Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related mRNA signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia. Nine RNA-Seq expression data sets were retrieved from the Gene Expression Omnibus database. DEGs were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. qRT-PCR was performed to validate the identified DEGs. By comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common miRNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data. The identified PPI network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.  A gene expression signature is a single or combined group of genes whose expression responds to a particular signal or changes in cellular status in a predictable way. Gene signatures are frequently extracted from a set of DEGs by comparing two groups, such as cell lines under different treatment conditions. Gene expression signatures can therefore be used as surrogate markers to comprehend the complexity of pathway activation.[1]. Hypoxia affects tumor behavior and facilitates tumor progression and metastasis, leading to resistance to conventional chemo- and radiotherapy[2]. Therefore, identifying the key genes regulating cancer cell behavior during hypoxia is essential for developing anticancer agents that efficiently kill tumor cells under hypoxic conditions.Oxygen deprivation occurs in almost all solid tumors. A shortage of oxygen is the consequence of inadequate oxygen delivery via inefficient tumor vasculature[3-5]. However, their findings only represent the genetic characteristics of specific tumor cells during hypoxia. In this study, we used RNA-Seq datasets of nine different hypoxic-conditioned cancer cell lines to find hypoxia-related mRNA signatures. Since human cancer cell lines are widely used for better understanding of cancer biology, cancer cell characterization, and anticancer drug discovery[6], we selected the available RNA-Seq datasets of cell lines to explore the effect of hypoxia on gene expression profiles. A growing number of studies have identified DEGs during hypoxia in different cancer cell types using RNA-Seq analysis[7]. Kulshreshtha and colleagues[8] described a functional link between hypoxia and miRNA expression. They indicated that miRNAs profile are regulated by hypoxia in a variety of cell types, and their dysregulation is associated with many cancers, making their signature a potential prognostic biomarker[9]. In the present study, common DEGs along with their hub genes among the nine different cancer cell lines were screened during hypoxia. Then we investigated a PPI network and predicted a miRNA-targeted gene network, which might provide a basis for further studies. Our aim was to discover the molecular mechanism underlying the effect of hypoxia and provide potential prognostic markers.MiRNAs play a central role in regulating gene expressionRaw biological data and differential RNA expression analysis [10]. The counts were imported into software R v. 3.4.0 using the tximport R package v. 1.4.0, and the DEGs were identified with a | log2 fold change | ≥1 and a false discovery rate <0.05 using the DESeq2 package in R v. 3.2.3. The UpSetR package in R was employed to ﬁnd common genes between diﬀerent datasets[11]. The default values were employed for all the packages.Raw RNA-Seq data of nine hypoxia-conditioned cancer cell lines were retrieved from the Sequence Read Archive (www.ncbi.nlm.nih.gov/geo). Among these datasets, GSE131378 contained four samples of hypoxic-conditioned and four samples of normoxic-conditioned A549 cells, while GSE72437 consisted of five samples of hypoxic-conditioned and five samples of normoxic-conditioned BeWo cells. Moreover, GSE78025, GSE81513, GSE84167, GSE13967, GSE149132, and GSE160491 contained three samples of hypoxic-conditioned and also three samples of normoxic-conditioned U78-MG, HCT116, MCF-7, ASPC-1, T47D, and BCPAP, respectively. GSE131379 also comprised of two samples of hypoxic-conditioned and three samples of normoxic-conditioned Hela cells. SAMtools was used to extract raw sequencing reads. The read quality was examined using FastQC version 0.11.2, and low quality bases and adaptor sequences were removed using Trimmomatic version 0.32; the expression level of each transcript was then quantified in transcripts per million using KallistoFunction enrichment analysis[12-14]. The Evolutionary Relationships (PANTHER) was also used to determine protein class over-representation[15], and p < 0.05 represented statistical significance. We used Database for Annotation, Visualization, and Integrated discovery (DAVID)  for GO functional analysis and KEGG pathway analysis of DEGsConstruction of aPPI network [16]. The protein interaction network was visualized using NetworkAnalyst  and Cytoscape (3.9.1)[16]. To evaluate the nodes in the PPI network, we adopted several topological measures, including degree (k), MCC, BC, and CC. Since degree (k), BC, and MCC are often used for detecting the hub in a network[17-19], we determined hub genes based on connectivity degree (number of interactions) >10, MCC, and BC using Cytohubba on Cytoscape. Interactions between the common DEGs and other proteins would be useful to fully understand their biological roles. In this study, 23 common DEG PPI network were constructed by Retrieval of Interacting Genes . Moreover, 23 common DEGs were integrated into the International Molecular Exchange Consortium database (https:// www.imexconsortium.org/) to identify the hub genes information in PPI networkMiRNA interactions analysis[20] and TarBase[21] (both version 8.0) were employed to collect the miRNA-gene interaction data. Topological analysis based on degree and betweenness centrality as key topological parameters was performed utilizing NetworkAnalyst. To identify the miRNA-mRNA target interactions, miRTarBaseCell culture for qRT-PCR validationGAPDH, LRP1, ALDOA, and PLOD2 to determine their expression in cancer cell lines  under hypoxic or normoxic conditions. Cells were purchased from the National Cell Bank of Iran . Cells used in the experiment were cultured in DMEM supplemented with 10% FBS and incubated in a humidified incubator with 5% CO2 at 37 °C.To validate our findings, we selected four hub genes, including Cancer cell adaptation to hypoxia2) for 4 h and then incubated in a standard culture environment (5% CO2 and 95% air) at 37 °C for 48-72 h. Cells were treated twice weekly, and hypoxic-conditioned cell lines were generated after 20 exposures to hypoxia[22]. Cells were seeded in a T25 flask and cultured in DMEM medium supplemented with 10% FBS. The cells were repeatedly incubated in hypoxic conditions in an Anoxomat chamber  was used for RNA isolation from the cells during normoxia and hypoxia. RNA samples were reversely transcribed to complementary DNA by the QIAGEN Reverse Transcription Kit . Subsequently, the quantification of cDNA was performed by the qRT-PCR method using SYBR Green Master Mix (Amplicon). The reaction conditions were conducted at 95 °C for 10 min, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The Statistical analysisp value was considered statistically significant when it was less than 0.05.Statistical analyses were performed using the student’s t-test with GraphPad Prism 8 software . The Differential RNA expression analysisEGLN3, ANGPTL4, GPR146, C4orf47, KCTD11, CA9, PPFIA4, PLOD2, HK2, and TMEM. Interestingly, all of these genes were upregulated in the hypoxic-conditioned cancer cell lines.RNA sequencing data from the nine different hypoxic-conditioned cancer cell lines  were analyzed, and 23 common DEGs were identified , includiFunctional categories and pathway analysisPPFIA4, PDK1, and PLOD2), scaffold/adaptor protein (KCTD11), transfer/carrier protein (LRP1), transmembrane signal receptor (GPR146), cytoskeletal protein (HK2), extracellular matrix protein (EFEMP2), intercellular signal molecule (ANGPTL4), metabolite interconversion enzyme , and regulatory protein (KDM3A). GO analysis, which covered the three GO categories , was performed using DAVID. DEGS were enriched significantly in different GO terms, including hexose metabolic process (ontology: BP), monosaccharide binding (ontology: MF), and mitochondrial pyruvate dehydrogenase complex (ontology: CCO); the results are summarized in p < 0.05 was selected. Moreover, seven pathways were significantly enriched based on KEGG pathway analysis, including HIF-1 signaling pathway, fructose and mannose metabolism, glycolysis/gluconeogenesis, carbon metabolism, cholesterol metabolism, central carbon metabolism in cancer, and biosynthesis of amino acids , MCC, and BC . In order to screen the PPI network’s interactions with other proteins, which provide important clues about their functions, the PPI network was integrated into the International Molecular Exchange Consortium database. A PPI network composed of 448 nodes and 531 edges was obtained . Twelve , and BC . Gene regulatory network analysis The key miRNAs  were identified based on network topological properties . Additionally, our results indicate miR-92-3p can regulate a large number of mRNA targets (n = 88), as shown by the PPI network .Quantitative real-time PCR for DEGs In order to validate the DEGs identified by RNA-seq analysis, four hub genes, including GAPDH, LRP1, ALDOA, and PLOD2, were selected for analysis via qRT-PCR under normoxic and hypoxic conditions. Primers were designed based on available sequences to amplify the specific altered genes. Primer sequences are shown in [24]. Therefore, exploring the role and impact of mRNA and miRNA in cancer cells, especially during hypoxia, could be helpful in cancer diagnosis and treatment. Because hypoxic cells are likely to be resistant to chemo- and radiotherapy, it is of high importance to identify the key hypoxia-inducible genes and resistance mechanisms for efficient therapeutic intervention. Moreover, it is well established that miRNA plays a central role in regulating the various biological pathways. Functional enrichment analysis revealed that the hexose metabolic process, response to hypoxia, and glucose metabolic process were significantly changed. According to KEGG enrichment analysis, 23 common genes were enriched in the HIF-1 signaling pathway, including fructose and mannose metabolism, glycolysis/gluconeogenesis, carbon metabolism, cholesterol metabolism, central carbon metabolism in cancer, and biosynthesis of amino acids. Since it is believed that proteins with more interactions have higher chances of being involved in the essential PPI[25], the PPI network was constructed and GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, and PDK1 were identified as the hub genes. In the current study, we conducted bioinformatics analysis to identify the candidate key genes and biological pathways among nine different cancer cell lines exposed to hypoxic conditions. Data was extracted from GSE131378, GSE72437, GSE78025, GSE81513, GSE131379, GSE84167, GSE13967, GSE149132, and GSE160491 datasets, among which 23 common DEGs were screened. To our surprise, all the common DEGs were upregulated in all the nine hypoxic-conditioned cancer cell lines. In order to gain some insight into how hypoxia affects the expression of genes at the molecular level, GO and KEGG pathway enrichment analyses were carried outGAPDH, LRP1, ALDOA, and PLOD2) for qRT-PCR validation in A549, U78-MG, HCT116, Hela, and MCF-7 cells under normoxic and hypoxic conditions. Expression patterns of four genes generated by qRT-PCR were consistent with RNA-seq data. Consistently, several studies have found that hypoxia-related genes such as GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, and PDK1 are upregulated during hypoxia[26-28].To support our findings, we selected four hub genes [40]. [41] showed that miR-335-5p can promote apoptosis in prostate cancer cells and may be used as a biomarker in the treatment of this disease. Upregulation of miR-6807-5p was reported in glioma specimens[43]. Dysregulation of miR-6764-5p was also identified in pituitary adenomas[44]. MiR-92-3p and miR-122-5p have been identified as the markers of hypoxic environments. MiR-92-3p can be used as a potential therapeutic target in patients with metastatic colorectal cancer. MiR-455-5p is dysregulated in many tumor cells, while miR-1915-3p and miR-124-3p could inhibit apoptosis, resulting in cancer progression. It has been exhibited that miR-1915-3p may play a role in the progression of gastric cancer and may have a potential therapeutic application in gastric cancer. Contradictorily, miR-484 could promote apoptosis by targeting Apaf-1[51], and miR-23b-3p and miR-615-3p could act as either tumor suppressors or oncogenes, which mainly depends on their context.To predict the correlation of common DEGs with miRNA, a DEG-miRNA network was constructed . These m[22].In summary, the present study identified hypoxia-related gene signatures among the hypoxia-conditioned cancer cell lines using RNA-Seq. Our analysis revealed the common hub genes and key pathways in cancer cells under hypoxic conditions. Moreover, we predicted a miRNA signature, among which miR-335-5p had the highest betweenness centrality during hypoxia. To our knowledge, for the first time, our results demonstrate that miR-6807-5p and miR-6764-5p are dysregulated under hypoxic conditions. However, further molecular biological experiments are required to confirm the function of the identified miRNA associated with hypoxia. The results of the present study may provide future directions in identifying the presence of cancer and determining the characteristics of cancer. For instance, hypoxia is a characteristic feature of cancer, and the hypoxia signature identified in this study, as well as predicted miRNAs might be helpful to detect the hypoxic state of cancer cells. Hypoxia is common in majority of malignant tumors and an attractive therapeutic target. As hypoxia targeted treatment are effective in patients with the most hypoxic tumors, hypoxic signature might be useful for developing proper treatment, such as engineered oncolytic viruses that could be utilized to control or regulate the biological interactions responsible for the functioning or malfunctioning of cancer cells during hypoxiaNot applicable. The raw data supporting the conclusions of this article are available from the authors upon request.SS: conceived, designed the analysis and performed the analysis; GB: performed bioinformatics analyses; AA: drafted or provided critical revision of the article; KA: conceived and designed the study, supervised the data analysis and interpretation. All authors have read and approved the final manuscript.None declared.funded as Ph.D. student project by Pasteur Institute of Iran, Tehran.This study was Supplementary Fig. 1


O-gallate, genistein, quercetin, and resveratrol. These polyphenols have similar chemical and biological properties in that they can act as antioxidants and exert the anticancer effects via cell signaling pathways involving their reactive oxygen species (ROS)-scavenging activity. These polyphenols may also act as pro-oxidants under certain conditions, especially at high concentrations. Epigenetic modifications, including dysregulation of noncoding RNAs (ncRNAs) such as microRNAs, long noncoding RNAs, and circular RNAs are now known to be involved in the anticancer effects of polyphenols. These polyphenols can modulate the expression/activity of the component molecules in ROS-scavenger-triggered anticancer pathways (RSTAPs) by increasing the expression of tumor-suppressive ncRNAs and decreasing the expression of oncogenic ncRNAs in general. Multiple ncRNAs are similarly modulated by multiple polyphenols. Many of the targets of ncRNAs affected by these polyphenols are components of RSTAPs. Therefore, ncRNA modulation may enhance the anticancer effects of polyphenols via RSTAPs in an additive or synergistic manner, although other mechanisms may be operating as well.Growing evidence has been accumulated to show the anticancer effects of daily consumption of polyphenols. These dietary polyphenols include chlorogenic acid, curcumin, epigallocatechin-3- O-gallate (EGCG), genistein (GEN), quercetin (QUE) and resveratrol (RES), which are six major polyphenols in our dietary life and are found in vegetables, fruits, and beverages. Preclinical and cell-based studies have supported their anticancer effects and provided a mechanism of action for these polyphenols [A number of epidemiological studies have provided evidence for the anticancer effects of daily polyphenol intake ,3,4,5,6.yphenols ,2,3,5. RWe have provided updated information from human studies supporting the anticancer effects of consumption of green tea, coffee, red wine, soybeans, and curry and discussed the involvement of miRs in polyphenol action mechanisms . PreviouIn this review, we further discuss the miR-modulating effects of polyphenols, which have been reported in studies using one or two of the six dietary polyphenols . FurthermiRs are defined as small single-stranded molecules (approximately 20 to 25 nucleotides) and can regulate gene expression at the transcriptional and post-transcriptional levels, leading to modulation of beneficial health effects exerted by these polyphenols in diseases including cancer ,8.One or two of the miRs up- and down-regulated by six polyphenols for which studies have been reported are listed in LncRs are more than 200 bases long and are species/tissue specific. LncRs can interact with DNA, RNA, and proteins to regulate a wide range of biological processes ,150. RecThere are two major categories of lncRs, which are defined as oncogenic lncRs such as MALAT1, HOTAIR, SOX2-OT and H19, and tumor suppressing lncRs such as MEG3, PANDAR, GAS5, and TUG1 according to their pathological features . EvidencGAS5Salmonella typhimurium-induced intestinal diseases, Tan et al. [For reasons unknown, information on CGA’s actions on lncRs is quite limited. In a study on CGA’s preventive effects on n et al.  found thTUG1In a study on a role of CGA in protection against oxidative stress injury in glaucoma, Gong et al.  found thAK294004In nasopharyngeal carcinoma (NPC) cell lines (CNE-2), a microarray analysis showed that expression of a number of lncRs was changed by X-ray irradiation and that the expression of 116 ncRNAs was restored by CUR . The resGAS5Dendrosomal CUR treatment of MCF7, MDA-MB231 and SKBR3 cells increased the expression of Tusc7 and GAS5 . GAS5 doH19Kujundzić et al.  found thIn tamoxifen-resistant MCF-7 BCa cells, CUR decreased the expression levels of the epithelial marker E-cadherin, increased the expression levels of mesenchymal marker N-cadherin, and decreased H19 expression . OverexpKCNQ1OT1The cisplatin-resistant colorectal cancer (CRC) HCT8/DDP cells exhibited higher expression levels of oncogenic KCNQ1OT1 compared to non-resistant cells. Zheng et al.  found thLINC00691In papillary thyroid cancer B-CPAP cells, CUR decreased cell proliferation, promoted apoptosis, and inhibited LINC00691 expression . CUR admlinc-PINTMicroarray experiments with acute lymphoblastic leukemia cells from the patients showed that 43 lncRs were aberrantly expressed as compared to healthy donor blood cells . qRT-PCRMEG3Alghanimi and Ghasemian  showed tNRB2Yu et al.  found thPANDARCUR increased PANDAR expression in CRC DLD-1 cells and silencing of PANDAR increased apoptosis and attenuated cell senescence by stimulating expression of PUMA . KnockdoPVT1The EZH2, a subunit of polycomb repressive complex 2 (PRC2), is known to have an important role in drug resistance. EZH2 interacts with several lncRs including PVT1 to modulate EMT and cancer stemness related to drug resistance. Yoshida et al.  found thRORShao et al.  demonstr+/CD133+ human prostate cancer (PCa) stem cells derived from the PCa cell lines Du145 and 22RV1, CUR treatment resulted in the inhibition of cell growth and invasion, and cell cycle arrest along with decreased expression of stem cell marker proteins such as Oct4 [In CD44 as Oct4 . In addi as Oct4 ,173.UCA1In A549 cells, CUR inhibited cell proliferation and cyclin D1 expression, and enhanced cell apoptosis . CUR inhXISTSun et al.  found thAF085935Sabry et al.  showed tLINC00511Zhao et al.  found thNEAT1CTR1 is known to promote cisplatin internalization in tumor cells. Jiang et al.  found thCancer stem cells have been implicated as a major player in tumor metastasis, tumor recurrence, and chemotherapy resistance. CTR1 is associated with cisplatin resistance. Jiang et al.  found thSOX2OT variant 7Wang et al.  found thOther studiesThe results of lncR microarray analysis revealed that EGCG treatment of lung cancer cells caused significant alterations in a total of 960 lncRs and 1434 mRNAs . Among tHOTAIRIn PCa cells, PC3 and DU145, GEN downregulated oncogenic HOTAIR and upregulated miR-34a . LuciferImai-Sumida et al.  showed tTTTY18GEN suppressed CRC cell growth and migration and promoted apoptosis . GEN dowMALAT1By data mining including computational analysis, Li et al.  identifiQUE treatment decreased the cell viability of HUVEC cells and downregulated the expression of MALAT1 and MIAT . Since MNEAT1Sheng et al.  found thSNHG7Chai et al.  found NSUCA1In BCa MCF-7 cells, QUE inhibited cell proliferation and induced cell cycle arrest at G2 phase . INXS isAK001796Yang et al.  found thDLEU2Kay et al.  demonstrH19In GCa SGC7901 cells, 200 µM RES was shown to increase expression of MEG3, PTTG3P and BISPR and decreased expression of GAS5 and H19 . RES at MALAT1RES inhibited invasion and metastasis of CRC LoVo cells and downregulated MALAT1 . RES’s sNEAT1Geng et al.  found hiPCAT29RES upregulated PCAT29 expression and attenuated its downregulation induced by IL-6 . KnockdoOther studiesIn glioma U87 and U251 cells, RES upregulated NEAT1, MIR155HG, MEG3, and ST7OT1 during induction of apoptosis . Since NIn colon adenocarcinoma HT-29 cells, the results of qRT-PCR indicated that RES decreased the expression of CCAT1, CRNDE, H19, HOTAIR, PCAT1, PVT1, and SNHG16, and upregulated CCAT2, MALAT1, and TUSC7 . AlthougCircRs are associated with cancer development by modulating miRNAs involved in cell proliferation, migration, and carcinogenesis . CircRs CUR’s downregulation of circ_102115circ_PRKCA, circ_FNDC3B, and circ_0078710, Microarray analysis of the radioresistant nasopharyngeal carcinoma cell line CNE-2 and normal cell lines identified 1042 upregulated and 1558 downregulated circRs . CUR wasXu et al.  found thIn RCa tissues and cells, circ_FNDC3B levels were upregulated and miR-138-5p was downregulated . CUR supCUR inhibited the proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cell lines HCCLM3 and Huh7 and induced apoptosis . CUR dowGEN’s downregulation of circ_0031250GEN inhibited cell viability and downregulated oncogenic circ_0031250 in NSCLC H292 and A549 cells . Circ_00QUE’s upregulation of circ_R 8:93786223|93822563In CRC HCT 116 cells, QUE inhibited cell proliferation and induced apoptosis. Zhang et al.  examinedAnticancer effects of dietary polyphenols, CGA, CUR, EGCG, GEN, QUE, and RES have been shown in many human, animal, and cell-based studies. These results may explain those of epidemiological studies demonstrating the reduced cancer risk of a variety of cancer types by consumption of coffee, green tea, soybeans, wine, and curry. A cohort study conducted by Wang et al. reported that the amount of total polyphenols ranged from 8.88 to 47.44 mg/day in the lowest and highest quintile, respectively, among 38,408 middle-aged and old women . SeveralThese polyphenols can modulate the expression/activity of several RSTAP components by increasing the expression of tumor-suppressive ncRNAs and decreasing the expression of oncogenic ncRNAs in general. Thus, modulation of ncRNAs may additively or synergistically enhance the anticancer effects of these polyphenols via RSTAPs.Although six dietary polyphenols have similar chemical properties, GEN and RES, and EGCG can act uniquely as phytoestrogens and a 67kDa laminin receptor, respectively, in anticancer effects ,215. TheIn a recent comprehensive review, Fujimura et al.  have dishttps://pubmed.ncbi.nlm.nih.gov/ (accessed on 24 November 2022)) and Web of Science (http://webofknowledge.com/WOS (accessed on 24 November 2022)), there is a limitation that this review has not covered all of findings related to the present theme.Since this review is written based on the data from two databases: PubMed (


Primary care-based preconception care (PCC) has the potential to improve pregnancy outcomes, but the effectiveness is unclear.To evaluate the effectiveness of primary care-based PCC delivered to reproductive-aged females and/or males to improve health knowledge, reduce preconception risk factors, and improve pregnancy outcomes.A systematic review of primary care-based PCC.Ovid MEDLINE, Cochrane CENTRAL, Embase, Web of Science, Scopus, and CINAHL were searched for randomised controlled trials (RCTs) published between July 1999 and May 2021. Two reviewers independently evaluated article eligibility and quality.n = 8), intensive education (multiple sessions) (n = 9), supplementary medication (n = 7), and dietary modification (n = 4). Brief education improved health knowledge in females (n = 3) and males (n = 1), reduced alcohol/tobacco consumption (n = 2), and increased folate intake (n = 3). Intensive education reduced spontaneous pregnancy loss (n = 1), alcohol-exposed pregnancies (n = 2), and increased physical activity (n = 2). Supplementary medication increased folate intake (n = 4) and dietary modification reduced pre-eclampsia (n = 1) and increased birth weight (n = 1). Only nine articles reported on pregnancy outcomes, with a range of interventions used; of these, four reported improvements in pregnancy outcomes. Most RCTs were of low quality (n = 12).Twenty-eight articles reporting on 22 RCTs were included. All but one focused on females. Interventions included brief education (single session) (Primary care-based PCC including brief and intensive education, supplementary medication, and dietary modification are effective in improving health knowledge and reducing preconception risk factors in females, although there is limited evidence for males. Further research is required to determine whether primary care-based PCC can improve pregnancy outcomes. However, less is known regarding the effectiveness of primary care-based PCC.8Preconception care (PCC) refers to interventions delivered before conception that modify preconception risk factors and reduce the burden of adverse pregnancy outcomes such as low birth weight, spontaneous abortion, and preterm birth.9–11 PCC is often a low priority and not routine practice in primary care in many countries1,8,9,12 and almost all primary care-based PCC interventions are directed towards women.8–10,12–18As the first point of healthcare contact, primary care providers are ideally placed to provide PCC; however, the effectiveness of primary care-based PCC interventions is unclear.19 and sperm quality,20 PCC directed towards reproductive-aged males may also improve pregnancy outcomes. Since the previous review investigating the effectiveness of primary care-based PCC interventions,8 a number of studies evaluating the effectiveness of PCC interventions in primary care have been published. In the current study therefore a systematic review was conducted to evaluate the effectiveness of primary care-based PCC interventions delivered to reproductive-aged females and/or males to improve health knowledge, reduce preconception risk factors, and improve pregnancy outcomes. This builds on the previous review published in 2016 that was limited to females, and which included randomised controlled trials (RCTs) published between July 1999 and July 2015.8As modifiable risk factors including smoking and alcohol consumption may also have an impact on men’s reproductive health21 and the review was registered with PROSPERO (CRD42021235499) as described in the protocol.22 Search terms were developed (Supplementary Table S1) and Ovid MEDLINE, Cochrane CENTRAL, Embase, Scopus, CINAHL, and Web of Science were searched. The pilot search for this review showed heterogeneity of outcomes measured across the studies, therefore search terms relating to outcomes were not included in the search strategy. Reference lists of included articles were manually screened for additional studies. Articles were included if the study:reported on the effectiveness of PCC in primary care;included reproductive-aged males and/or females (18–45 years);was an RCT;was written in English;was published in a peer-reviewed journal between July 1999 and May 2021; andincluded but was not limited to the outcomes outlined in The Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidelines were followedet al.23 Only RCTs were included as they are the reference standard for studying causal relationships between interventions and outcomes.24Articles were excluded if the study: included pregnant females or focused on fertility. The start date was selected following the end of the search of an earlier review by Korenbrot 25 tool with six domains: sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessors, incomplete outcome data, and selective reporting bias. Studies were classified as high quality , moderate quality , or low quality (high risk-of-bias in at least one domain or unclear risk-of-bias in multiple domains).All results of database searches were saved in Covidence systematic review software, Veritas Health Innovation, Melbourne, Australia for management. Duplicates were removed and two authors independently screened all titles, abstracts, and full texts of articles for eligibility; discrepancies were reviewed by a third author to reach consensus. Two authors independently evaluated the quality of the RCTs using the Cochrane risk-of-bias (ROB 2.0)7,8 and extracted data (Supplementary Table S2).8,12,18 The pilot search for this review also showed that meta-analysis cannot be undertaken because of the heterogeneity of the outcomes investigated across the different studies. The magnitude of the difference between the control and intervention groups are presented as reported in the RCTs. For dichotomous data, relative risks, odds ratios (OR) with 95% confidence intervals (CI), are presented and for continuous data the mean difference before and following the intervention and/or P-values are reported.One of the authors created a data extraction form based on previous reviews on PCCn = 8),26–33 Iran (n = 8),34–41 Vietnam (n = 5),42–46 China (n = 2),47,48 the Netherlands (n = 2),49,50 India (n = 1),51 Australia (n = 1),52 and Sweden (n = 1).53 Studies recruited females who were: aged 18–35,40 aged 18–45,26,30 able to conceive,28,33,37 planning to have children,41,47,48,51 planning pregnancy within 9 months,27 a year,31,35,36,38,39,43,44,46 2 years,29 1–5 years,49,50 or after the delivery of their first baby,32,42,45,52 and one study recruited males.53 The majority recruited females planning for pregnancy (n = 16).27,29,31,35,36,38,39,41,43,44,46–51 The type of intervention(s), provider, and outcome(s) measured varied across studies. In some studies the providers were healthcare professionals30,32,43,44,46–49,50–53 whereas in other studies non-healthcare professionals26–29,33–42,45 were trained to deliver the intervention (the latter referred to hereafter as ‘trained facilitators’).Out of 4622 articles, 1684 duplicates were removed and 134 full-text articles were evaluated for eligibility after title and abstract screening. After full-text screening, 28 articles were included, reporting on 22 RCTs . No addi26,35–37,49,50,52,53 intensive education (multiple education sessions),27–29,33,34,38–41 supplementary medication,43,44,46 dietary modification,42,45,48,51 and multiple interventions.30–32,47 Studies reported on the effectiveness of the intervention on pregnancy outcomes,29,42,44–46,48,50,51,52 health knowledge,37,38,50,53 and/or preconception risk factors.26–29,30–36,40,47,50 Study characteristics and quality are summarised in Supplementary Tables S2 and S3. According to the ROB 2.025 tool, only two of the RCTs were of high quality , eight were of moderate quality , and the majority were of low quality .The PCC interventions were categorised into brief education (a single education session),26,35–37,49,50,52,53 In four of these, healthcare professionals, including GPs,49,50 nurse–midwives,53 and midwives, directed the sessions.52 The other four were directed by non-healthcare professionals, including trained facilitators26,35,36 or the researcher.37 Healthcare professional-directed brief education improved pregnancy outcomes in one of two studies investigating this,50,52 improved health knowledge, and reduced preconception risk factors in females49,50 and males.53Eight articles reported on the effectiveness of brief education (a single session).50 This study also reported increased self-reported folate intake 50 and reduced alcohol use during the first trimester , and self-reported maternal anxiety (OR not reported).49 In the second study, midwife-directed counselling during a home visit did not affect the incidence of preterm birth or low birth weight during subsequent pregnancies.52In the one study that reported improved pregnancy outcomes, GP-directed counselling about preconception risk factors decreased adverse pregnancy outcomes including miscarriage, extra-uterine pregnancy, perinatal death, preterm birth, low birth weight, and congenital abnormalities.53 In this RCT, reproductive life plan-based (RLP) counselling by nurse– midwives increased men’s self-reported awareness of preconception lifestyle risk factors 3 months post-intervention (P<0.0001).53 The RLP tool provides individuals with information concerning reproductive health. After RLP counselling 76% of males in the intervention group reported a positive experience of the counselling, and 77% had received new information.Among the 22 RCTs, only one study involved males.26,35–37 Three studies reported on the effectiveness of trained facilitator-directed group workshops on preconception health and folate-focused education.26,35,36 Of these, group workshops in one study had a positive impact on healthy behaviours such as increasing physical activity (P<0.01)36 and the other two reported increased self-reported intake of folate-rich food.26,35 Likewise, a researcher-directed 5–10 min RLP counselling session increased self-reported knowledge of folate intake (P<0.001).37Non-healthcare professional-directed brief education reduced preconception risk factors in females in the four studies investigating this.27–29,33,34,38–41 In all of these, sessions were directed by non-healthcare professionals, including trained facilitators27–29,33,34,38–41 and the researcher.41Nine articles reported on the effectiveness of intensive education.38 reduced spontaneous pregnancy loss,29 or reduced preconception risk factors in all studies investigating this.27–29,33,34,38–41 In one study, group counselling sessions on preconception risk factors increased self-reported health knowledge of preconception lifestyle risk factors .38 In another, weekly counselling sessions on health responsibility for 6 months, followed by monthly sessions until delivery, reduced spontaneous pregnancy loss  and increased self-reported weight loss before pregnancy (P<0.001).29Non-healthcare professional-directed intensive education improved health knowledge,27 Similarly, two motivational interviewing sessions that aimed to increase participants’ commitment to change hazardous alcohol use reduced risk of alcohol-exposed pregnancies across 9 months .33A 14-week counselling programme around hazardous drinking reduced alcohol-exposed pregnancies at 3-, 6-, and 9-month follow-ups.P = 0.019).28 In another study, 6-weekly motivational interviewing sessions increased self-reported moderate (P = 0.01) and vigorous (P = 0.02) physical activity,40 and increased self-reported weight loss post-intervention .34 Two studies investigated the short-term effect of preconception risk factor counselling on maternal stress levels.39,41 In one study, trained facilitator-directed counselling reduced self-reported maternal stress 4 and 8 weeks post-intervention.39 In the other, researcher-directed counselling improved stress management 1 month post-intervention.41Six 2-hour sessions on preconception risk factors also increased self-reported physical activity  or iron were compared with females receiving folate only (control). Preconception multiple micronutrient supplementation did not affect the prevalence of low birth weight or preterm birth,46 but a follow-up analysis reported high prenatal and postpartum maternal ferritin levels in the groups supplemented with multiple-micronutrients or iron and folate and these females gave birth to infants with greater iron stores. However, the clinical significance is unclear, as anaemia prevalence did not differ between groups.44Three articles from one RCT reported on the effectiveness of supplementary medication delivered by village health workers.P<0.05).43Furthermore, maternal depressive symptoms were low during pregnancy and early postpartum, and there was no difference between the groups. Although the underlying mechanisms are unclear, among females at risk of depression, maternal depressive symptoms were lower in the first and second trimesters of pregnancy in the groups receiving multiple-micronutrients or iron and folate compared with the control group , provided from >90 days before pregnancy until delivery of baby, in addition to their usual diet, increased infant birth weight (P = 0.046).51 This may have resulted from higher micronutrients, energy, and/or protein levels in the snack provided to the intervention group when compared with the control group.51 Additionally, specialist provision of a diet comprising at least 100 g of mushrooms daily from preconception to the 20th week of gestation reduced gestational hypertension (P = 0.023), preeclampsia (P = 0.014), gestational weight gain (P = 0.017), excessive gestational weight gain (P = 0.032), and gestational diabetes (P = 0.047).48 In the other two articles, non-healthcare professional trained facilitators42,45 delivered macronutrient supplementation from preconception to term. This increased maternal protein, iron, zinc, folate, vitamin A, and B12; however, it did not affect infant birth weight42 or infant growth up to 24 months of age.45Four articles reported on the effectiveness of dietary modification.30–32 or intensive47 education. Interventions involved provision of folate education via a 15-min GP computerised session,31 brief counselling by a gynaecologist,30 monthly counselling by village doctors,47 or brief counselling by paediatric clinicians.32 All studies reported improvements in self- reported folate intake30–32,47 and one study reported reduction in self-reported binge drinking and smoking.32Four studies reported on the effectiveness of multiple interventions including supplementary medication such as folate supplementation along with brief8 Most articles in the current review were of low quality and the type of interventions, populations, providers, and outcomes varied substantially between the different studies.To the authors’ knowledge, this is the first systematic review of primary care-based PCC that includes males and also the first to consider the role of the provider in the delivery of primary care-based PCC. Results from 28 articles reporting on 22 RCTs were included incorporating an additional 17 articles published since the last review.A number of important findings were identified in this review. First, both brief and intensive education on preconception health improved health knowledge and reduced preconception risk factors for females, suggesting either method could be utilised by primary care providers to deliver PCC education. However, the duration of brief education , intensive education , and the timing of PCC education delivery  varied considerably between studies, so it is not clear which are the most effective.48 and increasing birth weight51 in two studies;48,51 however, the studies were of moderate to low quality and more high-quality evidence is required. Multiple interventions including brief or intensive folate education along with folate supplementation increased self-reported folate intake in all studies investigating this,30–32,47 reiterating that primary care providers should encourage supplementary medication, including folate supplements, and intake of folate-rich foods, to all females during the preconception period.Second, dietary modification improved pregnancy outcomes by reducing pre-eclampsia53 Fourth, although in 10 of the studies the intervention was delivered by healthcare professionals, in the majority of studies (n = 12) the intervention was delivered by non-healthcare professionals. In almost all (n = 11) of these latter studies, improved health knowledge,38 reduced preconception risk factors,26–28,33–36 or reduced spontaneous pregnancy loss were reported.29 These results suggest that primary care-based PCC are effective in improving health knowledge and reducing preconception risk factors; trained facilitators could help improve access to PCC in systems that are already struggling to provide care. Finally, although nine studies were found that reported on pregnancy outcomes, only four reported improvements.29,44,48,50 It is unclear whether this is related to the strength of the intervention being delivered or the intervention itself, therefore more evidence is required to understand the effectiveness of primary care-based PCC on improving pregnancy outcomes.Third, although findings suggest that brief education improves health knowledge among males, more research is required as this is based on only one study.This review was not restricted to a particular region or country, and therefore provides a broad international perspective on the effectiveness of primary care-based PCC interventions. Five databases were systematically searched for literature; however, relevant articles may still have been missed because of the search strategies employed. By limiting the eligibility criteria to RCTs, non-RCTs investigating primary care-based PCC were excluded that may have reported improved pregnancy outcomes.8 and 24 additional articles since the last Cochrane review published in 2009.18 Similar to another review of PCC in community settings,4 the current review found that primary care-based PCC interventions are effective in improving health knowledge, increasing here folate intake, and reducing alcohol consumption.This review included 17 additional articles published since the review published in 2016Given the effectiveness of PCC education delivered in primary care at reducing risk factors, brief or intensive PCC education should be mainstreamed for reproductive-aged females and males in primary care.54 medical and lifestyle health indicators. Also there is a lack of understanding about how high-risk females can be systematically identified in primary care. Research investigating how to best identify these females in primary care is therefore warranted.None of the RCTs in this review targeted PCC for females at high risk of poor pregnancy outcomes based on pre-existingLastly, in all studies providing intensive PCC education, interventions were delivered by trained facilitators or researchers. Future studies could explore the role and potential impact of primary care providers, including GPs, nurses and midwives, and trained non-healthcare professionals delivering intensive PCC education.In conclusion, primary care-based PCC including brief/intensive education, supplementary medication, and/or dietary modification improved health knowledge and reduced preconception risk factors among females, irrespective of the provider. Brief education may also improve health knowledge in males, although more research is needed.Given the limited number of studies reporting on pregnancy outcomes, further research is required to determine whether primary care-based PCC is effective in improving pregnancy outcomes.


Naringenin (Nar) is one of major citrus flavonoids predominantly found in grapefruit and orange. In vivo studies have demonstrated Nar potential as a normolipidemic agent capable to reduce circulating cholesterol in hypercholesterolemic rabbits, rats, and patients, suggesting a new role for this molecule in cardiovascular disease prevention. Although Nar cholesterol-lowering effects are known, the underlying mechanisms have not yet been elucidated. Interestingly, Nar binds to the estrogen receptors (ERs), modulating both transcriptional and membrane-initiating signals. Although estrogen and ERs are deeply involved in lipid metabolism, no data are available regarding a putative role of these nuclear receptors as mediators of the hypocholesterolemic effect exerted by Nar. Thus, the aim of this work was to study the involvement of ERs in Nar-induced modulation of cholesterol metabolism. Results obtained in HepG2 cell line demonstrate that Nar can modulate the molecular network of cholesterol homeostasis. However, these effects were only partially dependent on the activity of estrogen receptor α. As a whole, our data highlight new molecular mechanisms by which Nar influences cholesterol metabolism, opening a new scenery about dietary impact on human health. Coronary heart disease (CHD) is the major cause of morbidity and mortality in Western industrialized countries. Every year, cardiovascular disease (CVD) kills approximately 16 million people. In the US, 43% of deaths are attributable to CVD, and in 2019, an estimated 558,000 deaths were caused by coronary heart disease, and 109,000 deaths were caused by ischemic stroke [A positive correlation among elevated blood levels of low-density lipoprotein cholesterol (LDL-C), atherosclerosis, and coronary heart disease has been assessed; therefore, dyslipidemia is one of the main risk factors leading to CHD. Prevention of CHD remains a goal for researchers working with health or pharmaceutical care. In primary prevention, several modifiable risk factors should be considered for controlling this disease, such as the combination of smoking, hypertension, hyperlipidemia, obesity, sedentary life, and dyslipidemia. However, it is quite difficult to control cholesterol levels and meet the aggressive targets for plasma LDL-C concentrations by modifying daily habits alone. Thus, pharmacological intervention is often necessary. Three main classes of compounds are used in current clinical practice to treat dyslipidemia: statins, a primary therapeutic tool, fibrates, monoclonal antibodies, synthetic interfering RNA (siRNA), and bile-acid sequestrants or resins ,3. BecauIt has been recognized that a high intake of fruits and vegetables protects against chronic diseases. Among fruits and vegetables, the protective relationship between the consumption of citrus fruits or juices and the risk of ischemic stroke has been demonstrated . In addi−7 M, Nar induces the increase of LDLR, which is suggestive of an enhanced LDL cholesterol uptake  of AMP-activated kinase (AMPK), the major HMGCR kinase at least in the liver, and the level of protein phosphatase 2A (PP2A) catalytic subunit, which activates HMGCR by de-phosphorylation [−6 M for 24 h) increases AMPK phosphorylation state , a 72-kDa protease highly expressed in the liver under the control of SREBPs. Once secreted, PCSK9 binds LDLR, inducing the redistribution of the receptor from the cell surface to lysosomes; indeed, PCSK9 appears to change the trafficking of the LDLR, diverting the internalized LDLR to degradation in lysosomes and preventing them from being recycled to the cell surface . Figure Because Nar modifies the protein network of cholesterol metabolism maintenance, we investigated the putative ERα involvement in these changes. Thus, the ERα pure antagonist ICI 182,780 was administered to HepG2 cells 15 min before Nar treatment and, subsequently, HMGCR protein content and phosphorylation state, as well as LDLR expression, were analyzed. ICI 182,780 is an inhibitor of both ER subtypes; to investigate the specific involvement of ERα, subtype HepG2 cells were treated with Endoxifen (a selective ERα inhibitor) 15 min before Nar stimulation. Results show that, like ICI 182,780 treatment, Endoxifen pretreatment prevents Nar-induced HMGCR modulation A, whereaNar-induced AMPK activation was hindered by ICI 182,780 administration A. On theIntriguingly, the Nar effect on nSREBP1 and Insig-1 was independent on ERα A,B, wherEating habits and metabolic disorders are associated with negative effects on the risk factors for cardiovascular diseases, as they can induce increased cholesterol and triglycerides plasma levels. In the last years, natural food components have attracted considerable interest in treating hypercholesterolemia. In general, plant foods provide support for cardiovascular disease prevention due to their low amount of fats and the presence of bioactive compounds, which may act as cholesterol-lowering agents through different mechanisms. Among these molecules, Nar shows hypolipidemic properties both in vivo and in vitro ,35,36. IOur results show that Nar administration to HepG2 cell line reduces HMGCR activation state and increases LDLR protein expression, justifying the in vivo lipid-lowering properties exerted by this flavanone ,37,38,39To focus our attention on the putative role exerted by ERs in Nar-induced modulation of proteins belonging to cholesterol regulatory network, we used a pharmacological approach by using ICI 182,780, a pure ER antagonist, and Endoxifen, which specifically inhibits ERα. The results demonstrate that, differently from LDLR, Nar-induced HMGCR regulation is dependent on ERs. Interestingly, the regulation of proteins involved in HMGCR phosphorylation, AMPK and PP2A, is dependent and independent on ERs, respectively. Turning the attention on long-term regulation, our results demonstrate that only Nar-dependent Insig-2 increase is influenced by ERs. This feature is supported by previous data establishing a role for Insig-2 as a link between estrogen protective effects and cholesterol homeostasis. In particular, alignment sequence analysis showed that rat Insig-2 gene presents an estrogen response elements-like (ERE-like) within intron 2, suggesting that Nar binding to ERs is able to increase Insig-2 protein level as well as estrogen . To dateAll materials used were obtained from commercial sources and of the highest quality available. All materials with no specified source are obtained from Merck .2 in RPMI-1640 medium, containing 10% (v/v) fetal calf serum, L-glutamine (2 mM), gentamicin (0.1 mg/mL), and penicillin (100 U/mL). Cells were passaged every 3 days and media changed every 2 days. HepG2 cells were grown to 70% confluence in flasks then stimulated with vehicle , Naringenin , for 24 h in presence or in absence of pre-stimulation for 1 h with the ER inhibitor ICI, 182,780 (10−6 M) or the specific ERα inhibitor Endoxifen (10−6 M).HepG2 cells were used as experimental model. Cells were routinely grown in air containing 5% COv/v), and pelleted by centrifugation. Pellets were re-suspended in PBS containing 0.1 M sucrose, 0.05 M KCl, 0.04 M KH2PO4, 30 mM EDTA, pH 7.4, and sonicated. The protein concentration was determined by the Lowry method [Cells were washed twice in phosphate buffer saline (PBS), harvested with trypsin  SDS, protease inhibitor cocktail , then boiled for 2 min. Solubilized proteins (10 µg) were resolved by SDS–PAGE at 100 V for 1 h and then electrophoretically transferred to nitrocellulose for 80 min at 100 V and 48C. The nitrocellulose was treated with 3% (w/v) bovine serum albumin (BSA) in Tris Buffered Saline  and then probed over night with the antibodies listed in Samples, obtained as previously described, were solubilized in 0.25 mM Tris–HCl, (pH 6.8) containing 20%  software for Windows.Each mean was derived from four different experiments performed in duplicate. The statistical analysis consisted of unpaired t test when two means were compared, or ANOVAs followed by the Tukey’s multiple comparisons test when more than two means were compared.


The mechanical properties of cells are important in tissue homeostasis and enable cell growth, division, migration and the epithelial-mesenchymal transition. Mechanical properties are determined to a large extent by the cytoskeleton. The cytoskeleton is a complex and dynamic network composed of microfilaments, intermediate filaments and microtubules. These cellular structures confer both cell shape and mechanical properties. The architecture of the networks formed by the cytoskeleton is regulated by several pathways, a key one being the Rho-kinase/ROCK signaling pathway. This review describes the role of ROCK (Rho-associated coiled-coil forming kinase) and how it mediates effects on the key components of the cytoskeleton that are critical for cell behaviour. The biomechanical properties of cells are inextricably linked to their intracellular structural components, such as the cytoskeleton . InternaThe main components of the cytoskeleton include microfilaments , intermediate filaments and microtubules, which connect to each other, cellular organelles and the cell/nuclear membrane. They are biopolymers and organized into a three-dimensional network that resists deformation but can rearrange in response to internal or external forces and are thus responsible for maintaining the integrity of the cellular components. In general, there are four main functions of the cytoskeleton: organization of cellular contents; connection of cellular constituents to the external environment; mechanical resistance to deformation and generation of forces to move the cell or change its shape  2,19]. ,19. 2,19lP) [lP = BS/kBT, where BS is bending stiffness, kB is the Boltzmann constant and T is the absolute temperature [lP of microfilaments is 10–17 μm, the lP of intermediate filaments is less than 0.3–1.0 μm, and the lP of microtubules is more than 1 mm [The three types of cytoskeletal components differ from each other, both chemically and physically. Generally, the most significant difference is their rigidity, which can be represented by the persistence length (lP) . The perlP) . If a poperature . Filamenhan 1 mm . This shlP ~ 10–17 μm) polymers of actin (~375 amino acids with a molecular mass of ~42-kDa) folded into a U-shaped double helix structure. Each actin monomer has four subdomains and a bound adenine nucleotide, either ATP, ADP-Pi, or ADP, and a bound divalent cation, Mg2+ [Microfilaments, or more specifically actin filaments, are semiflexible  than microfilaments and microtubules. Their diameter is from 8 to 12 nm, intermediate in size between microfilaments and microtubules [Intermediate filaments play an important role in cell mechanics, signaling and homeostasis. They belong to a superfamily of highly conserved, α-helix-rich fibrous proteins that are encoded by approximately 65–70 different genes ,82,83,84otubules , and theotubules . Type I otubules ,32,87,88otubules , whereasotubules ,89. Typeotubules ,85. Typeotubules ,47. The otubules ,56,90.Intermediate filaments are vital determinants of intracellular organelle organization and form a delicate network in the cytoplasm, encapsulating the nucleus and radiating toward the plasma membrane . They arlP > 1 mm and are stiffer than microfilaments and intermediate filaments. They are relatively brittle and can be fractured more easily than intermediate filaments [−24 to 32 × 10−24 Nm2 [Microtubules are cylindrical polymers of α and β-tubulin dimers. There are multiple genes encoding tubulin protein isotypes ,96. Accoilaments . The pub0−24 Nm2 . Microtu0−24 Nm2 ,100,101.0−24 Nm2 . On the 0−24 Nm2 . They gr0−24 Nm2 . This hy0−24 Nm2 . Microtu0−24 Nm2 . This is0−24 Nm2 . In orde0−24 Nm2 . For exa0−24 Nm2 . In epit0−24 Nm2 .In addition to these three main cytoskeleton components, many other proteins are associated with the cytoskeleton, such as spectrin, kinesin, plectin, emerin, Sad1 and UNC-84 proteins, Klarsicht, ANC-1, and Syne homology proteins, and they are important for cellular mechanics. . TogetheOther kinases, such as PI4P and protein kinase C, are also important in regulating interactions of the cell membrane proteins and protein scaffolds involved in vesicle budding, and cytoskeletal organization ,198. In Rho GTPases are key regulators of several cellular processes, including cytoskeletal shape, gene expression, cell cycle progression, cell polarity and cell migration ,201,202.https://www.ncbi.nlm.nih.gov/gene/6093#gene-expression, accessed on 21 December 2022) whereas ROCK2 is located on chromosome 2  and its mRNA is expressed abundantly in adipose tissue and the colon  [Rho-associated kinase ROCK is one of the most important effectors downstream of Rho GTPase  Figure . Human Rer 2022) ,221. ROCer 2022) , LIM1/2 er 2022) , myosin er 2022) , ERM [22er 2022) , adduciner 2022) , calponier 2022) , myristoer 2022) , elongater 2022) , troponier 2022)  and profer 2022) . ROCK pher 2022) . ROCK aler 2022)   . Moreoveer 2022) . Howeverer 2022) . Also, Rer 2022) .ROCK proteins regulate different types of intermediate filaments via phosphorylation. For example, ROCK proteins can breakdown and separate the intermediate filaments via phosphorylation. It also upregulates the cell stiffness by interacting with keratin intermediate filaments ,238,239.There are six genes that either enhance (disabled 2 (Dab2), synaptopodin 2 (Synpo2) and thymus cell antigen 1, theta (Thy1)) or inhibit (cysteine and histidine-rich domain (CHORD)-containing, zinc-binding protein 1 (Chordc1), heart of glass (Heg1), and Ras interacting protein 1 (Rasip1)) ROCK activity. For example, Dab2 (on chromosome 15), which plays an important role in cell proliferation and differentiation, is capable of upregulating RhoA/ROCK signaling . SYNPO2 ROCK inhibitors act to suppress the ROCK pathway through multiple mechanisms. They can relax the trabecular meshwork through inhibiting the actin cytoskeleton contractile tone. They can induce beta-catenin nuclear translocation and inhibit cell migration or block ATP-dependent phosphorylation therefore inhibiting ROCK1 and ROCK2 and can inhibit Rho GTPases from binding to ROCK ,254,255.Although ROCK 1 and ROCK 2 are highly homologous kinases and are both involved in the Rho/ROCK signaling pathway, their target substrates and physiological/pathological activities differ. Unfortunately, most ROCK inhibitors cannot selectively target one or other of the two isoforms; such inhibitors would be very useful. Dimerization is required for the RhoE activation of ROCK 1 , and altThe cytoskeleton gives cells their shape, structure and mechanical support and plays an essential role in cellular mechanics. ROCK is an effector of the small Rho GTPase and when activated influences many cellular functions and processes by regulating different elements of the cytoskeleton. Many cellular and physiological functions are mediated by ROCK and its activity is often elevated in some disorders, making them good targets for the development of new drugs. ROCK inhibitions have been found to effectively manage several diseases in humans and animal models. Progress has been made towards understanding how non-selective ROCK inhibitors work for several diseases and conditions and efforts should be made to develop ROCK inhibitors with improved specificity and sensitivity.


CIC-rearranged sarcoma”, and the “Primary intracranial sarcoma, DICER1-mutant”. Other entities (such as rhabdomyosarcoma) have remained in the current WHO classification because these tumor types may present specificities in the CNS as compared to their soft tissue counterparts. Based on an extensive literature review, herein, we will discuss these newly recognized entities in terms of clinical observation, radiology, histopathology, genetics and outcome, and consider strategies for an accurate diagnosis. In light of this literature analysis, we will also introduce some potentially novel tumor types.The fifth edition of the World Health Organization Classification of Tumors of the Central Nervous System (CNS) now includes mesenchymal tumors that occur uniquely or frequently in the CNS. Moreover, this version has aligned the terminology of mesenchymal tumors with their soft tissue counterparts. New tumor types have been added, such as the “intracranial mesenchymal tumor, FET-CREB fusion-positive”, the “ CIC-rearranged sarcoma, and primary intracranial sarcoma, DICER1-mutant. Despite this increase in histomolecular deciphering, and because of this modified nosological organization within the classification, the proportion of mesenchymal, non-meningothelial tumors in the spectrum of all CNS tumors has artificially decreased . The WHO CNS5 is based on the cell of origin  and the advances of genetic and epigenetic data. The WHO CNS5 considerably modified the section on mesenchymal, non-meningothelial tumors. Indeed, this new version covers only tumor types that have special histopathological or molecular features, and occur uniquely in the CNS, or because they are relatively common in the CNS as compared to other tissues. On the one hand, many tumor types, which are common in soft tissue and only exceptionally found in the CNS , and have been present since 2000, have been removed from the current classification. On the other hand, new histomolecular entities have been added, like the intracranial mesenchymal tumor, FET::CREB fusion–positive, IMT, FET::CREB fusion-positive tumors develop predominantly in supratentorial sites (78% of reported cases) –29 but czonula occludens, zonula adherens, suggesting an arachnoidal origin for tumor cells [These tumors are multinodular and well-circumscribed from the brain parenchyma, frequently surrounded by a fibrous pseudocapsule. Dense lymphoplasmacytic cuffing at the tumor periphery or intratumoral lymphoplasmacytic infiltrates are typically observed. The stroma may be collagenous (with amianthoid fibers), myxoid or mucin-poor Fig. A, B 22,, 25. Theor cells , 25. TheEWSR1 (97% of reported cases) [FUS was only reported in one case [TAF15 gene has been reported to date. These data make the fluorescence in situ hybridization for EWSR1 a potentially useful diagnostic tool when histology is in line with a diagnosis of IMT  in fusions encountered in IMT [EWSR1::CREB1 fusion). Whereas no methylation class exists in the DKFZ classifier of CNS tumors (v12.5), two recent studies have shown that IMT, FET::CREB fusion-positive are characterized by a distinct epigenetic profile from other tumors of the CNS, but do not represent an homogeneous methylation class [The molecular hallmark of IMT is represented by a fusion of a FET gene with genes from the CREB (cAMP response element) family genes. In the FET family genes, fusions reported in IMT encompass mostly d cases) , 26–35 wone case  and no fIMT Fig. D. From ton class , 25. Oneon class . FurtherThe WHO CNS5 established the following essential diagnostic criteria for IMT, FET::CREB fusion-positive: 1/ primary intracranial location; 2/ variable morphological features including spindle cells, mucin-rich stroma, haemangioma-like vasculature, or epithelioid cells in a mucin-poor collagenous stroma; 3/ demonstration of a FET::CREB family fusion.CIC-rearranged sarcomas of the CNS occur in supratentorial sites (85% of reported cases) [Most d cases) –46 whered cases) , 46–48. d cases) , 44, 46.d cases) –48. Howed cases) –48. Therd cases) –48. Radid cases) –49. Tumod cases) , 44, 49 d cases) , 43, 47.d cases) , 43–48, d cases) , 47–49.CIC-fusions [CIC-rearranged sarcomas may express, only focally or in a subset of tumor cells, markers of various differentiations  [CIC::NUTM1 fusions express NUT protein [CIC-rearranged sarcomas is still unknown, the fact that soft tissue and CNS tumors share the same DNA-methylation profiling is suggestive of a common mesenchymal origin [These tumors are well-circumscribed from the brain parenchyma. They are mainly composed of diffuse sheets or lobules of undifferentiated round cells, epithelioid or even rhabdoid cells , 46–49. -fusions –10. A co-fusions , 46–49. -fusions , 49. Whee actin) , 46–48. e actin) , 46–48. e actin) , 51, WT1e actin) , 47, 49 e actin) . Sarcoma protein , 45. Thel origin .CIC::DUX4 fusion is encountered in 95% of CIC-rearranged sarcomas of the soft tissue, the molecular spectrum of CNS cases seems to be larger with different fusion partners: CIC::LEUTX (29%) [CIC::NUTM1 (29%)[CIC::DUX4 (18%) [ATXN1::DUX4 (12%) [ATXN1::NUTM1 (6%) [CIC gene (6%) [CIC::LEUTX and CIC::NUTM1 have also been reported in a subset of CIC-rearranged sarcomas [ATXN1 gene seem to be only encountered in CNS tumors. Whatever the type of fusion, a recent study has evidenced that CIC-rearranged sarcomas and ATXN1-rearranged sarcomas of the CNS share the same DNA-methylation signature and are not distinct from their soft tissue counterparts [Whereas TX (29%) , 40, 48,TM1 (29%), 45, CICX4 (18%) , 46, 47,X4 (12%) , 49, ATXTM1 (6%) , and a fsarcomas , 53, fusterparts . HoweverCIC gene fusion; 2/ predominant round cell phenotype; 3/ mild nuclear pleomorphism; 4/ variable admixture of epithelioid and/or spindle cells; 4/variably myxoid stroma; 5/ variable CD99 and frequent ETV4 and WT1 expression. The DNA-methylation profile for CIC-rearranged sarcoma is a desirable diagnostic criterion.The WHO CNS5 listed the following essential diagnostic criteria: 1/ evidence of a DICER1-mutant is almost exclusively encountered in supratentorial sites (92% of reported cases) [DICER1-mutant seems to present as a solid and cystic mass, with hemorrhage and leptomeningeal attachment, hypointensity on T1-weighted sequences, hyperintensity on T2-weighted images and a heterogeneous enhancement after gadolinium injection. Further series, including a radiological description of proven primary intracranial sarcoma, DICER1-mutant is needed to confirm these features. While the prognosis for patients with DICER1-mutant primary intracranial sarcoma remains to be determined, the literature data (36 cases) showed that 33% of patients presented local recurrences and that 86% of them are alive at the end of follow-up [As its name suggests, the primary intracranial sarcoma, d cases) –63. Howed cases) , 58, 64.d cases) –64. Howed cases) –64. The d cases) –64. Radid cases) , 59–63. ollow-up , 59–64. ollow-up .DICER1-mutant primary intracranial sarcomas are mainly well-circumscribed tumors from the brain parenchyma and may present a leptomeningeal component. Histopathologically, they are pleomorphic or composed of spindle cells arranged in fascicules or a patternless growth [DICER1-mutant primary intracranial sarcomas may present a complete or mosaic loss of H3K27me3 [DICER1 alterations have been reported in the literature  [s growth , 65, 66.s growth , 65, 66.s growth , 65, 66.s growth , 62, 63.s growth , 57–64. s growth , 61, 64.s growth , 59–63. H3K27me3 . TLE1 imH3K27me3 , but thetations) –69, but tations) , 71.DICER1 alteration is encountered in 98% of reported case [DICER1-mutant primary intracranial sarcomas are characterized by a biallelic alteration of the DICER1 gene combining a hotspot missense mutation on one allele and a truncating mutation  on the other [DICER1, as part of DICER1 syndrome, and rare cases have been reported with a familial history of cancers  [DICER1-mutant present a high level of tumor mutational burden. Indeed, associated with DICER1 alterations, recurrent mutations in the MAP-kinase pathway  and TP53 gene have been reported in 64% [DICER1-mutant primary intracranial sarcoma harbors a distinct DNA methylation profile in the v12.5 version of the CNS tumor classification [DICER1 mutation remains undetermined.A ted case –64 . DIhe other , 63, 66.cancers) , 72. Prid in 64% , 63, 72.DICER1 mutation (either germline or somatic). For unresolved lesions, the DNA-methylation profile for primary intracranial sarcoma, DICER1-mutant is mandatory.The current WHO classification has listed the following essential diagnostic criteria: 1/ primary intracranial sarcoma; 2/ pathogenic PAX3 and FOXO1 genes  or a posterior fossa mass (1 case) [FOXO1 gene fusion in diagnostically difficult cases is needed.Very few CNS cases in the literature have detailed histopathological and molecular characterizations. As reported in soft tissue, primary CNS alveolar rhabdomyosarcomas present fusions implicating nes Fig. M–P [73–7nes Fig. O [75, 76SMARCB1-deficient. This last subtype mainly concerns children (86% of reported cases) with a median age of 7 (varying from 1 to 42 years-old) [SMARCB1-deficient are associated with a poor outcome with high rates of metastases (30% of reported cases) and death (43% of reported cases) [SMARCB1 deficiency showed that poorly differentiated chordomas, SMARCB1-deficient constitute a different cluster [Chordomas have reappeared as a specific taxonomic category in the current WHO classification . This noars-old) –93. Therars-old) –93. Theyars-old) –93. Histars-old) –93. A suars-old) , 78, 93.d cases) –93. To d cluster . The WHOGJA4 mutation, recently reported in other vascular lesions from different organs  [Angioleiomyomas are well-known in the soft tissue and are classified within pericytic (perivascular) tumors in the WHO CNS5  –98. MoreNTRK gene fusions have been described in a wide variety of CNS and soft tissue tumors, including the provisional tumor type “spindle cell neoplasm, NTRK-rearranged” (SCN-NTRK), added to the 2020 WHO Classification of Soft Tissue Tumors. Because of histopathological and molecular overlaps with other soft tissue entities, controversy remains concerning the lineage and terminology of SCN-NTRK. Rare CNS primary presentations of SCN-NTRK have been reported in the literature [CDKN2A homozygous deletion) features and formed a unique and new methylation cluster . This increased complexity reflects our current understanding of biological features of CNS tumors. However, great effort is now necessary to 1/ compare them to their extra-CNS counterparts; 2/ to more precisely characterize the clinical and radiological aspects and outcomes of these new tumor types, and eventually 3/ to determine a grading (no grade is currently associated with these novel tumor types) to adapt therapeutic approaches in the future.


GELP gene family, we applied genetic evolution and molecular simulation techniques to explore molecular mechanisms in cotton species. A total of 1502 GELP genes were identified in 10 cotton species. Segmental duplication and differences in evolutionary rates are the leading causes of the increase in the number and diversity of GELP genes during evolution for ecological adaptation. Structural analysis revealed that the GELP family has high structural diversity. Moreover, molecular simulation studies have demonstrated significant differences in the properties of the binding pockets among cotton GELPs. In the process of adapting to the environment, GELPs not only have segmental duplication but also have different evolutionary rates, resulting in gene diversity. This diversity leads to significant differences in the 3D structure and binding pocket properties and, finally, to functional diversity. These findings provide a reference for further functional analyses of plant GELPs.Cotton is one of the most important crops in the world. GDSL-type esterases/lipases (GELPs) are widely present in all kingdoms and play an essential role in regulating plant growth, development, and responses to abiotic and biotic stresses. However, the molecular mechanisms underlying this functional diversity remain unclear. Here, based on the identification of the  RMS2, an endoplasmic reticulum-localized GDSL lipase gene, has a significant role in the male fertility regulatory network in rice , a lipid hydrolysis enzyme, typically have a Ser-His-Asp active site near the N-terminus . Because in rice . Additio in rice . Three G in rice . Many GEand rice . A studyis seeds . GDSL esis seeds , and oves plants . Moreoves plants .Arabidopsis . Although genome-wide identification of GELP genes in cotton has been performed, it has been limited to those in Gossypium hirsutum, unlike the systematic analysis of 10 cotton species [five diploid species  and five allotetraploid species (At/Dt)] performed in this study for evolutionary and functional aspects  background, were used. A Trans1-T1 phage-resistant chemically competent cells were purchased from TransGen Biotech . The vectors pEASY-T3 and pRI 101-AN were used for cloning and expression, respectively.In this study, the sea island cotton cultivars PS-7, Yuehai 9, and GELP candidate genes in 10 Gossypium species: G. herbaceum (A1) (G. arboreum (A2) (G. thurberi (D1) (G. raimondii (D5) (G. turneri (D10) (G. hirsutum (AD)1 (G. barbadense (AD)2 (G. tomentosum (AD)3 (G. mustelinum (AD)4 (G. darwinii (AD)5  and have been described in a previous report  , G. arboeum (A2) , G. thureri (D1) , G. raimdii (D5) , G. turnri (D10) , G. hirsum (AD)1 , G. barbse (AD)2 , G. tomeum (AD)3 , G. mustum (AD)4 , and G. ii (AD)5 . The proii (AD)5 . The GELs report .GELP genes, the Hidden Markov model (HMM) profiles of the GDSL conserved domain  from the Pfam database (http://pfam.xfam.org/) were used to scan the local database. Candidate GELPs were further verified using SMART (http://smart.embl-heidelberg.de/) and CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) online tools. Genes lacking complete GDSL-type esterase/lipase domains were excluded, splice variants were discarded, and only the first variant was retained for further analysis.To identify the cotton https://web.expasy.org/protparam/). The WoLF PSORT program (https://wolfpsort.hgc.jp/) was used to predict protein subcellular localization . Signal peptides were identified using SignalP 5.0 .Physicochemical properties, including protein molecular weight (MW), instability index (II), and theoretical isoelectric point (pI), were predicted using the ExPASy server  (1000 replicates each) (https://itol.embl.de/) were used to modify the phylogenetic tree  . iTOL totic tree .http://meme-suite.org/) was used to identify conserved motifs in the GDSL-type esterase/lipase proteins. Default parameters were used, with the exception that the maximum E-value of the motifs was set to 0.01.MEME software v5.3.0 (GELP genes were obtained from genome annotation files. Gene duplication events were obtained using the Multiple Collinearity Scan Toolkit (MCScanX)  . Ka, Ks,MCScanX) .GELP genes from these cotton species and A. thaliana were used in the analysis. To identify orthologous gene clusters, each cotton species was individually assessed and compared with each other, as well as with Arabidopsis.Orthologous proteins from ten cotton species were identified using OrthoVenn2 . All theGELP genes were obtained by searching the cotton genome database, and the online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict cis-regulatory elements.The 2 kb sequences upstream from the translational start sites of all To obtain information about the tertiary structure of GELPs, based on the phylogenetic tree, 16 protein structures were computed using two prediction methods: AlphaFold2  and homo® Structure Database (https://www.lipidmaps.org/). CDOCKER is a grid-based molecular docking method that uses CHARMm for molecular docking. Before starting the docking process, the 3D structures were optimized using the Prepare Protein protocol. After all ligands were minimized in the receptor molecule via the in situ ligand minimization program, docking between the minimized ligands and the receptor was performed using the CDOCKER program in the default mode. The results of molecular docking were evaluated based on the –CDOCKER_INTERACTION_ENERGY score and the non-bonding interactions of three conserved catalytic residues. Molecular dynamics were analysed using the Discovery Studio software.To better understand the function of cotton GELPs, we performed molecular docking using Discovery Studio software for substrate specificity. The lipid structures were obtained from the LIPID MAPSGELP genes. To analyse the expression patterns of cotton GELPs under different conditions, including growth and development, various biotic and abiotic stresses and the transcriptome data  were used to examine the relative expression patterns of cotton GELPs. Abiotic stress consisted of cold, hot, salt, and polyethylene glycol (PEG) exposure at various time intervals . The different tissues included roots, stems, leaves, thorns, petals, stamens, pistils, calyces, ovules, and fibres. The biotic stresses on cotton aphids and Aspergillus flavus were also investigated. Several time points were assessed after cotton aphid infestation . A heat map was generated for relative expression analysis using TBtools software, which was normalized by Log2  values, followed by hierarchical clustering.Gene expression profiling provides vital evidence for investigating the biological functions of the The sea island cotton materials Yuehai 9 and PS-7 were cultured using Hoagland nutrient solution hydroponics. The hydroponic seedlings were divided into two parts and treated with NaCl for short-term stress and long-term stress.-1, while the control group was continuously treated with the same amount of Hoagland nutrient solution. The leaf and whole root samples of the treatment and control groups were collected at 0, 4, 8, 12, and 24 h, frozen in liquid nitrogen, and stored at -80°C until use.Short-term stress: When cotton seedlings grow to two leaves and one heart, seedlings with consistent growth are selected and divided into five groups, with three biological repeats set for each group. The treatment group was treated with NaCl at a concentration of 300 mmol·L-1 NaCl, whereas the control group was continuously irrigated with the same amount of Hoagland nutrient solution. They were divided into two groups, and each group was set with three biological replicates. When cotton seedlings grew to two leaves and one heart, samples of leaves and whole roots in the treatment and control groups were collected, frozen in liquid nitrogen, and stored at -80°C until use.Long-term stress: From germination, the treatment group was continuously treated with 150 mmol·LGbaGELP142D in the leaves and roots of Yuehai 9 and PS-7 were performed according to Zhao et al.’s method , and the relative expression of s method . The priGbGELP142D was significantly upregulated in response to salt stress in both sea-island cotton cultivars. Total RNA from the leaves was extracted using the EASYspin Plus Plant RNA Kit RN37 (Aidlab Biotechnology) and cDNA synthesis was performed using the M-MLV RTase cDNA Synthesis Kit (TaKaRa Company). A pair of primers (GbGELP142D was designed based on its open reading frame (ORF). The resulting amplicon was ligated into the pEASY-T3 vector and confirmed using DNA sequencing.According to our transcriptomics (not published) of the sea-island cotton cultivars PS-7 and Yuehai 9 under salt stress, GbGELP142D was cloned into pRI 101-AN, and the vector was transferred into Agrobacterium tumefaciens strain GV3101. Transgenic Arabidopsis plants were created through Agrobacterium-mediated transformation using the floral dipping method , five allotetraploid species (At/Dt), and A. thaliana, respectively. A and D were used as postfixes after the names of GELP genes for the A genome or At sub-genomes, and D genome or Dt sub-genomes, respectively.A total of 1502 darwinii Figure 1GELPs from these 10 species were renamed GarGELP1A-GarGELP104A, GbaGELP1A-GbaGELP202, GdaGELP1A-GdaGELP206D, GheGELP1A-GheGELP100A, GhiGELP1A-GhiGELP206, GmuGELP1A-GmuGELP213D, GraGELP1D-GraGELP85D, GthGELP1D-GthGELP77D, GtoGELP1A-GtoGELP207D, and GtuGELP1D-GtuGELP102D , the total number of positively charged residues (Arg+Lys), instability index, whether they are stable/unstable, grand average of hydropathicity (GRAVY), aliphatic index, signal peptide prediction, number of predicted TMHs, subcellular localization, and chromosome locations are listed in The subcellular localization prediction showed that the identified GELPs were located in all parts of the cell. In each cotton species, >64% GELPs contained signal peptides, >29% GELPs contained one potential transmembrane-spanning domain, and 1–4 GELPs had two potential transmembrane-spanning domains in each cotton species Table S2GELP genes and their phylogenetic relationships in the 10 cotton species and Arabidopsis, IQ-TREE2 was used to build an unrooted phylogenetic tree. Consistent with previous studies on Arabidopsis  in the 1502 GELPs. Conserved motifs  of typical GDSL esterase/lipase proteins  were revealed, except motifs 36 and 71 for block I in subclade IIIC.To investigate All four clades contained 62 of 140 motifs. Furthermore, each clade (except clade I) contained specific motifs that represented its own subclade functions Table S4GELP genes were unevenly distributed on every chromosome in cotton. However, there were only two GELP genes (GbaGELP202 and GhiGELP206) in the scaffolds. Specifically, the GELP genes from the A1 and A2 genomes were mainly located on Chr A05, A07, A08, and A10; GELP genes from the D1, D5, and D10 genomes were primarily located on Chr D05, D07, D08, and D11; and GELP genes from five allotetraploid species were distributed mainly on Chr A05, A07, A08, A11, D05, D07, D08, D11, and D13 .Synonymous substitutions (Ks), non-synonymous substitutions (Ka), and Ka/Ks were calculated for the selected types of duplicated 0.5 to 1 Table S5GELP genes derived from the D5 genome formed orthologous pairs with all five Dt sub-genomes, 74 GELP genes derived from the A1-genome formed orthologous pairs with all five At sub-genomes, and 84 GELP genes derived from the A2 genome formed orthologous pairs with all five At sub-genomes. Gene duplication between the A/D and At/Dt sub-genomes in the five allotetraploids revealed that the Ka/Ks ratios of orthologous pairs were different among the five allotetraploids . The identified orthologous gene clusters and their overlapping regions in the 11 species are shown in A. thaliana. In addition, there were 102 orthologous gene clusters which were solely composed of genes found in the cotton species, whereas A. thaliana had seven exclusive orthologous gene clusters. Most clusters were recorded from (AD)5, followed by (AD)2, (AD)4, (AD)3, (AD)1, A2, D10, A1, D5, and D1. These results revealed that polyploidization resulted in the evolution of new cotton-specific orthologous gene clusters.To assess polyploidization events in the evolution of A. thaliana separately. Between 1144 and 1176 orthologous genes were identified in the five allopolyploid species, 843, 864, 670, 745, and 858 orthologous genes were identified in A1, A2, D1, D5, and D10, respectively, whereas 354 orthologous genes were identified in A. thaliana 1, and (AD)4; four in-paralogs were detected in (AD)5; 234 in-paralogs were found in A. thaliana; but no in-paralogs were detected in other species. Between 11 and 14 singletons were identified in the five allopolyploid species, and 1 to 3 singletons were observed in the A and D genomes. The findings mentioned above indicate that the GELP gene family diverged across different species during evolution and was more likely to perform distinct functions.We also constructed orthologous gene clusters between each cotton species as well as with thaliana Table S7cis-elements in conjunction with transcription factors. To explore the possible roles of GELPs in plant hormones, stress stimuli, and development, PlantCARE was used to analyse putative cis-elements in the promoter region (2.0 kb upstream ATG). This study investigated numerous putative cis-acting elements (71 types). Of these, five abiotic stress-responsive elements , 13 hormone-responsive elements , cell cycle regulation (MSA-like), circadian control (circadian), differentiation of the palisade mesophyll cells (HD-Zip 1), endosperm expression (GCN4_motif/AACA_motif), flavonoid biosynthetic genes regulation (MBSI), meristem expression (CAT-box), root-specific (motif I), phytochrome down-regulation expression (Unnamed_1), seed-specific regulation (RY-element), wounding and pathogen responsive (W box/WUN-motif), MYB, MYC, and light-responsive cis-elements. Overall, cis-elements related to abiotic stress, hormones, light-response, growth, and development were widely present in the promoters of GELP genes in the 10 cotton species. These results suggest that these GELP genes play essential roles in regulating growth and development, as well as in regulating responses to different sources of stress in cotton.The transcription level of a gene is controlled by https://www.rcsb.org/). Sixteen 3D structures of GELPs from each subclade were predicted using the homology modelling method. According to the primary sequence identity and crystal resolution models, 1–3 template crystal structures with six templates  were used for homology modelling in this study for the 16 GELPs  database using the BLAST algorithm  were used to determine the active sites of GELPs. From the molecular docking results, it can be inferred that the larger values of –CDOCKER_INTERACTION_ENERGY represent a better affinity of the derivative for GELPs. Ligands with –CDOCKER_INTERACTION_ENERGY values greater than 0 were used to analyse the substrate specificity of the GELPs. Molecular docking screens indicated that the 16 GELPs contained 2526 ligands and 797 unique lipids. These lipids included 17 classes, including fatty esters, fatty acids and conjugates, fatty aldehydes, oxygenated hydrocarbons, and fatty alcohols, of which numbers were greater than 7%. We also found that GdaGELP128D in clade I contained 40 unique lipids, whereas GdaGELP59A in clade IV had 490 ligands. After docking, GhiGELP172D of clade IIC had zero candidate molecules with docking scores above 0. The ligands docked to the GELPs were mostly fatty acyls, such as (3Z)-buta-1,3-diene-1,1,4-tricarboxylic acid, 10-hydroxy-2Z,8Z-Decadiene-4,6-diynoic acid, 6-hydroxy-2-hexynoic acid, iodoethanoic acid, and 3-hexynoic acid. All four clades collectively had 136 unique lipids, and each clade contained specific lipids   Figure 5GELP genes across tissues. The results revealed that the expression of many GELP genes varied significantly among different tissues. For example, Gh_A05G1329 and Gh_D10G0307 showed significantly upregulated expression at 3 DPA and 5 DPA in the ovule, and 5 DPA and 10 DPA in the fibre  decreased with increasing treatment time. However, only a few genes (Ghir_D08G023300) exhibited elevated expression levels . In addition, Ghir_D06G014530 was differentially expressed under biotic stresses (cotton aphid and aflatoxin from the fungus A. flavus). After analysing the data, the orthologous genes of GbaGELP142D are Gh_D06G1406 and Ghir_D06G014530 in G. hirsutum. From these results, it can be speculated that GbaGELP142D plays an important role in salt stress resistance.Moreover, the expression of GbaGELP142D was performed in the sea island cotton materials Yuehai 9 and PS-7, which were treated with NaCl short-term stress and long-term stress, respectively. It can be observed in GbaGELP142D in the roots and leaves of Yuehai 9 was not significantly different. However, after 24 h of NaCl treatment, the expression of GbaGELP142D in the leaves of Yuehai 9 increased 27.98 times compared to that in the roots. It can be observed in GbaGELP142D in the roots and leaves of PS-7 was the largest and the difference was significant, and the expression of GbaGELP142D in roots was 7.19 times than that in leaves. With the extension of NaCl stress time, the expression of GbaGELP142D in the roots and leaves of PS-7 decreased rapidly, reaching a minimum at 8 and 12 h of NaCl stress, respectively.The qRT-PCR analysis of GbaGELP142D in the leaves of island cotton Yuehai 9 and PS-7 was 230 and 31 times higher than that in the roots, respectively  were selected by qRT-PCR to examine salinity resistance  values suggest increased flexibility, whereas low RMSF values reflect decreased flexibility. RMSF analysis of the best compounds revealed that the amino acid residues of the binding pocket showed a flexible conformation. Based on the analysis mentioned above, we presumed that GELP genes are present in plants. Different numbers (65–194) of GDSL genes have been reported in different plants , the Hawaiian Islands (Gto), South America (north-eastern Brazil) (Gmu), Central and South America, the Caribbean, and the Pacific (Ghi and Gba)  . These rn cotton . TherefoGELP gene family contain largely cis-acting motifs related to plant growth and development, hormones, biotic stress, and abiotic stress. Subcellular localisation prediction showed that GELP genes were present in all parts of the cell. More than 64% of GELPs contain signal peptides and >29% of GELPs contain one potential transmembrane domain. The expression patterns of a large number of GELP genes were specifically expressed in different tissues or developmental stages and were differentially expressed under different stresses  and biotic (cotton aphid attack) stresses. It has previously been shown that GELPs exert essential functions in many physiological and biochemical processes . Recently, as a novel machine learning approach, AlphaFold2 has been used to predict protein structure based on the amino acid sequence and has made landmark advances in protein structure prediction (in vitro and in vivo (The reasons for the difficulty in determining a suitable substrate are as follows: (1) Unlike other lipases, GDSL lipases have a flexible active site in their structure that leads to the binding of different substrates, resulting in more diverse enzyme activities . (2) Homediction . (3) Manediction . Therefo in vivo .GELP gene family in cotton. The 3D structures of the 16 GELP lipase proteins were generated using AlphaFold2 and homology modelling. The results highlighted notable differences in the structures of the 16 GELPs. Similar results were reported by To overcome this challenge, we used molecular simulation techniques to analyse the 3D structure of GELPs and assess the properties and conformational changes of the binding pocket based on the phylogeny and genetic analysis of the The GELPs in subclade IIIC had unique structures. For example, typical GDSL were located in motifs 36 and 71 in block I. The 3D structures and substrates showed larger differences compared to the other subclades. Therefore, it can be inferred that GELPs in subclade IIIC have special functions.GDSL lipases have a flexible active site in their structure that leads to the binding of different substrates, resulting in more diverse enzyme activities. For example, because of the flexibility of the substrate-binding pocket of TEP-I, it can bind different substrates in optimal conformations for catalysis, making it an extremely versatile enzyme, such as a lysophospholipase, arylesterase, protease, and thioesterase . The binTherefore, to characterise the binding pocket to uncover the diverse functions of GELPs, it was found that the properties of the binding pockets include not only the volume, shape, hydrophobicity, electrostatics, and chemical fragment interactions, but also dynamic properties such as stability, continuity, and correlation . The proThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/JW, HZ, YQ, JH planned and designed the study. JW and HZ performed experiments. JW wrote the manuscript. HZ and PY provided principal funding. We have read, edited, and approved the manuscript for publication. All authors contributed to the article and approved the submitted version.