein, and increased YAP phosphorylation, which leads to its degradation in the cytoplasm  in some cell types . YAP is ytoplasm . The polytoplasm . Therefoytoplasm , 51. In P. aeruginosa pneumonia model is associated with an increase in pro-inflammatory cytokines   and downregulation of pro-inflammatory cytokines, while data from other groups has shown an opposing function of Piezo1 where activation of Piezo1 in a murine , IL-1β) , 46, 49.2+ that was insufficient to result in the mitochondrial dysfunction and necrosis associated with pancreatitis. Only after activation of TRPV4 by Phospholipase A2 (PLA2) was there a sustained increase in Ca2+ which could result in cell death  through TRPV4, while Piezo channels sense excessive, damaging levels of mechanical strain (18% at 0.5 Hz for 8 hours) . In pancll death . Activatequences . These dAlong with ion channels, other molecular families play important roles in mechanotransduction and mechano-responses in immune cells, such as the cytoskeletal proteins, actin and myosin, focal adhesions, selectins, and integrins. Due to the focused nature of this review, we direct you to more comprehensive reviews in this area , 54–56. Integrins are transmembrane, heterodimeric proteins, with α and β subunits, which connect the extracellular matrix with the cytoskeleton and mediate intracellular signaling , 60. TheGiven the known interaction between TRPV4, integrins, and toll-like receptors (TLRs), it is worthy to mention the importance of epithelial/endothelial barrier function in the host response to infection. For example, integrin αvβ3 on epithelial/endothelial cells has been shown to be integral to the containment of herpes simplex virus (HSV). Integrin αvβ3 directs HSV to the acidic endosome pathway by re-localizing nectin-1, a cellular protein which mediates the entry of HSV . Integrivia cytoskeletal rearrangement, is essential to immune cell function. Upon activation of TLRs and G-protein coupled receptors (GPCR) by chemokines and LPS, phagocytes exit their resting state and undergo actin remodeling to become primed for interaction with phagocytic targets  leads to increased permeability, rapid accumulation of inflammatory cell infiltrates, and pulmonary edema  and infectious (e.g. pneumonia) inducers of lung injury , 70. In ry edema , 72. Thery edema . In a mutivation .P. aeruginosa  . The safOngoing study of mechanobiology in innate immune cells offers further understanding of human disease and opportunities for potential therapeutics. Future work is needed to target mechanosensitive channels or adhesion molecules to control intracellular signal transduction and modulate disease, with particular emphasis on examining Piezo1 and TRPV4 interactions during inflammation. While clinical trials in humans have thus far failed to show efficacy, opportunities to target specific cell types or interacting partners may yield greater efficacy. Future therapeutic targets may be identified from further understanding of downstream signaling pathways, with important areas of future study including autoimmune, allergic, infectious, and malignant conditions. Research has only begun to elucidate the mechanisms involved in innate immune cell mechanotransduction and their potential involvement in a variety of human diseases leads to vast opportunities for ongoing study.EMO, MAO, and RGS contributed to the conception and writing of the manuscript. AP, BDS, and LMG provided critical revisions to the article. All authors contributed to the article and approved the submitted version.This work was supported by NIH grants (HL132079) to BDS, (R01HL-133721 and R01HL-158746) to MAO, and (K08HL-133380 and R01HL-155064) to RGS, the Ann Theodore Award to MAO and RGS, and the SMARRT T32 which is funded by the National Institutes of Heart, Lung, and Blood grant, T32HL-155005. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. 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.


N = 1354 women; Mage= 31.89 years, SD = 11.14) filled in the Contour Drawing Rating Scale, the Emotional Overeating Questionnaire, the Eating Motivation Survey, the Mindful Eating Questionnaire, and a COVID-19-related stress measure and sociodemographic survey. The cluster analysis technique revealed four distinct clusters: (a) Cluster 1 (N = 314): healthy body weight, no COVID-related stress, and low body dissatisfaction (M = 1.19); (b) Cluster 2 (N = 131): overweight, no COVID-related stress, and high body dissatisfaction (M = 2.41); (c) Cluster 3 (N = 597): healthy body weight, COVID-related stress, and low body dissatisfaction (M = 1.27); (d) Cluster 4 (N = 312): overweight, COVID-related stress, and high body dissatisfaction (M = 2.84). Generally, our outcomes partially support our hypothesis, as higher levels of some types of disordered eating were observed in women who were overweight with COVID-related stress and high body dissatisfaction (Cluster 4) as compared with women with healthy body weight, no COVID-related stress, and with low levels of body dissatisfaction (Cluster 1). Our results indicate that both body weight status, as well as COVID-19-related stress and body dissatisfaction, may contribute to the intensity of disordered eating. During future epidemic-related quarantines, this may be an argument in favor of organizing support regarding emotional functioning, body image, and eating behaviors, particularly for the most vulnerable groups—including overweight and obese women.We hypothesized that women who are overweight, experiencing COVID-19-related stress, and with high body dissatisfaction would have significantly greater disordered eating than those of healthy weight, without stress, and with low body dissatisfaction. Participants ( There have been a lot of data published recently on the impact of the coronavirus pandemic on health ,2,3. TheThe results so far indicate that for many people, COVID-19 was (or still is) a strong source of stress ,22,24, aReferring to the category of disordered eating, it should be mentioned that this is a very broad category including, among others, emotional (over)eating and a low level of mindful eating. Emotional (over)eating can be defined as the tendency to regulate emotional states by eating (too much) food ,34,35. IWhen writing about eating behaviors, it should also be mentioned that we can evaluate them by examining the motivation underlying particular food choices . ReferriTo sum up, although knowledge about COVID-19-related stress, body weight, and eating behaviors/disorders has increased in diverse populations ,13,22,25Our cross-sectional study was approved by the University Ethics Committee (no. 2021/2/2E/2) and conducted in accordance with the international standards of research ethics . From January to March 2021, Polish women were recruited via an online advertisement  and flyers . After contacting the researchers, all women were made aware that participation in this study was anonymous and voluntary. Next, they were asked to read informed consent to participate in the study. After becoming acquainted with all the information, if they expressed their willingness to participate in the study, they signed this informed consent online. Next, women completed an online survey (populated on Google Forms), which was accessed via a link sent to participants. Participants were not remunerated.M = 31.89, SD = 11.14).One thousand five hundred and ninety-four people volunteered for the study. Two hundred and forty participants had significant missing data gaps or were outliers . Finally, our sample included 1354 Polish women who ranged in age from 18 to 72 years .To assess how often people eat for health reasons, to control body weight, and to regulate emotion, participants completed the Eating Motivation Survey . The 45-recognition = 0.70, αawareness = 0.82).To assess mindful eating, participants completed the Mindful Eating Questionnaire . The 20-To assess COVID-19-related stress, participants were asked “Is the current epidemiological situation related to COVID-19 stressful for you?” and they could choose to respond: “No” (0) or “Yes” (1) (a dummy-coded variable).2) was calculated based on self-reported weight and height. Based on the World Health Organization guidelines [2, and (b) overweight (pre-obesity and obesity): BMI ≥ 25 kg/m2.Participants were asked about gender, weight, height, age, sexual orientation, and ethnicity. Body mass index .A two-step cluster analysis (with Schwarz’s Bayesian criterion) was chosen to identify clusters based on BMI, COVID-related stress, and body dissatisfaction. This analysis is appropriate for samples larger than 200 and for both categorical and continuous variables . One-wayM = 67.02, SD = 13.04), (b) height from 146 to 188 cm , and (c) body mass index (BMI) from 18.50 to 47.96 kg/m2 (M = 24.23 SD = 4.40). Nine hundred and eleven women (67.28%) had normal body weight, and 443 women (32.72%) were overweight.The analysis included 1354 Polish women who ranged in: (a) body weight from 45 to 130 kg (N = 314): healthy body weight, no COVID-related stress, and low body dissatisfaction (M = 1.19); (b) Cluster 2 (N = 131): overweight, no COVID-related stress, and high body dissatisfaction (M = 2.41); (c) Cluster 3 (N = 597): healthy body weight, COVID-related stress, and low body dissatisfaction (M = 1.27); (d) Cluster 4 (N = 312): overweight, COVID-related stress, and high body dissatisfaction (M = 2.84).The cluster analysis technique revealed four distinct clusters : (a) CluOne-way analysis of variance (ANOVA) showed the significant effect of clusters on emotional overeating, two eating motives , and mindful eating . p = 0.076.Referring to the most important findings, our results indicated that Cluster 4  had significantly greater disordered eating than Clusters 1  and 3  and these significant differences were found with regard to emotional overeating, health motive, and affecting regulation motive of eating, and to one aspect of mindful eating—recognition. In turn, for the second aspect of mindful eating—awareness—the differences between Cluster 4 and Cluster 3 were at Cluster 4 differed significantly from Cluster 2  in terms of emotional overeating and affect regulation motive for eating. Moreover, Cluster 1 also had a lower level of emotional overeating and affect regulation motive of eating than Cluster 3, and Cluster 3 had significantly higher health motive for eating and awareness than Cluster 2. Interestingly, in relation to weight control motive for eating, we did not find any significant differences between Clusters 1, 2, 3, and 4, and Clusters 1 and 2 did not differ in terms of any of the analyzed variables.Generally, our outcomes partially support our hypothesis, as higher levels of some types of disordered eating were observed in women who were overweight, with COVID-related stress and high body dissatisfaction as compared with women with healthy body weight, no COVID-related stress, and with low levels of body dissatisfaction. Our results indicate that body weight status, as well as COVID-19-related stress and body dissatisfaction, may contribute to the intensity of disordered eating.Our study examined patterns of weight status, COVID-19-related stress, and body dissatisfaction in a sample of Polish women during the COVID-19 pandemic. Four clusters were discovered. By analyzing the most important findings, significant differences (in terms of most of the assessed behaviors and eating motives) were found between women who were overweight who were simultaneously stressed by COVID-19 and had a high level of body dissatisfaction (Cluster 4), and women with a healthy body weight, who experienced no stress from COVID-19 and had a low level of body dissatisfaction (Cluster 1). Women from Cluster 4 (compared to Cluster 1) significantly more often declared that their motive for undertaking eating behavior during COVID-19 was the regulation of emotions . This tThe importance of COVID-19-related stress for more disordered eating in a sample of Polish women may also be indicated by some of our other results. This is evidenced by significantly higher results in affect regulation motive of eating and emotional overeating in Cluster 4 compared to Cluster 2 , and similar differences between Clusters 1  and 3 . Thus, both when comparing clusters composed of overweight women and women with a healthy body weight, emotional (over)eating is stronger in groups that are stressed by COVID-19. This may mean that although this tendency among women with a healthy body weight is not as strong as among those who are overweight ,45, it ip < 0.01 and “awareness” p = 0.076). This could mean that even though both groups (Clusters 3 and 4) are stressed by COVID-19, more disordered eating patterns have emerged in women who are overweight and have high levels of body dissatisfaction. Furthermore, even when we compared Clusters 2 and 3 (differing in their experience of COVID-19-related stress), it turned out that in the group with high body dissatisfaction, who were overweight and had no COVID-related stress (Cluster 2), we observed a more disturbed eating  compared to the group with low body dissatisfaction, healthy body weight, and COVID-related stress (Cluster 3).When planning some interventions, there are other important aspects to consider—body image and body weight. In the context of our research, it turns out that if women did not accept their silhouettes (and the related body weight), then their eating behaviors were more disordered. This is evidenced by the differences described above between Cluster 4 and Cluster 1, as well as the differences between Clusters 4 and 3 and between Clusters 2 and 3. The differences between Clusters 3 and 4 show that women who are overweight, with COVID-19-related stress and high body dissatisfaction  were more likely to eat under the influence of emotions and physical pain, less often likely to eat taking into account health and less often likely to eat mindfully  managing stress, emotions, and physical pain in a more adaptive way than by (over)eating; (b) learning to accept the body, taking into account negative emotional states such as stress and anxiety, which could be aggravated by COVID-19 and its limitations; (c) learning more adaptive forms of managing one’s own body weight than through disordered eating; (d) developing and consolidating a diet model in which one of the primary eating motives is health.Our findings are important because understanding how COVID-19 experiences and people’s individual predisposition (associated with body shape and weight) differentially relate to eating behaviors has direct implications for tailoring prevention efforts and treatment. Our study shows that COVID-19-related lockdowns and related consequences  may affect eating behaviors. During future epidemic-related quarantines, this may be an argument in favor of organizing support regarding emotional functioning, body image, and eating behaviors, particularly for the most vulnerable groups—including overweight and obese women. Taking such actions seems to be important in the light of reports which show that disordered eating can intensify over time and lead to the development of a full syndrome of eating disorders . And, asOur study also had some limitations: (a) study design: a cross-sectional study; (b) participants: only female volunteers; (c) measures: only self-reported data, one-item COVID-related stress measurement, Cronbach’s alpha for “recognition” = 0.70, and, with regard to some measures, there is no validation on the Polish sample. It is therefore necessary to plan further studies that take into account these limitations (e.g., longitudinal studies, women and men, and comparison of their functioning, objective measurement of body weight and height, and validated instruments for measuring of stress  and othe


Previous studies have highlighted the important role of vitamin D and calcium pathway genes in immune modulation, cell differentiation and proliferation, and inflammation regulation, all closely implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).CYP2R1, CYP24A1, and CYP27B1) are associated with the risk of NAFLD.This study aims to investigate whether 11 candidate single nucleotide polymorphisms (SNPs) in vitamin D and calcium pathway genes , rs2248359 variant genotypes , and CYP27B1 rs4646536 variant genotypes  were associated with an elevated risk of NAFLD. In combined effects analysis, we found that NAFLD risk significantly increased among patients carrying more rs2296241-A, rs2248359-T, and rs4646536-T alleles (ptrend = 0.049). Multivariate stepwise analysis indicated that age, visceral obesity, ALT, γ-GT, hypertriglyceridemia, hypertension, low HDL-C, hyperglycemia, and unfavorable alleles were independent predictors of NAFLD . The area under the receiver operating characteristic curve was 0.789 for all the above factors.After adjusting for age, gender, and metabolic measures, we identified that CYP24A1  and CYP27B1 (rs4646536) were associated with NAFLD risk in Chinese Han population, which might provide new insight into NAFLD pathogenesis and tools for screening high-risk population.The polymorphisms of vitamin family genes  Non-alcoholic fatty liver disease (NAFLD) has emerged as a major chronic liver disease, afflicting more than one-quarter of adults worldwide . PreviouThe mechanisms underlying NAFLD are far from clear . PreviouVitamin D mediates immune-inflammatory and metabolic processes . Numerou2D3 requires two sets of hydroxylation. Vitamin D hydroxylases play a prominent role in this process  and cytochrome P450 family 27 subfamily A member 1 (CYP27A1) genes. 25(OH)D3 is then hydroxylated in the kidney and synthesized into 1,25(OH)2D3 in other organs, which is catalyzed by the 1α-hydroxylase encoded by cytochrome P450 family 27 subfamily B member 1 (CYP27B1) gene. On the other hand, 1,25(OH)2D3 can be decomposed by 1,25-hydroxyvitamin-D3-24-hydroxylase encoded by cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene, maintaining the balance of vitamin D metabolism in vivo. Several genetic studies have reported that common single nucleotide polymorphisms (SNPs) in vitamin D pathway genes are associated with low-level serum 25(OH)D, including vitamin D receptor (VDR), vitamin D-binding protein (VDBP), CYP2R1, CYP24A1, and CYP27B1  polymorphisms on NAFLD susceptibility.The activation of 1,25(OH) process . Vitamin CYP27B1 . Genome- CYP27B1 . Moreove CYP27B1 . TherefoCYP2R1, CYP24A1, and CYP27B1) with the risk of NAFLD among Chinese Han population. Our findings may deepen our understanding of NAFLD and provide strategies for the screening, prevention, and individualized treatment of NAFLD patients.The aim of this study was to investigate the correlation of candidate SNPs in vitamin D and calcium pathway genes .The participants of this case-control study were recruited from a community  from July to September 2018. NAFLD was diagnosed based on 8 update . DiagnosThose patients diagnosed with NAFLD were grouped. The non-NAFLD controls were collected from the same community during the study period and randomly assigned to the control group. The constituent ratios of gender and age between case and control groups were considered similar, according to the result of frequency matching. Included were patients (1) signing informed consent; (2) aged between 18 and 60 years. Excluded were those: (1) taking antihypertensive, antidiabetic, lipid-lowering, or hypouricemic agents within 24-h before physical examination; (2) with infection, acute or chronic gastrointestinal diseases, autoimmune diseases, or malignant tumors; (3) with history of other viral hepatitis, alcoholic liver disease, or primary liver cancer; (4) having excessive alcohol consumption ; (5) receiving a liver transplant within the previous year or had complications of advanced liver disease ; (6) with drug-induced fatty hepatitis; or (7) with a history of psychiatric disorders.After literature review, we assumed that the frequency of gene mutation in the general population was 10–30%, odds ratio (OR) was 1.5, two-sided test α was 0.05, and power of test (1-β) was 80%. Sample size was estimated by NCSS-PASS 11.0 software . The sample size of 1,114 NAFLD cases and 1,909 controls in this study was large enough to guarantee the production of reliable results.The study was performed in accordance with the World Medical Association Declaration of Helsinki on ethical principles in medical research involving human subjects and was approved by the Institutional Ethics Review Committee of Nanjing Medical University . Written informed consent was obtained before blood test and genetic analysis.via the antecubital vein in the morning. The serum and blood cells in each blood sample were separated and frozen at −80°C within 2 h, until further serological tests and genotyping assays.The demographic and clinical characteristics of all participants were collected from self-designed questionnaires and electronic medical records. All participants underwent abdominal ultrasound and blood biochemical tests. Five-milliliter ethylenediaminetetraacetic acid (EDTA) anticoagulant venous blood was collected from the fasting participants CYP2R1, CYP24A1, and CYP27B1). The selection processes were as follows: (1) downloading the genotype databases of CYP2R1, CYP24A1, and CYP27B1 in Han Chinese in Beijing (CHB) from the 1,000 Genomes Project database1; (2) importing the genotype databases into the Haploview software . Parameters: Hardy-Weinberg (H-W) p-Value cutoff = 0.05; minor allele frequency (MAF) = 0.05; r2 ≥ 0.8. At this point, 41 tagging SNPs (tagSNPs) were generated; (3) searching for related studies, including (I) relevant research in the Chinese population; (II) studies on NAFLD, hypertension, type 2 diabetes, hyperlipidemia, metabolic syndrome, etc. Definitively, 11 candidate SNPs  were selected for further genotyping.We searched for target SNPs with potential biological function in three candidate genes  for isolating genomic DNA from EDTA-anticoagulated blood samples. Genotyping assay was performed with a TaqMan allelic discrimination assay on the Light Cycler 480 II Real-Time PCR System . Detailed information on primers and probes was shown in 2; (2) viewing the scores of all genetic variation sites in the Regulome DB database,3 and SNP scoring 1–3 might act in transcriptional regulation; (3) checking whether the genetic variation sites were located on the histone modification peak through the UCSC Genome Browser database4; and (4) using the RNA fold Web Server5 to predict the effect of genetic variation of positive SNP on the secondary structure.To further explore the potential functions of gene variants, we performed bioinformatics analysis using some online database as follows: (1) determining the genetic variation-specific location sites on the chromosome and transcriptional regulation information with NCBI dbSNP6 Distributions of demographic and clinical characteristics among case and control groups were compared by the Chi-square (χ2) test , Student’s t-test, or Mann-Whitney U-test (for continuous variables). Logistic regression analysis with adjustment for gender, age, aspartate aminotransferase (AST), ALT, γ-GT, triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) was used to calculate odds ratio (OR) and 95% confidence interval (95% CI) for assessing the associations of genotypes with the risk of NAFLD. Each SNP was analyzed using codominant model , dominant model (mutant homozygous type + heterozygous type vs. wild type), recessive model (mutant homozygous type vs. heterozygous type + wild type), and additive models (mutant homozygous type vs. heterozygous type vs. wild type). False discovery rate (FDR) corrections were used for multiple comparisons, considering the pFDR-value ≤ 0.25 as modest confidence that the correlation represented a positive result  was analyzed using the Cochran-Armitage trend test. Subgroup analysis was performed for positive SNPs, and Q-test was performed to calculate the heterogeneity between subgroups. Multivariate stepwise logistic regression analysis was used to determine the independent predictive factors for NAFLD. A receiver-operating characteristic curve (ROC) was used to represent the risk prediction model for NAFLD, with the area under the receiver operating characteristic curve (AUROC) indicating its predictive power. A two-tailed test with a p-value < 0.05 was regarded as statistically significant in all analyses.All data analyses were performed using IBM SPSS Statistics for Windows 23.0  and R software v3.4.3.e result . The comp > 0.05). However, there were significant differences in body mass index (BMI), waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure (DBP), TG, TC, HDL-C, low-density lipoprotein cholesterol (LDL-C), glucose (GLU), γ-GT, ALT, AST, direct bilirubin (DBIL), and total bilirubin (TBIL) .A total of 3,023 participants were enrolled in our study, including 1,114 NAFLD cases and 1,909 controls. The distribution of demographic and clinical characteristics in two study groups is summarized in CYP24A1-rs2296241-A allele , CYP24A1-rs2248359-T allele , and CYP27B1-rs4646536-T variant  significantly improved the risk of NAFLD. They were also significant after FDR correcting for multiple comparisons .The genotype distributions of the 11 SNPs between the two study groups and the results of the logistic regression analysis are shown in p = 0.039; adjusted OR = 1.438, 95% CI = 1.038–1.993, p = 0.029, respectively). The more unfavorable alleles, the higher NAFLD risk, suggesting a significant locus-dosage effect of combined alleles on NAFLD risk (ptrend = 0.049). Moreover, participants carrying more than one unfavorable allele of relevant SNPs had a 36.8% higher risk of NAFLD, compared with people without an unfavorable allele (p = 0.028).The combined effects of rs2296241-A, rs2248359-T, and rs4646536-T on susceptibility to NAFLD were evaluated by counting the number of unfavorable alleles from these three SNPs, as shown in p = 00.022), γ-GT ≤ 50 U/L , non-low HDL-C . Moreover, heterogeneity test discovered no statistical significance in all the subgroups .Afterward, stratified analysis was conducted to further evaluate the combined effects of rs2296241-A, rs2248359-T, and rs4646536-T . We founCYP24A1 rs2296241-A, CYP24A1 rs2248359-T, and CYP27B1 rs4646536-T). The result showed that age , visceral obesity , ALT , γ-GT , hypertriglyceridemia , hypertension , low HDL-C , hyperglycemia , and unfavorable alleles  were independent influencing predictors of NAFLD in A stepwise regression model was established comprising gender, age, visceral obesity, ALT, γ-GT, hypertriglyceridemia, hypertension, low HDL-C, hyperglycemia, and combined unfavorable alleles  encoding hydroxylase in the vitamin D metabolic pathway. No relationship was found between CYP2R1 and NAFLD. Similar negative results were found in previous studies among different populations , gender was not significant, but the remaining nine were independent risk factors of NAFLD. These results are almost consistent with NAFLD guidelines and some previous studies. Abnormal ALT and γ-GT levels, hypertriglyceridemia, hypertension, low HDL-C levels, and hyperglycemia have been recognized as predictors of metabolic syndrome . Age andCYP24A1-rs2296241, CYP24A1-rs2248359, and CYP27B1-rs4646536 were 4, 4, and 1 days, respectively, which indicated that these loci have strong potential functions, regulate the expression of CYP24A1 and CYP27B1 by changing multiple regulatory motifs, and interfere with protein-binding activity  and CYP27B1 (rs4646536) variants are associated with a high risk of NAFLD in the Chinese Han population. The combination of unfavorable SNPs and metabolic-related indicators shows high efficiency in predicting the risk of NAFLD. These findings might provide new insight into NAFLD pathogenesis and a new tool for early screening of high-risk population.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Ethics Review Committee of Nanjing Medical University . The patients/participants provided their written informed consent to participate in this study.JW designed and organized the study. MXW, RZ, MW, LZ, YD, ZT, HLW, WZ, and YC contributed to the planning, designing, and analyses of the data collection and quality control. MW, RZ, MW, LZ, YD, and ZT performed data reduction and statistical analysis. HLW and WZ provided materials and analysis tools. MXW, RZ, and JW wrote and critically revised the manuscript. All authors read and approved the final 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.