MTDH (Q86UE4), AKT1 (P31749), PTPN1 (P18031), MYC (P01106), GSK3B (P49841), RHOA (P61586), and PIK3CG (P48736) and put forth several novel interactors, with AKT3 (Q9Y243), NCOR2 (Q9Y618), PIK3R2 (O00459), SMAD4 (P61586), and TGFBR1 (P36897). Double-stranded RNA duplexes of the strongest interactors were found to exhibit higher binding affinity with AGO. In conclusions, the study has explored the role of miR-122 in CRC and has identified a closely related group of genes influencing the prognosis of CRC in multiple ways. Further, these genes prove to be targets of gene silencing through RNA interference and might serve as effective therapeutic targets in understanding and treating CRC.The role of microRNA 122 (miR-122) in colorectal cancer (CRC) has not been widely investigated. In the current study, we aimed to identify the prominent gene and protein interactors of miR122 in CRC. Based on their binding affinity, these targets were chosen as candidate genes for the creation of miR122–mRNA duplexes. Following this, we examined the miRNA-mediated silencing mechanism using the gene-silencing complex protein Argonaute (AGO). Public databases, STRING, and GeneMANIA were utilized to identify major proteins and genes interacting with miR-122. DAVID, PANTHER, UniProt, FunRich, miRwalk, and KEGG were used for functional annotation, pathway enrichment, binding affinity analysis, and expression of genes in different stages of cancer. Three-dimensional duplexes of hub genes and miR-122 were created using the RNA composer, followed by molecular interaction analysis using molecular docking with the AGO protein. We analyzed, classified, and scrutinized 93 miR-122 interactors using various bioinformatic approaches. A total of 14 hub genes were categorized as major interactors of miR-122. The study confirmed the role of various experimentally documented miR-122 interactors such as  The discovery of non-coding RNAs and their function as disease regulators has shifted the outlook of viewing RNAs from mere expression mediators to a biomolecule that aids the evolution of complex organisms. The past decade has witnessed a constant rise in non-coding RNA research, including the widely studied microRNAs (miRNA), a group of non-coding gene expression regulators that inhibit protein biosynthesis by binding to specific mRNAs and initiating the cleavage of primary miRNA that in turn adheres to the RNA-induced silencing complex (RISC) for beginning the downstream processes ,2. This TRIM29, involved in PI3K/AKT regulation, Bcl-W, and CCNG1 involved in apoptosis and cell cycle regulation [AEG-1 (MTDH), CDK8, AKT, PCNA, NFKB, and PI3K [Serum and plasma miRNAs are regarded as promising biomarkers in the field of oncology . One sucgulation . Other igulation , the XIAgulation , and ALDgulation . Similarand PI3K . Furtherand PI3K . Argonauand PI3K . The appp-value ≤ 0.05 were identified comprising twenty-three and nine genes  analysis consisted of six categories including cellular component (CC), biological process (BP), molecular function (MF), transcription factor (TF), site of expression (SE), and clinical phenotype (CP). The CC analysis A exhibitAKT1, PIK3CG, PIK3R2, AKT3, GSK3B, MYC, RHOA, TGFBR1, SMAD4, NCOR2, MAPK1, MAPK11, PTPN1, and MTDH , a total of 14 genes were scrutinized as Hub genes, this includes and MTDH .AKT1, AKT3, GSK3B, MAPK1, MAPK11, MTDH, MYC, NCOR2, PIK3R2, and SMAD4 to be present at the nucleus  was performed with the AGO protein. The interaction of miR-122–PIK3CG with AGO exhibited a carbon–hydrogen bond at ARG173 and MET172, electrostatic bonds at ARG173 and GLU122. The miR-122–RHOA–AGO complex interacted with conventional hydrogen bonds at LYS39, ARG395: HE, ARG395:HH21, and ARG396:HH21, while the miR-122–SMAD4–AGO complex demonstrated hydrogen linkages at LYS39 and LYS212, electrostatic bonds at ASP48, and hydrophobic bonds at LEU210 and MET400. Further, the miR-122–PTPN1–AGO complex exhibited a strong hydrogen bond at LYS402 and LYS739, electrostatic bonds at GLU46, and a hydrophobic interaction at TRP211. Finally, the miR-122–MTDH–AGO complex exhibited hydrogen bonds at ARG90:H and ARG90:NH2, electrostatic interactions at MET400, and hydrophobic interactions at ILE86. The presence of hydrogen bonds in the protein–nucleic-acid interaction further highlights the role of the AGO protein in gene regulation through miRNA and in RNA interference .miR-122 has been regarded as a potential tumor suppressor miRNA in various types of cancers including gastric cancer, rectal cancer, hepatocellular carcinoma, gallbladder cancer, uveal melanoma, and breast cancer ,24. The The mRNA interactors of miR-122 were primarily obtained from the mirNet database , but themiR-122 has emerged as an anti-proliferative and apoptotic component in cancerous cells, given its association with the PI3K/Akt signaling pathway . ConfirmBiological pathways play a major role in the regulation of all cellular processes including cancer progression and metastasis. The molecular pathways identified in CRC include chromosomal instability pathways, CpG methylator phenotype pathways, and microsatellite instability (MSI). Each pathway has been associated with a series of genes that tend to either upregulate or downregulate at different stages of cancer . The genIn total, nine of the fourteen hub genes, i.e., AKT1, AKT3, GSK3B, MAPK1, MYC, PIK3CG, PIK3R2, SMAD4, and TGFBR1 were present in both the STRING and GeneMANIA databases, analysis of MCODE further classified six of the hub genes, AKT1, AKT3, MAPK1, MAPK11, RHOA, and SMAD4 to be present in cluster one A, the laThe topology of the CRC proteins spans from transmembrane proteins such as TMEM180  to extraThermus thermophilus HB27) as the expression system was Escherichia coli, and was further modelled using Discovery Studio Visualizer 3.5 suite prior to molecular docking [The AGO protein serves as a primary regulator of RNAi by modulation of the post-transcriptional gene expression . In the  docking . The abi docking . The gen docking . FollowiAKT1, AKT3, GSK3B, MAPK1, MAPK11, MYC, NCOR2, PIK3R2, RHOA, SMAD4, and TGFBR1 ), a web-based tool for miRNA centric network building and analyses [The list of possible interactors of the microRNA-122, namely, genes and transcription factors (TF) along with their immediate interactors using experimentally validated STRING entries, were screened, and enriched from miRNet 2.0 ) [PANTHER classification was employed to predict the protein class of miR-122 interacting genes ,50. Kyotr 2021)) ,54. The r 2021)) .https://genemania.org/ (accessed on 10 February 2021)) [http://strin g-db.org (accessed on 10 February 2021)) [A gene interaction network was visualized using the curated list of miR-122 interactors through Cytoscape version 3.8.0 using GeneMANIA (y 2021)) ,57 and ay 2021)) . The MCLy 2021)) . Module y 2021)) .p value of ≤0.05 was maintained in all cases. The consortium of transcription factors and the interacting mRNAs were visualized using the Cytoscape 3.8.0 software and further, the interrelationship of transcription factors was predicted using STRING app in Cytoscape [The Enrichr online tool was used to analyze the major transcription factors interacting with the gene set using the TRANSFAC_and_JASPER_PWMs category of the database. A statistically significant ytoscape .Hub genes from identified clusters and enriched pathways were plotted based on the degree of connectivity  through http://mirwalk.umm.uni-heidelberg.de/ (accessed on 10 February 2021)), containing a repository of miRNA-target binding site analysis was used to identify the binding regions of miR-122 with the hub genes. The miR-122–mRNA interaction affinities were analyzed based on the binding site at 3’ UTR, coding sequence, and 5’ UTR regions, starting and ending regions of the binding targets, and binding site length [The MiRWalk 2.0 database (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi (accessed on 24 August 2022)) [http://rnacomposer.cs.put poznan.pl/ (accessed on 24 August 2022)) [The secondary structure of the miRNA and the target genes were designed using the RNAfold web server (t 2022)) . Followit 2022)) .The AGO protein, the major gene regulator was extracted as a three-dimensional structure from Protein Data Bank . Further structural refinement was carried out in Discovery Studio Visualizer 3.5 suite ,66.The protein–nucleic acid docking was carried out using the Haddock server. The RNA duplexes of the major interactors of miR-122  were selected based on the binding affinity scores and were docked with the AGO protein. The most prominent geometrical shape complementary score was produced by the Haddock algorithm. Further molecular interactions were analyzed using the Discovery Studio Visualizer 3.5 suite ,67.In the current study, we identified the diverse range of miR-122 interactors from several public databases. After further classification, we proceeded to analyze the protein class, subcellular localization, molecular function, biological function, cellular components, the associated pathways, and analysis of networks interacting with miR-122 and its interactors using a range of bioinformatics tools. The synergy of miR-122 interactors and the corresponding pathways was established. Further studying the subcellular localization of the major interactors and miR-122 confirmed their presence in the nucleus. Through analysis of the KEGG pathway, the miR-122 interactors chiefly fall under the categories “Pathways of Cancer” and “Colorectal Cancer”. In addition, the network analysis of the interacting proteins, genes, and transcription factors has led to scrutinization of fourteen hub genes that have a higher degree of interaction with miR-122. Five of these fourteen genes exhibited promising interaction sites with miR-122; hence, miRNA–mRNA duplexes were created with the genes and miR-122. These genes including PIK3CG, RHOA, SMAD4, PTPN1, and MTDH served as candidate genes for gene expression analysis through molecular docking analysis with the AGO protein. Hence, through the current research, we emphasize the role miR-122 bears as a major interactor and modulator of genes associated with CRC.


Psoriasis vulgaris (PV), as a primarily Th17-driven inflammatory skin disease with proven inflammation-induced accumulation of sodium chloride in the skin, merits our interest in the role of oxidative stress in the pathogenesis of PV, as well as in the possible beneficial effects that could be achieved through modulation of dietary salt intake and antioxidant supplementation.Although oxidative stress is recognized as an important effector mechanism of the immune system, uncontrolled formation of reactive oxygen and nitrogen species promotes excessive tissue damage and leads to disease development. In view of this, increased dietary salt intake has been found to damage redox systems in the vessel wall, resulting in endothelial dysfunction associated with NO uncoupling, inflammation, vascular wall remodeling and, eventually, atherosclerosis. Several studies have reported increased systemic oxidative stress accompanied by reduced antioxidant capacity following a high salt diet. In addition, vigorous ionic effects on the immune mechanisms, such as (trans)differentiation of T lymphocytes are emerging, which together with the evidence of NaCl accumulation in certain tissues warrants a re-examination of the data derived from in vitro research, in which the ionic influence was excluded.  Psoriasis is a chronic, self-perpetuating inflammatory skin disease with apparent disease-related systemic comorbidities  that significantly reduces quality of life, and places a high socioeconomic burden on those affected by it . A speciPsoriasis has a relatively high incidence and prevalence worldwide, with significant differences between countries, reflecting their geographical location and socioeconomic status . AccordiIn recent years, significant advances in understanding the etiopathogenesis of the disease have led to the development of better treatment options, most notably biologics targeting tumor necrosis factor (TNF)-alpha(α) or the interleukin(IL)-23/IL-17 axis . HoweverStreptococcus spp.), obesity, and some drugs  to the onset of the disease i amplitude has been observed in response to the addition of muscarinic receptor agonist methacholine, as well as histamine [2+]i response, suggesting that the impairment of Ca2+ signaling may not be a direct effect of O2●−. Conversely, chronic exposure to Tempol added to the drinking water before and during HS feeding increased the methacholine-induced amplitude of [Ca2+]i in HS rats. Zhu et al. suggested that the possible explanation for this difference in acute and chronic Tempol exposure on the Ca2+ signaling pathways in HS diet might be due to the damage caused by the continuously elevated O2●− to G protein/receptor-mediated signaling in endothelial cells in the aortas of rats fed a HS diet [2+]i in endothelial cells, an effect attenuated by the addition of ROS scavengers, the mechanisms of HS dietary load on this attenuated amplitude of the [Ca2+]i increase in the rat aorta have yet to be elucidated [Even though oxidative stress in HS diet primarily affects vascular homeostasis through the reduction of NO bioavailability, as demonstrated previously, it also may affect alternative sites in the cell, such as nucleic acids, proteins and lipids, and consequently lead to a change in several biochemical and physical processes . For exaistamine . Interes HS diet . Given tucidated ,78. Furthermore, short-term HS diet in young healthy individuals has been linked to elevated levels of 8-iso-prostaglandin F2α (8-iso-PGF2α), a biomarker for oxidative stress, which is formed through non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA), such as arachidonic acid ,79. Impo2●−. In addition, an in vitro study on lamina propria mononuclear cells (LPMC) showed enhanced expression of TNF-α and IL-17a in the presence of increased NaCl compared to LPMC in the absence of increased NaCl concentrations. Furthermore, this increase in expression of TNF-α and IL-17a was prevented by the addition of SB202190, a p38/MAPK inhibitor, leading to the conclusion that p38/MAPK has a role in the sodium-induced increased expression of TNF-α and IL-17a in LPMC [In an in vitro study using bifurcating flow-through HUVEC  cell culture mimicking arterial bifurcation, it was shown that high sodium conditions greatly increase endothelial cell susceptibility to the effects of TNF-α in conditions of non-uniform shear stress and that this effect of HS is concentration-dependent. Thus, incubation with increased sodium concentrations in vitro induced both increased expression of VCAM-1 and E-selectin and led to greater monocyte adhesion to endothelial cells in the model with non-uniform shear stress . However in LPMC . Still, As previously mentioned, psoriasis is a disease with genetic heredity, which still has not been fully explained. However, numerous exogenic trigger factors for psoriasis, well-known for their prooxidative influence, have been recognized, e.g., cigarette smoking, consumption of alcoholic beverages and narcotic drugs, physical and psychological stress, and infections and physical injuries . Psorias2O2 production in vitro and that this effect is dose-dependent [2O2, O2●− and NO have been reported in the skin of patients with psoriasis [2●− superiorly to HK [In a study on primary human keratinocytes, it was shown that TNF-α, a prominent cytokine in the pathogenesis of psoriasis, induces Hependent , which iependent ,93. Corrsoriasis . In addisoriasis ,95. In cly to HK . ∙2) and hydroxyl radical (HO∙), react with lipids having carbon-carbon (C-C) double bonds, e.g., PUFA [It is now widely recognized that enhanced lipid peroxidation, occurring in the setting of excessive ROS levels, plays a role in the pathogenesis of psoriasis ,97. Lipig., PUFA . The prig., PUFA ,100,101,g., PUFA . In factg., PUFA , linked g., PUFA . It has g., PUFA , even thg., PUFA . Furtherg., PUFA ,107,108,g., PUFA . Also, Mg., PUFA . In addig., PUFA . Furthermore, in patients with psoriasis, higher levels of ox-LDL have been detected in lesional skin than in non-lesional skin . AnotherFurthermore, ROS can also influence cellular signal transduction pathways, also known to play an important role in the pathogenesis of psoriasis, such as mitogen-activated protein kinase/activator protein 1 (MAPK/AP1), nuclear factor κB (NF-κB) and Janus kinase-signal transducers and activators of transcription (JAK/STAT) ,118,119.+-dependent protein deacetylase that removes acetyl groups from various proteins and also acts as a transcription factor, may be important in psoriasis. As SIRT1 endorses keratinocyte differentiation and is able to inhibit keratinocyte proliferation [In addition, levels of sirtuin 1 (SIRT1), a NADferation ,122, theferation ,123,124.feration . Dietaryferation . In addiferation . Members of MAPKs family (Ser/Thr protein kinases), which are included in redox-sensitive pathways and play a role in cellular signaling of oxidative stress, are the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and the p38 MAPK ,126,127.2, H2O2, NO and ONOO [In addition, the activity of p38 MAPK ,134, is and ONOO ,138,139.and ONOO . In addiand ONOO . There iand ONOO ,142,143.Furthermore, ROS leads to degradation of the inhibitory protein I-κB, which, due to the attenuated inhibition, causes greater transcriptional activity of NF-κB, which then further induces the expression of pro-inflammatory cytokines involved in the pathogenesis of psoriasis ,145,146.2O2 [Another redox-sensitive pathway important in psoriasis is JAK/STAT. STAT 1 and STAT3 are activated in fibroblasts in response to the addition of H2O2 . JAK/STA2O2 . Importa2O2 . As regards the antioxidative defense mechanisms in psoriasis, evidence seems to be controversial. On the one hand, the evidence from most studies supports a weakened antioxidative status, reporting decreased SOD and CAT levels, which negatively correlate with disease severity assessed by PASI ,158,159,23Na magnetic resonance imaging (MRI) compared to healthy controls, and they subsequently reaffirmed those findings by atomic absorption spectrometry measurements on skin biopsies ex vivo. In addition, sodium content in non-lesional skin was elevated and positively correlated with PASI. Interestingly, sodium content did not differ significantly between lesional and non-lesional skin [As discussed in the previous section, sodium can potentially cause oxidative stress through enhanced prooxidant and weakened antioxidant activity . Importanal skin . Since tnal skin . These fnal skin . There inal skin . Naïve T helper cells have an inherited capacity to adapt their maturation process after an antigen encounter in relation to their changing environment and the nature of antigenic challenge. Their tremendous plasticity is reflected in a growing family of Th subsets, including Th1, Th2, Tfh, Th17, Th9 and Th22 etc. . ClassicMoreover, recent studies have provided evidence for divergent post-activation fates of T-cells when local conditions related to cytokine milieu have changed. For example, presence of proinflammatory cytokines, IL-6 alone or in combination with IL-1β, can drive transdifferentiation of Foxp3+ regulatory T cells into either Th17 or Treg/Th17 cells, respectively . That wa+) and negatively charged chloride ions (Cl−). Free Na+ ions then rapidly establish noncovalent bounds with negatively charged tissue macromolecules, such as glycosaminoglycans and hyaluronic acid, thus preventing determination of their precise tissue concentrations [23Na) magnetic resonance imaging, have brought to light the fact that tissue sodium content dynamically adapts to dietary changes in salt intake and exhibits variable sodium tissue content depending on the type of tissue, sex, age, and inflammation status [It was long postulated that NaCl concentrations in body fluids and tissues are tightly regulated by the kidneys and the endocrine system. Assessment of serum sodium concentration in animals and humans under various physiological and pathological conditions showed that it is maintained within a narrow range ,179. Howtrations . Advancen status ,181,182.+ and produce vascular endothelial growth factor C (VEGFC) leading to lymph capillary hyperplasia/lymphangiogenesis and increased salt clearance from cutaneous deposits [Tissue particularly sensitive to dietary changes in sodium intake is the skin . Furtherdeposits . IllustrMo, monocytes; ILC3, innate lymphoid cell 3; Tc, cytotoxic T cell; MAIT, mucosa associated invariant T cell; pDC, plasmacytoid dendritic cell; mDC, myeloid dendritic cell; Treg, regulatory T cell; Th, T helper cell; Tem, effector memory T cell; GAG, glycosaminoglycans; VEGF-C, vascular endothelial growth factor C; IL, interleukin; TGFβ, transforming growth factor beta; γδT, gamma delta (γδ) T cells.Moreover, this mechanism seems to be important for blood pressure regulation during acute salt loading, since in an experimental animal model, VEGF-C receptor blockade led to an immediate blood pressure increase ,185. ThiIn the case of psoriasis, a prototypical TNFα–Th17 disease model, recent data demonstrated increased sodium and water retention in lesional and non-lesional skin, but only in patients with a moderate to severe form of the disease (PASI > 5), and not in those with a mild form (PASI < 5). Sodium accumulation correlated to the disease severity and frequencies of peripheral blood IL-17-secreting ɣδ T cells . ConsistC. albicans-specific Th17 responses depend on the presence of IL-1β, thus such effector cells respond to hypertonic NaCl conditions by amplification of Th17 cell–associated proinflammatory effector functions [Pioneering studies onionic-salt-induced effects on the Th17 cell differentiation also demonstrated increased Th cell pathogenic effector functions exerted by high NaCl . Namely,unctions , as illuThe effects of hypersaline conditions on Th17 differentiation and phenotype depend on the presence of pro-inflammatory cytokines such as IL-1β and IL-6. In addition, specific microbes possess differential ability to induce IL-1 production in antigen-presenting cells. Hence, increased NaCl conditions may have both pro-inflammatory and anti-inflammatory effect on the Th17 cells in response to invading microbes. For example, hypersaline microenvironment has been shown to increase the anti-inflammatory functions of S. aureus-specific Th17 lymphocytes, while it promotes the proinflammatory functions of C. albicans-specific Th17 cells; LL37, cathelicidin antimicrobial peptide LL37; HBD, human beta defensin; S100, calcium-binding protein S100; IL, interleukin; TGF-β, transforming growth factor β; FOXp3, forkhead box P3; ROS, reactive oxygen species; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; Th17, T helper 17 cells; Th1, T helper 1 cells; RORγt, retinoic-acid-receptor-related orphan nuclear receptor γ; Mo, monocytes.Taken together, the effects of hypersaline tissue microenvironments on the Th17 cells are divergent, both pro-inflammatory and anti-inflammatory, and dependent on the context, primarily on the presence of pro-inflammatory cytokines like IL-1β and IL-6. These findings have possible implications for therapeutic strategies in the treatment of chronic autoimmune diseases. Targeted abrogation of the IL-1 signaling pathway by IL-1- or IL-1R-blocking agents could induce anti-inflammatory Th17 responses in high-NaCl tissue microenvironments. Alternatively, tissue salt accumulation reduced through modification of dietary habits may possibly promote anti-inflammatory Th17 effector functions in patients with psoriasis and other autoimmune diseases.Since the same pro-inflammatory IL23/Th-17 axis is known to promote both psoriasis and cardiovascular diseases (CVD), psoriasis could be considered as an independent risk factor for development of CVDs  and increased cardiovascular mortality ,191. Endothelial dysfunction (ED), an early sign of CVDs, encompasses a number of pathophysiological conditions, ranging from initial localized mechanical injury to inappropriate, persistent, global endothelial activation. ED is characterized by vasoconstriction, pro-thrombotic and pro-inflammatory phenotype ,193, andOver the last 40 years, most studies on endothelial function in psoriasis have reported impaired macrovascular endothelial function in psoriasis patients, as assessed by flow mediated dilation (FMD) of brachial artery ,204,205,Surprisingly, there is little data on microvascular endothelial function in psoriasis patients, particularly data investigating the cutaneous microcirculation, whose impairment may play an important role in pathophysiology of cardiovascular and metabolic diseases, especially diseases specifically affecting the skin , as is tFew studies examined available biomarkers of endothelial function in psoriasis patients, and the results obtained were in line with the findings of functional vascular studies. Abdou et al. reported that psoriatic skin exhibited over-expression of endocan , reflecting the ongoing endothelial activation and neovascularization . SimilarAt present, there is limited systematic data evaluating the potential clinical implications of the above findings and investigating how biologic therapy may affect endothelial function and CV risk in psoriasis patients. Several studies have shown that TNF-alpha inhibitors improve endothelial function in psoriasis  provide evidence on significantly impaired macrovascular endothelial function in psoriasis patients, possibly induced by the modulation of inflammatory responses  and increased levels of oxidative stress. However, unexplored endothelial function in microcirculation , as well as a more precise assessment of the role of oxidative stress in the pathogenesis of endothelial dysfunction in psoriasis patients open up new avenues for future research. In the past decades, it has become clear that psoriasis, as a systemic inflammatory condition, may be accompanied by various systemic comorbidities, including psoriatic arthritis, cardiovascular and cerebrovascular diseases, hyperlipidemia, hypertension, obesity, diabetes and psychiatric disorders like depression and anxiety ,259. Amo18F-fluorodeoxyglucose positron emission tomography computed tomography (18-FDG PET/CT), which appears to have prognostic value for future CV events, was increased and associated with longer disease duration [Even though ample evidence from multiple studies shows that psoriasis patients have a higher burden of the so-called traditional CV risk factors, such as cigarette smoking, obesity, elevated blood pressure, insulin resistance or type II diabetes mellitus, and hyperlipidemia ,273,274,duration ,276,277.duration . Howeverduration .Moreover, it has been suggested that even in mild-to-moderate psoriasis there is a greater risk to develop severe cardiovascular events, since elevated carotid intima-media thickness (IMT), decreased flow mediated dilation (FMD) and greater levels of advanced oxidative protein products (AOPPs) have all been reported in psoriasis patients compared to non-affected individuals; but no difference in pulse wave velocity (PWV) has been observed. However, since the upper threshold for moderate psoriasis in this report was defined as PASI score of 50 and the mean PASI was above 18 , which iFurthermore, two different meta-analyses confirmed that there is a higher relative cardiovascular risk, particularly in younger individuals suffering from psoriasis, and in those with more severe clinical presentation ,283. It The importance of endothelial dysfunction also lies in the fact that this is a quantifiable condition reflecting early reversible damage to vascular homeostasis , precediIn a report from a prospective cohort study, flow-mediated dilation (FMD), a measurement technique for endothelial dysfunction, negatively correlated with cardiovascular events independently of other, traditional CV risk factors , underliSodium is an electrolyte crucial for numerous processes in the organism and is therefore considered to be an essential nutrient, necessarily present in small quantities in the human diet. However, in most Western and Asian countries, the dietary intake of sodium is markedly surpassing the maximum daily sodium intake of 2000 mg  recommended by the World Health Organization (WHO) guidelines established to prevent diet-related non-communicable diseases ,294,295.n-3 polyunsaturated fatty acids (PUFAs) resulted in clinical improvement in patients suffering from psoriasis [Diet and nutrition are generally considered to be a modifiable risk factor in the occurrence of many diseases, including autoimmune diseases . The idesoriasis ,304,305.As previously stated, while the serum sodium level is regulated to be closely maintained at around 140 mmol/L , it has Since LS diet can lead to the recovery of endothelial microvascular function in both normotensive and hypertensive individuals ,309, theLS intake could lead to the recovery of endothelial dysfunction, which is the first, but still reversible, step in the process of atherogenesis . This coIn addition, it has been shown that psoriasis patients have an unhealthy diet characterized by consuming more simple carbohydrates, total fat and n-6 PUFA, with less fibers, proteins, complex carbohydrates, fruits, vegetables and n-3 PUFA when compared to healthy controls ,312, whiVitamin E (α tocopherol) is a liposoluble antioxidant vitamin, known to prevent lipid peroxidation ,319, whiSince selenium is an essential component of selenoproteins, such as glutathione peroxidase (an important cellular antioxidant enzyme), its nutritional supplementation has been studied in psoriasis patients in a few studies . In addiIn addition, it has already been mentioned that the intake of n-3 PUFA, i.e., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which also have antioxidant properties, is lower in patients with psoriasis than in controls ,312,327.


Background: Neurodevelopmental disorder with dysmorphic facies and distal skeletal anomalies (NEDDFSA) is a rare syndromic disorder characterized by global neurodevelopmental delay, early-onset hypotonia, poor overall growth, poor speech/language ability, and additional common phenotypes such as eye anomalies, joint hypermobility, and skeletal anomalies of the hands and feet. NEDDFSA is caused by heterozygous pathogenic variants in the ZMIZ1 gene on chromosome 10q22.3 with autosomal dominant (AD) mode of inheritance. All the 32 reported cases with variants in ZMIZ1 gene had a genetic background in Caucasian, Hispanic, North African, and Southeastern Asian. Until now, there are no reports of Chinese patients with ZMIZ1 pathogenic variants.Methods: A 5-year-old girl was found to have the characteristic phenotypes of NEDDFSA. Array-Comparative Genomic Hybridization (array-CGH) and whole exome sequencing (WES) were applied for the trio of this female patient. Sanger sequencing was used to verify the selected variants. A comprehensive molecular analysis was carried out by protein structure prediction, evolutionary conservation, motif scanning, tissue-specific expression, and protein interaction network to elucidate pathogenicity of the identified ZMIZ1 variants.Results: The karyotype was 46, XX with no micro-chromosomal abnormalities identified by array-CGH. There were 20 variants detected in the female patient by WES. A de novo heterozygous missense variant  was identified in the exon 20 of ZMIZ1. No variants of ZMIZ1 were identified in the non-consanguineous parents and her healthy elder sister. It was predicted that G777E was pathogenic and detrimental to the spatial conformation of the MIZ/SP-RING zinc finger domain of ZMIZ1.Conclusion: Thus far, only four scientific articles reported deleterious variants in ZMIZ1 and most of the cases were from Western countries. This is the first report about a Chinese patient with ZMIZ1 variant. It will broaden the current knowledge of ZMIZ1 variants and variable clinical presentations for clinicians and genetic counselors. ZMIZ1, OMIM #607159) and proline-rich protein 12 . It produced two types of fusion genes, ZMIZ1-PRR12 and PRR12-ZMIZ1, which might be related to the occurrence of intellectual disability (ID) and neuropsychiatric alterations (ZMIZ1 variant (c.1497+2T > C), a heterozygous frameshift variant of OTUD6B (OMIM #612021) , and a 0.118 Mb 8q21.3 microdeletion involving OTUD6B  is a rare syndromic disorder characterized by global neurodevelopmental delay, hypotonia, poor overall growth, poor speech/language ability, and other common phenotypes such as eye anomalies, joint hypermobility, and distal skeletal anomalies of the hands and feet . A balanerations . Later, erations . In the erations . In 2021g OTUD6B .ZMIZ1  from a fetal brain cDNA library  of mice cortices (E14.5) resulted in impaired neuronal positioning with an accumulation in the ventricular and subventricular zones (VZ/SVZ) and intermediate zone (IZ) and a corresponding depletion in the upper cortical plate (CP). Therefore, ZMIZ1 variants were regarded as the causal genetic factors for NEDDFSA  in China, array-CGH and WES were carried out for a cohort of 54 patients with NEDD/ID living in Shenzhen, Guangdong Province, China. After comprehensive bioinformatic analysis, a de novo missense variant  was identified in the exon 20 of ZMIZ1 in a 5-year-old girl with mild development delay, mild intellectual disability, bilateral hip dysplasia, joint hypermobility, amblyopia in both eyes, strabismus in the right eye, and dysmorphic facial features. According to the criteria proposed by the American College of Medical Genetics and Genomics (ACMG)  , this vaZIMI1 variant. Due to the huge population, more patients with ZMIZ1-related disorder will be found in the near future.To our knowledge, this is the first case of Chinese NEDD/ID caused by a This study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. This study was approved by the Ethics Committee of the Shenzhen Baoan Women’s and Children’s Hospital. Written informed consent was obtained from each individual.Peripheral venous blood was collected from the 54 NEDD/ID patients and their parents. Genomic DNA was extracted using the TIANamp Blood DNA Kit  according to the manufacturer’s instructions.Array-CGH was performed using the Fetal DNA Chip (Version 1.2) designed by The Chinese University of Hong Kong (CUHK) . The chiOnly gains or losses that were encompassed by at least three consecutive oligomers on the array were considered. Then, the clinical relevance of observed chromosomal aberrations was estimated according to data found in the scientific literature and databases for each of the regions and genes involved, using the DECIPHER database  for knowWES was performed for family trios (trio-WES) without chromosomal abnormalities at MyGenostics or BerryGenomics Co. LTD. Briefly, the fragmented genomic DNAs were ligated with the 3ʹ end of the Illumina adapters and amplified by polymerase chain reaction (PCR). The amplified DNA was captured with Gencap Human whole Exon Kit (52M) at MyGenostics or with xGen Exome Research Panel v2.0  at BerryGenomics. The capture procedure was performed in accordance with the manufacturer’s protocol. Finally, the generated libraries were sequenced on Illumina HiSeq 2500 platform for paired-end sequencing.https://gatk.broadinstitute.org/hc/en-us). ANNOVAR was applied to annotate the generated VCF file . The workflow of the screening for causal variants was depicted in VCF file . Deletedn = 2504) (n = 6503)     , NHLBI E = 6503) , The Exo 60,706) , gnomAD  15,708) , and NHL 60,000) . The fun 60,000) , and PRO 60,000) . Pathoge 60,000) . The selHomo sapiens, Pan troglodytes, Gorilla Gorilla gorilla, Hylobates moloch, and Macaca fascicularis), one cattle (Bos taurus), one horse , one dog (Canis lupus familiar), three carnivores , three rodents , five reptiles , two birds , three amphibians , three fish , three arthropods , and four mollusks . The protein sequences were aligned by the ClustalW alignment algorithms of MEGA X  with long disorder setting to identify probable disordered regions using the IUPred2 model and disordered binding regions using the ANCHOR2 model (https://myhits.sib.swiss/cgi-bin/motif_scan) under default settings to search all known motifs in HAMAP (http://www.pondr.com/cgi-bin/depp.cgi) using 0.50 as the cutoff value  were analyzed using the online web server IUPred2A  was generated by STRING  under default settings. Gene Ontology (GO) analysis was performed on the nine members of the network in the GO knowledgebase (http://geneontology.org/) under default parameters.The effect of G777E on the structural change was predicted by the online protein structure and function prediction tool, I-TASSER (Iterative Threading ASSEmbly Refinement) under default parameters  for the ZMIZ1 was detected to have a pathogenic missense variant . This patient was a 5-year-old girl who was referred to our department because of psychomotor developmental delay. She was the second child of a non-consanguineous couple (There were 54 cases in our current NEDD/ID cohort collected from southern China. There were 48.15% (26/54) women and 51.85% (28/54) men. The mean age of women and men patients was 2.45 ± 1.15 and 2.67 ± 1.71, respectively. In these 54 samples, pathogenic variants were found in 33 patients, 5 with microdeletions and 28 with variants in protein-coding genes . The poss couple . The profacial features, the patient had epicanthus, ptosis, up-slanting eyelid fissure, wide eye distance, wide nose bridge, Cupid lip arch and low-set ear. Regarding skeletal abnormalities, she had short fingers and toes, conical fingers  test showed that after blink flash stimulation for both eyes, N75, P100, and N145 waves could be induced with good repeatability. However, the latency of the P100 and N145 waves on both sides was prolonged, which was slightly abnormal. The evaluations for audiology system, heart, and urinary system were normal. DR X ray film for hip joint anteroposterior projection at 4-years-7-months old showed that the left and right acetabular angles were about 22 and 25°, respectively . She wasNeuropsychological development assessment was performed for the patient at 5-years-1-month old using the Wechsler Preschool and Primary Scale of Intelligence Fourth Edition (WPPSI-IV) and the parent-rated Adaptive Behavior Assessment System II (ABAS-II) infant version. Her score on the full-scale intelligence quotient of WPPSI-IV was 75 . The verbal comprehension index, visual spatial index, perceptual reasoning index, working memory index, and processing speed index of WPPSI-IV were 77 , 83 , 79 , 76 , and 71 , respectively. The overall adaptive function score of ABAS-II was 77 . The scores of social skills, conceptual skills, and practical skills in the three composite areas of adaptive function were 71 , 84 , and 80 , respectively. According to the clinical evaluation, she was at the edge level of intellectual development.Whole exome sequencing was performed for the trio to identify possible genetic factors of the proband. After removal of adaptors and low-quality reads, the obtained total clean data obtained for the trio were 11,959.04 (Mb) for the proband, 15,681.80 (Mb) for the father, and 12,202.51 (Mb) for the mother . The tarANKRD36C, MUC2, MUC4, HRCT1, KLHL29, MYO15B, PER3, RPTN, TWIST1, and ZMIZ1) and 8 homozygous in 8 genes  (PER3 (OMIM #603427), TWIST1 (OMIM #601622), and DSPP (OMIM #125485) were also recorded in the OMIM database as disease-causing genes. However, the phenotypes caused by these genes were not in line with our female proband. Besides, two rare heterozygous variants  were annotated as “likely pathogenic” (PS2 + PM2 + PP2) and “pathogenic” (PS2 + PM1 + PM2 + PP2 + PP3), respectively.In this proband, 20 specific variants were selected. Twelve of them were heterozygous in 10 genes (LC35E2A) . AccordiKLHL29 leading to neurodevelopmental disorder, ZMIZ1 was considered as the most potential disease-causing gene. The c.2330G > A (p.G777E) was a heterozygous SNV in the exon 20 of ZMIZ1 gene (NM_020338)  were downloaded from NCBI GenBank and aligned by the ClustalW alignment algorithms of MEGA 11, the G777 was highly conserved in the animals during evolution (n = 2504), NHLBI Exome Sequencing Project (GO-ESP) (n = 6503), the Exome Aggregation Consortium (ExAC) , Genome Aggregation database (gnomAD) , and NHLBI Trans-Omics for Precision Medicine (TOPMED) . Since this amino acid changing variant was only identified in the patient, and not in her parents, it was regarded as “spontaneous.” According to the Probability of Loss-of-function Intolerance (pLI) analysis, the pLI value of ZMIZ1 was 1.000, which indicated ZMIZ1 being a haploinsufficient gene. It has been reported that ZMIZ1 could cause the occurrence of a rare neurodevelopmental disorder, neurodevelopmental disorder with dysmorphic facies and distal skeletal anomalies (NEDDFSA). Based on the recorded clinical phenotypes , c.540 + 20T > C and c.679G > A (p.A227T)   and conf_020338)  only in volution . G777E wvolution  and predenotypes , this pap.A227T) .The intrinsically disordered regions of ZMIZ1 protein (NP_065071) were analyzed using the online web server IUPred2A. Two functional globular regions were identified at two portions (aa2-110 and 575-820), which overlapped with the two important functional domains, Zmiz1 N-terminal tetratricopeptide repeat domain  and MIZ/SP-RING zinc finger , respectively. The predicted two globular regions and two intrinsically disordered regions are displayed in The distinct regions of ZMIZ1 were analyzed using the online software Motif Scan under default settings. Seven distinct regions were identified, one alanine-rich region , two proline-rich regions , one bipartite nuclear localization signal , MIZ/SP-RING zinc finger , and one copper binding octapeptide . All variants of ZMIZ1 were also recruited from the DECIPHER database  and the ZMIZ1 was expressed in the heart, thyroid, immune cells, ovary, retina, and brain, with the highest in the pineal  could cause the occurrence of a rare syndromic disease, neurodevelopmental disorder with dysmorphic facies and distal skeletal anomalies (NEDDFSA) with an autosomal dominant (AD) mode of inheritance. Currently, 32 patients with neurodevelopmental disorders have reported carrying pathogenic variants in the protein coding sequences of ZMIZ1 (n = 29) and chromosomal translocations involving ZMIZ1 (n = 3) (de novo missense pathogenic variant c.2330G > A (p.G777E) was detected in a 5-year-old girl. This patient presented the characteristic clinical phenotypes of NEDDFSA, such as neurodevelopmental delay, mild intellectual disability, hypotonia, language delay, dysmorphic facial features, joint hypermobility, and hand and foot anomalies, which were the common features of NEDDFSA  (Pathogenic variants of the zinc finger MIZ-type containing 1 ( (n = 3) . Among t NEDDFSA . As far 15C > T) . AlthougZMIZ1 was previously known as ZIMP10, RAI17, or KIAA1224. In 1999, Nagase et al. identified the gene ZMIZ1  from a fetal brain cDNA library . Currently, a total of 32 patients with 27 types of variants of ZMIZ1 (24 in protein-coding sequences and 3 translocations) have been identified globally. However, the underlying molecular mechanism of these variants has not been elucidated. Further experimental studies should be carried out to clarify these unknown fields to determine potential drug targets for the treatment of NEDDFSA.In conclusion, a


Treatment options for Coronavirus Disease 2019 (COVID-19) remain limited, and the option of repurposing approved drugs with promising medicinal properties is of increasing interest in therapeutic approaches to COVID-19. Using computational approaches, we examined griseofulvin and its derivatives against four key anti-SARS-CoV-2 targets: main protease, RdRp, spike protein receptor-binding domain (RBD), and human host angiotensin-converting enzyme 2 (ACE2). Molecular docking analysis revealed that griseofulvin (CID 441140) has the highest docking score  with main protease of SARS-CoV-2. Moreover, griseofulvin derivative M9 (CID 144564153) proved the most potent inhibitor with −9.49 kcal/mol, followed by A3 (CID 46844082) with −8.44 kcal/mol against M protease and ACE2, respectively. Additionally, H bond analysis revealed that compound A3 formed the highest number of hydrogen bonds, indicating the strongest inhibitory efficacy against ACE2. Further, molecular dynamics (MD) simulation analysis revealed that griseofulvin and these derivatives are structurally stable. These findings suggest that griseofulvin and its derivatives may be considered when designing future therapeutic options for SARS-CoV-2 infection. The COVID-19 pandemic has had an overwhelming long-term impact on healthcare systems globally. Despite vaccine developments and clinical research progress, the fast rate at which SARS-CoV-2 and its variants have spread has become a serious concern worldwide [Although extensive efforts have been made to manage this crisis, treatment of COVID-19 remains an urgent need and therapeutic options remained limited . So far,gsf gene cluster have been investigated among fungal genomes to identify essential genes involved in griseofulvin production [Considering the time and cost required to develop new drugs, computational drug repurposing is an effective strategy for developing new indications for approved drugs already available for clinical profiles ,9. Griseoduction .In addition to these inhibitory effects, griseofulvin may also have function-enhancing effects by binding to ACE2. ACE2 participates in the regulation of the renin–angiotensin–aldosterone system (RAAS) . AngioteMolecular docking provides preliminary information about small molecule interactions with receptors . MD simuThe proteins and ligand structures were optimized to be in their minimum energy state, which makes the molecule more stable. Molecules are most stable when their energy drops to a lower energy level . Figure The coronavirus main protease (Mpro) is essential for the proteolytic maturation of the virus. It has been examined as a potential target to stop the spread of infection by inhibiting viral polyprotein cleavage ,25. The −1.Based on the lower dock score in the catalytic pocket of Main protease, the derivative M9 with -9.49 kcal/mol binding energy was the most effective, as it strongly binds to SARS-CoV-2 main protease, as confirmed by the AutoDock analysis. Notably, it has been reported that this derivative can be used to treat precancerous hyperproliferative conditions . Our in The docking of receptor ACE2 with candidate ligands showed well established bonds with one or more residues in the active site of the receptor. The residues of Arg514, Glu402, Asn394, Ala348, Asp382, His401, Asp350, Gly394, and Phe400 form strong H-bond interaction with methylene and OH, while His378, Asp382, Asp350, Thr385, Ala348, Glu402, Glu398, and Asn394 are involved in weak van der Waals interactions. Close examination of the docking pose suggested a fluorine atom of A5 (PubChem ID 118254232), lined with the residues of Asp382 and Ala348. The molecular docking results also revealed that hydrophobic interactions of residues His401, Glu375, His378, Glu402, Glu398, His378, Ala 348, Arg514, and His345 are dominant interactions in the A1, A4, and A5 compounds, which have PubChem IDs 10574693, 118254131, 118254232, respectively.The compound A3 (PubChem ID 46844082) exhibited the lowest binding energy, with the value of -8.44 kcal mol−1, indicating the highest affinity to the ACE2 receptor . This co−1.The amino acids Arg454, Arg457, Lys458, Ser459, Ile468, Se469, Glu471, and Ile472 can form hydrogen bonds in the binding cavity. Molecular docking results suggest that the hydrophobic actions of residues Arg454, Phe456, Arg457, Lys458, Arg466, Asp467, Ser469, Glu471, Ile472, Tyr473, and Pro491 with griseofulvin analogues played a predominant role in all compounds except Rb9, with the best result for the hydrogen bond interaction. The Rb3 compound showed the highest binding affinity towards the RBD protein, with a docking score of −7.21 kcal mol−1 to the RdRp receptor  and M9 (2.79 Å) compounds . These v to them D.ACE2, a type I membrane protein, has a role in the renin–angiotensin–aldosterone system (RAAS), a key component in controlling hydroelectrolyte balance . SARS-CoThe RMSD trajectory measures protein deviation from the reference structure . The binRdRp plays an essential role in the replication and transcription cycle of RNA viruses, making this protein an appealing antiviral drug candidate for a broad range of viruses . Targete2), Rd2 , and griseofulvin  show no significant changes upon binding to RdRp . This indicates the slow rate of conformational changes in the protein, based on its solvent accessibility.Steady RMSDs with small changes for almost all complexes demonstrated the stability of the complexes throughout the simulation A. No sigThe receptor-binding domain in SARS-CoV-2 S protein binds to the human ACE2 receptor, enabling viral entry into the host cell ,42. The It is observed from The purpose of secondary structure is to identify protein structural characteristics. The average number of secondary structures in the complexes remained at approximately equal frequency over the last 20 ns of MD simulations, compared to protein alone, . This fiHydrogen bonds play an important role in the stability of the inhibitor–protein complex. To validate structural stability, the hydrogen bonds paired within 3.5 Å indicating the cutoff distance, as per the hydrogen bond, were calculated in a solvent environment during the 100 ns MD simulations . NotablyThe ligands with the greatest potential for inhibiting targeted receptors were analyzed for their drug likeliness through SwissADME . A compoThe ligands’ 3D SDF file formats were obtained from PubChem, and PubChem CIDs were also retrieved. The PubChem CID of the ligand is 441140 for griseofulvin, and the CIDs of 464 griseofulvin derivatives are listed in The 3D protein structures of the receptors were downloaded from the RCSB Protein Data Bank (PDB) as.pdb files. The PDB IDs of the M protease, RdRp, spike RBD, and ACE2 receptor were 6LU7, 6M71, 6M0J (chain E), and 6M0J (chain A), respectively. Chimera version 1.15  was used for protein structure optimization. The structures were subjected to energy minimization for 1000 steepest descent steps at a root means square gradient of 0.02 Å, with an Amber ff14SB force field . Due to The Computed Atlas of Surface Topography of proteins (CASTp) server was used to determine the binding sites of targeted proteins. The grid box was created around the identified binding pocket to perform virtual screening studies.https://ccsb.scripps.edu/mgltools/) was used for the virtual screening, using default settings. Ligands with the highest docking score were considered as the most stable conformation of the ligands regarding the receptor. Finally, eight hits with the highest docking score (most often negative) were selected for further analysis. Water molecules were manually removed from the PDB file prior to docking, and polar hydrogens were added. Molecular docking was performed for griseofulvin and thirty-two compounds using the Lamarckian genetic algorithm, conducted in AutoDock 4 [https://discover.3ds.com/discovery-studio-visualizer-download).PyRx 0.8  from MGLtoDock 4 . Out of + and Cl− counter-ions were added instead of solvent molecules. To reduce significant forces and relax the simulated systems, an energy reduction procedure was used in the initial step, followed by a relaxation process. The Nose–Hoover thermostat and the Berendsen barostat were employed to keep the temperature and pressure at 310 K and 1.0 bar, respectively [To validate the docking results, molecular dynamics simulation (MD) of the complex was performed. The single protein and two best protein–ligand and protein–griseofulvin complexes were analyzed using the GROMACS-2021 software package to carry out 100 ns simulations  with an ectively . The trawww.swissadme.ch/index.php) was used to obtain Lipinski’s rule of five parameters. Hepatotoxicity was also evaluated using the pkCSM server (http://biosig.unimelb.edu.au/pkcsm/prediction).The drug likeliness of a molecule is indicated by Lipinski’s rule of five parameters  . The SwiSeveral antiviral agents and other conventional treatments are being investigated as treatments for COVID-19, but therapeutic options remain poor ,57. The Our in silico study results of the tested compounds showed good-to-significant activity toward target proteins of SARS-CoV-2. Griseofulvin exhibited the highest docking score, with the most interactions in the form of hydrogen bonds to COVID-19 main protease (6LU7), which is essential for the replication and transcription of the novel coronavirus (SARS-CoV-2). However, favorable interactions with ACE2, RBD, and RdRb were also identified for griseofulvin.The molecular docking analysis revealed that, among the derivatives, M9, A3, Rb3, and Rd2, with docking scores greater than -7 kcal/mol, could act as the best inhibitors of SARS-CoV-2 main protease, ACE2, RBD, and RdRp, respectively. The structure of top ligands suggests that functional groups containing oxygen, such as the ether group in compounds M9, A3, and Rb3, may provide more possibilities for better interaction and higher affinity toward receptors. Moreover, structures with sulfonyl group in derivative M7, and fluorobenzene group in Rd2, could enhance the interaction between protein and ligand . The doc


MNX1 gene and generation of a fusion transcript in 50% of patients. Lack of research models has hindered understanding of t biology, which has historically focused on MNX1 overexpression rather than the cytogenetic entity itself. Here, we employed CRISPR/Cas9 to generate t in the human K562 cell line, and in healthy CD34+ haematopoietic progenitors where the translocation was not sustained in long-term cultures or through serial replating. In contrast, in K562 cells, t was propagated in self-renewing clonogenic assays, with sustained myeloid bias in colony formation and baseline depletion of erythroid signatures. Nuclear localisation analysis revealed repositioning of the translocated MNX1 locus to the interior of t-harbouring K562 nuclei — a known phenomenon in t patients which associates with ectopic overexpression of MNX1. Crucially, the K562-t model successfully recapitulated the transcriptional landscape of t patient leukaemia. In summary, we engineered a clinically-relevant model of t acute myeloid leukaemia with the potential to unravel targetable molecular mechanisms of disease.Acute myeloid leukaemia carrying the translocation t is an adverse-risk leukaemia uniquely observed in infants. Despite constituting up to 30% of cases in under 2-year-olds, it remains poorly understood. Known molecular features are ectopic overexpression of the  MNX1 gene, which is entirely relocated to der(12). The breakpoint on 12p13, on the contrary, disrupts the ETV6 gene on its 5’ portion [The t7;12)q36;p13) translocation is a recurrent chromosomal rearrangement uniquely associated with infant acute myeloid leukaemia (AML) [2 in approximately half of patients with t [Mechanisms of leukaemogenesis associated with t7;12) remain poorly understood. A ;12 remai t , but a t t .MNX1 gene [MNX1 overexpression [MNX1 in mouse haematopoietic stem cells (HSCs) did not result in expansion or increased survival capacity [MNX1 overexpression induced erythroid transcriptional programmes and reduced colony-forming capacity. In vivo, transplantations of MNX1 overexpressing bone marrow cells did not cause leukaemia in the recipients, but resulted in an accumulation of MNX1-overexpressing cells in the megakaryocytic-erythrocyte fraction, but not in the granulocytic-monocyte nor in the mature lymphoid compartments [A common feature among all t7;12) patients is overexpression of the ;12 patieNX1 gene , 11, whipression , 12, 13.capacity , and wasGenome editing technologies can be harnessed for generation of chromosomal abnormalities –16. The By delivery of CRISPR/Cas9 ribonucleoprotein (RNP) complexes targeting clinically accurate breakpoints described by Tosi et al.  and SimmMNX1 is already expressed in K562 cells [MNX1 overexpression in patients [MNX1 overexpression in t leukaemia, with relocalisation of the der(12) containing the translocated MNX1 gene to a more internal transcriptionally active position within the nucleus, where expression of MNX1 can be triggered [MNX1 expression  rearrangement [While 62 cells , the trapatients , 9, 11. sion in t;12 leukaion Fig. . Conversery Fig. , also insion in t;12 leukaosome 7q . Specifixpressed . The genangement , 22.MNX1/ETV6 and MNX1 overexpression in adult haematopoietic tissues, for which phenotypic changes could not be maintained through self-renewal [While the transformed nature of the K562 cell line did not allow assessment of the transforming capacity of t in this system, we observed a distinct phenotype associated with the K562-t. In clonogenic colony-forming assays on methylcellulose-based medium, K562-t produced a significantly higher proportion of colonies with a diffuse phenotype, at the expense of the more prevalent compact colonies of K562 control Fig. . K562 pr-renewal , 13.Fig.HBB and TAL1 in K562-t compared to K562 control , the differentiation capacity was not blocked by the presence of the translocation as seen by the upregulation of the three genes, albeit displaying higher fold changes compared to the control  and K562 control to induced erythroid differentiation assay by adding DMSO to the culture medium and assessing the expression of erythroid marker genes  Fig. 2C. At the rol Fig. . In thisrol Fig. , we demogenitors . In humacapacity , indicatMNX1 overexpression to transform adult haematopoietic cells [MNX1/t-associated transformation, or otherwise suggest that t selectively affects a cell type or developmental stage not efficiently represented in adult mouse or human haematopoietic tissues. The transformed nature of K562 cells, with or without contribution from the persistence of foetal developmental programmes, as evidenced by the nature of haemoglobins expressed [In order to understand t effects in untransformed haematopoietic cells, we introduced the translocation in adult CD34+ HSCPs. Despite being able to confirm its presence by PCR  , integrins (ITG), and the surface markers CD24 and CD84 , inv(16) and t, identifying 12 discriminative genes  recapitulated molecular signatures identified in t AML patients, we performed RNA sequencing analysis of three subclones each of K562-t compared to K562 control, and identified 436 upregulated and 116 downregulated genes by DR) Fig. . Gene OnDR) Fig. , as prevnts Fig. . Genes aD84 Fig. , which wD84 Fig. . AdhesioD84 Fig. . Indeed, recapitunes Fig. . Our modHOXA genes in t compared to MLL patients, which we did not find as differentially expressed in K562-t  were enriched for molecular functions of macromolecule binding Fig. . Several;12 were eukaemia , but notMLL rearrangements, normal karyotype = NK, t, and other) by identification of the common differential gene set  AML with MLL rearrangements [MNX1 was identified as a leading-edge gene, asserting its centrality to t biology  patients Fig. . We sougset Fig. . Similarset Fig. . Consistset Fig. . We alsongements , were noogy Fig. .Fig. 4CoMNX1 and production of a MNX1/ETV6 fusion transcript, albeit at a low transcriptional level that questions its biological relevance. Despite extensive FISH analyses in early studies [MNX1/ETV6 mRNA was achieved by targeting a similar genomic region to us on 7q36, but a distinct intronic region of ETV6. In our K562 model, a chimaeric transcript was not detected by potential gene fusion search by TopHat-Fusion on the RNA sequencing reads; an interesting observation given the 50% occurrence of chimaeric transcripts in patients [MNX1/ETV6 chimaera. Both the iPSC and K562 models of t showed similarities to patient-specific gene expression patterns, which strengthens the validity of CRISPR/Cas9 to generate disease models based on clinical genomic information.During the preparation of this manuscript, another group successfully recreated the t7;12) translocation in human induced pluripotent stem cell (iPSC) using CRISPR/Cas9 , with co studies –6, the e studies , the exp translocMNX1 overexpression and captures cell adhesion signatures putatively core to t oncogenic programmes. The model displays a granulocytic-monocytic phenotype compatible with the lineage affiliation of clinical t AML, and disrupts erythroid lineage programmes as described in MNX1 overexpression. We confirmed the inability of t to transform adult haematopoietic tissues, highlighting the requirement for additional oncogenic hits, or a specific cellular background which may explain the unique association of t with infants. Complementarily to earlier MNX1-based models [In summary, we generated cellular model of t translocation which recapitulates d models , 13, andd models , our t(7Supplementary material


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death globally. The cancer stem cells (CSCs) of HCC are responsible for tumor growth, invasion, metastasis, recurrence, chemoresistance, target therapy resistance and radioresistance. The reported main surface markers used to identify liver CSCs include epithelial cell adhesion/activating molecule (EpCAM), cluster differentiation 90 (CD90), CD44 and CD133. The main molecular signaling pathways include the Wnt/β-catenin, transforming growth factors-β (TGF-β), sonic hedgehog (SHH), PI3K/Akt/mTOR and Notch. Patients with EpCAM-positive alpha-fetoprotein (AFP)-positive HCC are usually young but have advanced tumor-node-metastasis (TNM) stages. CD90-positive HCCs are usually poorly differentiated with worse prognosis. Those with CD44-positive HCC cells develop early metastases. Those with CD133 expression have a higher recurrence rate and a shorter overall survival. The Wnt/β-catenin signaling pathway triggers angiogenesis, tumor infiltration and metastasis through the enhancement of angiogenic factors. All CD133+ liver CSCs, CD133+/EpCAM+ liver CSCs and CD44+ liver CSCs contribute to sorafenib resistance. SHH signaling could protect HCC cells against ionizing radiation in an autocrine manner. Reducing the CSC population of HCC is crucial for the improvement of the therapy of advanced HCC. However, targeting CSCs of HCC is still challenging. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death globally ,2. DespiThe etiologies of HCC are multifactorial, including viral infections, such as hepatitis B virus (HBV) and hepatitis C virus (HCV); nonalcoholic steatohepatitis; excessive alcohol consumption or cigarette smoking; and environmental toxins ,5,6. RegCancer stem cells (CSCs) are defined as a subpopulation of poorly differentiated cancer cells that can grow, regenerate and invade tissues ,9,10. ThIn this paper, we review the current literature regarding these main surface markers and molecular signaling pathways and the crosstalk and interactions among them, along with the role of CSCs in mitigating the effects of current therapies.Epithelial cell adhesion/activating molecule (EpCAM), a cancer antigen, is one of the most frequently reported markers of CSCs of HCC ,21,80. KYamashita et al. categorized HCC into subgroups according to EpCAM expression and AFP level . PatientCluster of differentiation 90 , a 25–37 kDa glycophosphatidylinositol-anchored cell surface protein, is another well-known HCC stem cell marker ,23,24. YGuo et al. compared patients with CD90-positive HCC and those with CD90-negative HCC and found that the overall survival rates at one, two and three years were 87.5% vs. 100%, 72.9% vs. 94.1% and 54.7% vs. 88.2%, respectively . Meta-anCD44, belonging to the transmembrane glycoprotein family, can be found in monocytes and neutrophils ,86. It pLuo et al., using a meta-analysis, reported that CD44 expression correlates with more advanced stages of HCC with a worse 5-year survival . The corCD133, a penta-span transmembrane protein, may play a pivotal role in membrane topology organization . CD133 aSome studies have reported a significant correlation between CD133 expression and the clinicopathological characteristics of HCC, including tumor grade, tumor stage, AFP serum level, malignant potential, a higher recurrence rate and a shorter overall survival ,32,33,34Recent studies have shown that the main molecular pathways involved in liver CSCs include the Wnt/β-catenin, TGF-β, SHH, P13K/Akt/mTOR and NotchWnt target genes, such as c-Myc and matrix metalloproteinases (MMPs), in order to facilitate HCC progression [CTNNB1) in HCC progression has been emphasized in some clinical studies. β-catenin signaling enhances the activity of cancer stem cells to promote HCC growth [c-MYC and cyclins A and E [Abnormal β-catenin activity is correlated with HCC in individuals with HCV and HBV infections . The actgression ,43,44,45gression . In upregression . This ovgression . The rolC growth . It may C growth ,91. Furt A and E .Some cross-interactions occur among Wnt/β-catenin signaling and different components of the tumor microenvironment (TME) of HCC . The TMECTNNB1) exist in about 20–40% of all patients with HCC [CTNNB1 gene encoding β-catenin and its overexpression could trigger the progression and migration of HCC [CTNNB1 mutations is correlated with a larger tumor size and macrovascular or microvascular invasion [Mutations in the β-catenin gene 1 + liver CSCs, possibly decreasing the self-renewal capacity of CSCs and suppressing tumorigenicity in vitro and in vivo .During the process from chronic inflammation to HCC, cancer cells grow in an environment enriched with extracellular matrix proteins. TGF-β may orchestrate the crosstalk between tumor cells and the host stroma, and they could also promote EMT ,55,58,95In HCC cells, when TGF-β induces EMT, it may trigger a switch to express stem cell genes and their potential for stemness, migration and invasiveness ,95. HoweTGF-β could activate CD133 expressions and suppress the expressions of DNA methyltransferase (DNMT) 1 and DNMT 3 beta, which could maintain DNA in a methylated state . The demThe SHH signaling pathway (SHH) plays a pivotal role in the proliferation and invasiveness of HCC cells ,50,51,52The activation of the PI3K/AKT/mTOR signaling pathway plays a role in the invasiveness of HCC, and it is frequently detected in immunohistochemical analyses of patients’ HCC tissues ,65,66,67The Notch family consists of evolutionarily conserved genes that encode for single-pass transmembrane receptors participating in stem cell maintenance ,75,76,77In mammals, the canonical Notch pathway includes four receptors  and two ligand families (jagged 1 and 2 and delta-like ligand (DLL) 1, 3 and 4) ,75. The Villanueva et al. found that Notch occurs in about one-third of cases of HCC . Notch sAmong the Notch receptors, only Notch 3 is present in the liver tissue during the middle embryonic stage . CompareCrosstalk among signaling pathways can occur ,56,100. Steinway et al. reported that the network modeling of TGF-β signaling in the EMT of HCC involves the activation of the SHH and Wnt pathways .The clinical significance of the co-expression and interaction among these different liver CSC markers has been studied. A significantly poor differentiation of HCC is found in individuals with liver CSC with the co-expression of CD44 and CD133 . CompariThe tumor microenvironment may also affect the extent to which liver CSCs facilitate the progression and maintenance of HCC ,103,104.Jeng et al. reported that SHH plays a role in the tumor microenvironment of HCC, while CSCs affect the resistance to the treatment modalities of HCC .Chemotherapy for HCC to improve survival is limited in its effectiveness ,107,108.Both overexpressed EpCAM CSCs and activated Wnt/β-catenin signaling CSCs could contribute to 5-FU treatment resistance ,110. CD150 dose of VIN or 5-FU [An activated SHH signaling pathway also affects chemoresistance . However or 5-FU . Then, t or 5-FU . The Not or 5-FU . Zhang e or 5-FU . The inh or 5-FU .Sorafenib, a multi-targeted receptor tyrosine kinase inhibitor used as a standard target therapy, may suppress various kinases , RAS/RAF/MAPK/ERK signaling pathways, etc.). However, the single use of sorafenib or the combination use of sorafenib with some chemotherapeutic agents failed to significantly increase patients’ survival ,121,122.The activation of SHH signaling is another possible mechanism for CD133-related sorafenib resistance. The cells induce sorafenib resistance via their overexpression of ATP-binding cassette (ABC) C1 (ABCC1) transporter ,115,125.Another possible mechanism for sorafenib resistance in CD133+/EpCAM+ CSCs is the Wnt/β-catenin signaling pathway . CompareEpCAM+ cells, another important liver CSC population, also play a crucial role in affecting sorafenib therapy. Guan et al. found that, after sorafenib therapy, the activation of the tuberous sclerosis complex protein kinase β (TSC-AKT) cascade increased from 38.5% to 58.7% in EpCAM+ cells . This avC. elegans protein SMA) to suppress SMAD3 phosphorylation and activate the IGF2BP3/AKT/mTOR pathway [In those with CD133+ liver CSCs, NANOG-dependent genes (Yap1 and Igf2bp3) could inactivate TGF-β signaling via the cytoplasmic retention of phosphorylated SMAD3  are currently ongoing ,139,140.The irradiation of mesenchymal stem cells (IR-MSCs), which pre-exist in the tumor microenvironment, could promote CD133+ cells in HCC . IR-MSCsBased on preclinical evidence, Bamodu indicated that phosphoinositide-dependent kinase-1 (PDK1) is an active driver of irradiation (IR) resistance following the activation of PI3K/AKT/mTOR signaling . Cancer Hong et al. suggested that a subpopulation of CD133+ liver CSCs could resist sublethal irradiation and enhance the invasion and migration of HCC cells . They foReducing the CSC population of HCC is crucial for the improvement of therapy for advanced HCC ,8. Drug The CSCs of HCC are responsible for growth, invasion, recurrence, drug resistance and radioresistance. Various surface markers and different signaling pathways affect the CSCs of HCC. There are co-expressions, interactions and crosstalk among them. Reducing the population of CSCs is crucial for the treatment of advanced HCC. However, challenging problems still remain.