Multiple sclerosis (MS) is the most demyelinating disease of the central nervous system (CNS) characterized by neuroinflammation. Oligodendrocyte progenitor cells (OPCs) are cycling cells in the developing and adult CNS that, under demyelinating conditions, migrate to the site of lesions and differentiate into mature oligodendrocytes to remyelinate damaged axons. However, this process fails during disease chronicization due to impaired OPC differentiation. Moreover, OPCs are crucial players in neuro-glial communication as they receive synaptic inputs from neurons and express ion channels and neurotransmitter/neuromodulator receptors that control their maturation. Ion channels are recognized as attractive therapeutic targets, and indeed ligand-gated and voltage-gated channels can both be found among the top five pharmaceutical target groups of FDA-approved agents. Their modulation ameliorates some of the symptoms of MS and improves the outcome of related animal models. However, the exact mechanism of action of ion-channel targeting compounds is often still unclear due to the wide expression of these channels on neurons, glia, and infiltrating immune cells. The present review summarizes recent findings in the field to get further insights into physio-pathophysiological processes and possible therapeutic mechanisms of drug actions. Oligodendrocytes (OLs) are ramified glial cells within the central nervous system (CNS) whose terminal processes generate myelin and enwrap neuronal axons. Myelin is crucial for the saltatory propagation of electrical impulses along nerve fibres, enabling rapid communication between networks in the CNS . In addiThus, a deeper understanding of OL functions during brain development, as well as during their regeneration in neurological disorders that involve OL and myelin loss, is crucial to understand their homeostatic functions within the CNS function and to identify new therapeutic targets for demyelinating diseases.During CNS development, OL progenitor cells (OPCs) are generated from neural stem/progenitor cells (NSPCs) in several regions in a precise spatiotemporal manner ,4. MultiOnly 5–8% of total glial cells are OPC, which are evenly distributed in white and grey matter  with difOligodendrogliogenesis and remyelination are crucial events for white matter reorganization . Among oIn order to study oligodendrogliogenesis, several markers are available to identify different steps of OPC maturation . Once coAmong the best known OPC markers are PDGFR-α, the receptor for PDGF-A, and the neuron-glial antigen 2 (NG2) proteoglycan ,17,18. P+ and K+ channels; de novo myelination were increased following spatial learning in the water maze test  changes, might locally influence NCX-mediated Ca2+ transients in OLs thus triggering local MBP synthesis in the vicinity of an active axon [In healthy conditions, Nav1.2 is expressed on immature, unmyelinated neurons, where it supports impulse transmission. However, this channel subtype is no longer detectable in adult mice myelinated neurons, where the closely related Nav1.6 channel supports saltatory conduction of nerve impulses at nodes of Ranvier . Nav1.2 s in EAE ,129. Thill death ,129. Durll death . Thus, rneration . Accordil injury . Of notel injury  and its cultures . Recent ive axon .Furthermore, an abnormal upregulation of mRNA and protein for Nav1.8 was found within cerebellar Purkinje cells in EAE mice and MS humans . This su+ channel inhibitor flecainide, the mechanism behind safinamide mediated protection was attributed to the inhibition of Navs [Wang et al. found that the expression of Nav1.5 was significantly increased in reactive astrocytes within MS lesions, while Nav1.1-Nav1.3 and Nav1.6 exhibited only minor changes , thus su of Navs ,55.+ channel blockers such as carbamazepine [In confirm, the use of Nav blockers, such as flecainide, phenytoin and, more recently, lamotrigine preserved both axonal integrity and electrical conduction in EAE mice. Importantly, these agents maintained conduction along the CNS and reduced disability scores in treated animals ,57. Phenmazepine .Although encouraging preclinical data, Nav-targeting compounds still miss clinical translation due to rebound effects after discontinuation of the drug. Only phenytoin differs from related compounds as both preclinical and clinical studies unveiled its neuroprotective properties, based on Nav blockade . For a s2+ signalling has been shown to regulate many oligodendroglial cell functions, including proliferation, migration, process extension, differentiation, and myelination [2+ can increase in NG2+ cells throughout several mechanisms, such as direct influx through plasma membrane voltage-gated Ca2+ channels (Cav) or ligand-gated channels [2+ stores [2+-permeable acid-sensing ion channel opening [Calination ,138,139.channels ,141, as + stores ,143 as w opening .2+ currents in OPCs from corpus callosum and mouse cortex [2+ currents through Cav appear to diminish with maturation of OLs from progenitors to mature cells in culture [Cav immunoreactivity was found in CNS white matter, located in oligodendroglial soma, projections and paranodal wraps  see als. Electroe cortex ,83,96,97 culture .2+ channel subunits in OPCs and subsequent downregulation in newly formed and myelinating OLs [+ cells [+ cells removes ∼75% of the Ca2+ elevation following depolarization [These electrophysiological results have been confirmed by RNA-sequencing transcriptome database that revealed high expression of L-type Cating OLs , indicat[+ cells . In partrization .Manduca sexta [Functional Cav are reported to be necessary for glial cell migration in vivo during olfactory glomeruli formation in the developing antennal lobe of sphinx moth ca sexta . In addica sexta , glial Cca sexta .2+ oscillations in the OPC soma, leading to accelerated migration and process formation [2+ transients is one of the mechanisms underlying AMPA-induced stimulation of OPC migration [2+ transients may affect the recycling of cell-adhesion receptors and induce the rearrangement of cytoskeletal components, which are essential for cell movement [In addition, data indicate that L-type Cav activation, probably mediated by PDGF, contributes to spontaneous Caormation . Furtherigration , as descmovement .+, used as a depolarizing stimulus to activate Cav, they are prompted towards maturation, as demonstrated by a more complex morphology and a significant increase in the expression of mature markers [2+ currents, significantly prevents OPC culture maturation [2+ entry, as well as Ca2+ release from intracellular stores, are essential mechanisms for PDGF-mediated mitotic action in OPCs [2+ entry by L-type channels modulates OPC division and cell maturation through independent intracellular pathways. Cav seem to be essential for cell cycle progression of in mitotic OPCs whereas, in post-mitotic pre-OLs, the same channels are playing an important role in cell maturation. In support to this hypothesis, it was demonstrated that a loss of Cav1.2 in oligodendroglial cells affects axonal contact in co-cultured cortical neurons and consequently inhibits the initial steps of myelination [When OPCs are grown in high extracellular K markers . At the turation . Accordituration . Interes in OPCs . A wideslination . Moreovelination ,101.corpus callosum of CPZ-treated mice, suggesting that these channels may play a key role in the induction and/or survival of newly generated OPCs after an insult [Importantly, it is likely that factors involved in physiological myelination also participate in remyelination of the injured CNS. In this regard, a significant increase in the activity of OPC L-type Cav was found in demyelinated n insult . Cav exp2+ influx through Cav was assumed to facilitate neurological impairment and histological damage in EAE mice and, by inference, MS [®) is prescribed to MS patients to treat neuropathic pain by targeting Cav [2+ overload in EAE lesions [Several evidences support the notion that aberrant Cav-mediated currents contribute to the pathophysiology of MS or EAE. Increased Caence, MS . Pregabating Cav  and, in  lesions . However lesions ,63. In a lesions  and its  lesions . 2+ channels was found in acutely injured axons, followed by rearrangement of the axonal membrane after injury [An abnormal redistribution of N-type Car injury . Since Nr injury , a similr injury  and the r injury .2+ channel  blockers Bepridil and nitrendipine had comparable beneficial effects in reducing neuroinflammation and axonal pathology on EAE mice [2+ overload activates the Ca2+-dependent protease calpain, leading to disruption of the cytoskeleton and to other structural and functional alterations of the axon [2+ homeostasis and prevention of intracellular Ca2+ overload might be responsible for the neuroprotective properties of this Cav blocker [In mice, it has been shown that, after knockout of Cav1.2, axonal myelination is inhibited and OPC maturation disturbed . HoweverEAE mice . When niEAE mice . RecentlEAE mice ,148. It the axon . Of note blocker ,69. + astrocytes did not prevent myelin damage during CPZ treatment. Interestingly, recent studies have reported that the Cav1.2 channel blockers nimodipine and verapamil exert their neuroprotective effects through anti-inflammatory properties , possibl2+ overload in neurons and immune cells. Nevertheless, there are no trials for clinical translation of Ca2+ channel blockers so far [Hence, it appears that Cav blockers might represent promising targets for demyelinating diseases as they concur to pathological intracellular Cas so far . For a srest) is near the calculated equilibrium potential for K+ (EK), i.e., −80 mV, suggesting that K+ channels account for the majority of the resting membrane conductance. The predominant K+ channel subtypes open at rest (‘leak’ channels) are the inward-rectifier Kir4.1 and two-pore (K2P) K+ channels [+ cells [+ whole cell recordings upon membrane potential hyperpolarization lower than –100 mV. These currents are blocked by low (200 µM) concentrations of extracellular Ba2+, an inhibitor of Kir channels [+ released during axonal firing, thus maintaining resting membrane potential and AP propagation [The OPC resting membrane potential  are also prominent in OPCs, where they are known to regulate cell proliferation and differentiation, and are subsequently downregulated during differentiation [+ cells display a non-linear current-to-voltage profile that is shaped by the activation of A-type (IA) and delayed-rectifier (IK) K+ channels. These currents have been extensively characterized in OPCs recorded from cell cultures or in brain slice preparations [A contribute to the initial ‘peak’ outward current during the depolarizing phase, due to their rapid activation and inactivation kinetics. Moreover, TEA-sensitive IK, which activate more slowly and do not inactivate, contributes to the sustained ‘steady-state’ current of the depolarizing stimulus [+ cells, white matter OPCs in P5-P10 mice present higher IK current densities, while IA density is comparable, resulting in a higher IK/IA ratio [+ conductances, in particular IK, in OPCs undergo a strong downregulation up to almost completely disappearance in mature OLs [K downregulation, there is a gradual increase in the expression of Kir, that represents the main conductance observed in mature OLs [2+-sensitivity of overall OPC conductance increases during maturation [Outward rectifying voltage-gated Kntiation ,123  channel KCa1.1 is highly expressed in NG2+ cells [2+-dependent, are expressed in cultured NG2+ cells [+ channels may also be important in maintaining oligodendroglial cell functions and integrity, including Kir2.1, Kir7.1 and TASK1 channels [The large conductance Ca2+ cells . This co2+ cells . Other Kchannels ,112,113.As mentioned above, deleting Kir4.1 channels in mice causes impaired myelination and OL maturation during development, leading to neuronal degeneration . In conf+ channel expression and distribution along the axon are reported. Indeed, a reduction of Kv1.2, Kv1.4, and Kv2.1 has been demonstrated in EAE mice, correlating with disease severity [A blocker 4-AP enhances axonal conduction [A is widely diffused along demyelinated axons and contributes to MS symptoms [+ outflow from axons causing extracellular K+ homeostasis outbalance that postpones the K+ reversal potential to more positive values. Consequently, a gradual depolarization of neuronal membrane occurs with a consequent inactivation of Nav leading to impaired action potential propagation. Hence, Kv block may help preserving signal conduction. Furthermore, as axonal K+ outflow leads to intracellular K+ depletion causing water loss and disinhibition of proapoptotic enzymes [+ homeostasis. Of note, a recent paper demonstrated that, although Kv1.4 deficiency decreases OPC proliferation in vitro, it does not influence de- or remyelination in the CPZ model [via glial cells. Since the authors did not observe any change in the CPZ model, it is tempting to speculate that the benefits observed in the EAE model are not only due to remyelination, but also to a reduced impact of immune system on the CNS, as discussed previously [In myelinated axons, Kv1.1 and Kv1.2 are located underneath the myelin sheath in the paranodes or internodal regions , but, afseverity . Moreoveseverity . In contseverity . These fseverity . In accoseverity , and trenduction . As IA isymptoms , in 2010symptoms . The mec enzymes , these cPZ model . Moreoveeviously ,159. ForOPC proliferation and myelination are coordinated by communication with axons and neurons within the circuit ,162,163.2+ increase, Ca2+ signalling has also been postulated to participate to OPC development, as described above [Increasing evidence shows that glutamate modulates OPC proliferation, migration and myelination during development ,193. Sined above ,195,196.+ channel activity [Initial in vitro studies suggested that glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors (AMPAR and KAR) block prevents OPC proliferation and maturation by inhibiting Kactivity ,173,174.activity ,177, wheactivity . Indeed,activity , a resulactivity . Intriguactivity .Glutamate released by excitatory neurons may also serve as a chemoattractant, stimulating OPCs migration toward their target destination in the developing brain . Indeed,Kip1 and p21Cip1 [Kip1 and p21Cip1 represent an “internal clock” or timing component of OL lineage cell progression, leading to stalling at the G1-S phase transition by dissociating cyclin-cdk complexes [Furthermore, it has been demonstrated that the molecular mechanisms underlying glutamate signalling in oligodendrogliogenesis involve elevated levels of the cell cycle inhibitors p27 p21Cip1 , whose eomplexes . NMDARs, probably located extrasynaptically, are enriched on cell processes and myelin sheaths of OLs , which mThe complexity of molecular mechanisms involved in myelination reported here highlights how glutamate signalling in OPCs may depend on developmental stage and brain area.A and GABAB receptors (GABAARs and GABABRs), while no data are present to date about a role of the third GABAR subtype, the ionotropic GABACR [Oligodendroglia express GABAc GABACR .AR signalling to NG2 cells in the hypoxic brain results in increased OPC proliferation, delayed OL differentiation and causes dysmyelination in mice [AR antagonist bicuculline whereas, conversely, blockade of GABA catabolism or uptake reduced NG2 cell numbers and increased the formation of mature OLs both in control and hypoxic mice [AR activation that has been recently confirmed by Cisneros-Mejorado using magnetic resonance imaging in a rat demyelination model consisting in ethidium bromide (EB) injection into the cerebellar peduncle [ARs, reduced the number of OL lineage cells in organotypic cerebellar slices but also inhibited myelination in this limited in vitro model [AR in myelination could be attributed to in vivo [Zonouzi et al. observed that a decreased GABA in mice . Similarxic mice . These rpeduncle . On the ro model . This di in vivo ,180 vs.  in vivo  experimeAR, whose sustained expression depends on the interaction with axons and thus in isolated OL cultures is downregulated during the differentiation process [BRs over time [BR agonist baclofen leads to an increase in proliferation and migration by decreasing cAMP levels in cultured OPCs [BR agonist baclofen stimulates OPC differentiation by increasing OL process branching and myelin protein expression via Src phosphorylation [In contrast to GABA process ,205, OLsver time ,206. Intred OPCs . Moreovered OPCs ,206. In rylation . ARs and GABABRs exert opposite roles on OPC proliferation, whereas both receptors enhance myelination. Overall, it appears that GABA1Rs, A2ARs, A2BRs and A3Rs) were identified [Purinergic signalling plays an important role in OL lineage cells both in development and in re-myelination during homeostatic and allostatic conditions ,169,207.entified .1-7, are expressed by OPCs and OLs, with most robust evidence for the P2X7R subtype [7R activation mediates a rise in intracellular Ca2+, through direct influx by the channel pore, and activates multiple intracellular pathways, including MAPK, PKC, and PI3K, all involved in the regulation of OPC proliferation, differentiation and myelination. In addition, P2X7R also participates to OL damage and myelin loss during ischemia or neuroinflammation [7R in OPC migration has been outlined by Feng et al. who demonstrated that high concentrations of ATP or the P2X7R agonist BzATP increased the number of migrating OPCs in vitro, an effect abolished by pre-treatment with the P2X7R antagonist oxidized ATP [7R stimulation by extracellular ATP causes intracellular Ca2+ overload resulting in EAE-induced OL death [P2XRs, further divided into P2X subtype . P2X7R aammation ,185. Theized ATP . Howeverized ATP , exaggerOL death .1,2,4,6,11,12,14 [1R subtype mediates intracellular Ca2+ raise, through Gq-mediated inositol-phosphates pathway, which facilitates OPC migration and affects proliferation and differentiation [1Rs, induced OPC migration and inhibited OPC proliferation in purified cell cultures and in cerebellar tissue slices, and all these effects were blocked by the P2Y1R antagonist MRS2179 [12R, known to be highly expressed in OLs, is strikingly downregulated in the cerebral cortex of post-mortem MS brains and, importantly, decreased P2Y12R immunoreactivity in proximity to the lesions directly correlates with the extent of demyelination [+/O1+ cells, its overexpression impairs final OL differentiation and myelination in vitro [post-mortem MS brains [P2YRs, further divided into P2Y11,12,14 , also plntiation . Indeed, MRS2179 . In addilination ,189. Of lination  and difflination ,191 in pin vitro . Of noteS brains . + channel regulation, as these channels are known to control cell cycle progression and/or migration by regulating membrane potential and cell volume [1Rs, adenosine inhibits OPC proliferation and enhances their differentiation and axonal myelination in OPC-DRG co-cultures [2ARs [2BRs [3R subtype has been reported to induce OL apoptosis [Regarding adenosine, all four subtypes of G protein-coupled adenosine receptors have been identified in OPCs, where they regulate migration, proliferation, and differentiation . One mecl volume ,109,213,cultures . Howeveres [2ARs  and/or ARs [2BRs , it inhiRs [2BRs ,170,214,Rs [2BRs . The A3RK currents and thus on OPC maturation, brain A1R, A2AR and A2BR might represent new pharmacological targets for demyelinating pathologies such as MS, stroke and brain trauma.Overall, the effects mediated by adenosine on oligodendrogliogenesis and myelination may depend on the developmental stage or by the environmental conditions during demyelinating pathologies. However, due to the important actions of adenosine on ITaken together, data indicate ion channels--expressed either on neurons or glia--play a key role in demyelinating pathologies such as MS. Regarding oligodendrogliogenesis, ionic channel expression changes during maturation ,29,30,31During demyelinating insults, this tuning of ion channel expression is dysregulated. In MS and EAE lesions, an overexpression of Nav1.2, 1.6, 1.8 ,90,91 waCav2.2 is also upregulated in demyelinating areas , but itsA K+ channel blocker Dalfampridine improves walking in MS patients and was approved by the FDA in 2010 [A mislocation of Kv is described in demyelinating areas of the MS brain . Of note in 2010 . 7R, lead to direct increase of intracellular Ca2+ that facilitates OPC migration [ARs and GABABRs, even if contradictory results have been described on their role in OPC proliferation. Lastly, adenosinergic receptors are expressed on oligodendroglial cell lineage and operate a fine tuning of oligodendrogliogenesis (2AR and A2BR, delay OPC differentiation by reducing K+ currents [1R, as well as the purinergic-like GPR17 subtype, improves OPC differentiation and myelination [Ion channels are also modulated by neurotransmitters, e.g., glutamate, GABA and purine receptors. Ligand-activated ionic channels, as AMPAR, NMDAR and P2Xigration ,195,196,ogenesis . The Gs-currents ,118,170.lination ,192,206.scenario of neuro-glial interaction, mediated by either or both ion channels and neurotransmitter receptors, should be considered to better understand the mechanisms leading to dysmyelination in CNS pathologies, and will be critical in evaluating the rationale for future MS treatments.The above data demonstrated the presence of a faint ionic channels/neurotransmitters receptors balance, whose interference is implicated in several neurodegenerative pathologies, including MS. The complex


Finally, 734 chickens and 321,314 SNPs remained after quality control and removal of the sex chromosomes, and these data were used to carry out a GWAS analysis. A total of 470 significant single-nucleotide polymorphisms (SNPs) for 43 of the 68 traits were detected and mapped on chromosomes (Chr) 1–6, -9, -10, -16, -18, -23, and -27. Of these, the significant SNPs in Chr1, -4, and -27 were found to be associated with more than 10 traits. Multiple traits shared significant SNPs, indicating that the same mutation in the region might have a large effect on multiple growth or carcass traits. Haplotype analysis revealed that SNPs within the candidate region of Chr1 presented a mosaic pattern. The significant SNPs and pathway enrichment analysis revealed that the MLNR, MED4, CAB39L, LDB2, and IGF2BP1 genes could be putative candidate genes for growth and carcass traits. The findings of this study improve our understanding of the genetic mechanisms regulating chicken growth and carcass traits and provide a theoretical basis for chicken breeding programs.Chicken growth traits are economically important, but the relevant genetic mechanisms have not yet been elucidated. Herein, we performed a genome-wide association study to identify the variants associated with growth traits. In total, 860 chickens from a Gushi-Anka F Maynard Smith and Haigh  demonstr2 resource population. The results deepen our understanding of the genetic architecture of growth and carcass traits in chickens.Growth traits play an important role in animal production and breeding programs. Growth traits are quantitative traits that are controlled by multiple genes, and it is difficult to make breakthrough progress with conventional breeding methods. Kemper, Visscher The animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University under approval number 17-0118. All experiments strictly followed the guidelines of this committee.2 chicken resource population used in this study was established by the Henan Innovative Engineering Research Center of Poultry Germplasm Resource for genomic studies. The F2 resource population consisted of four cross-bred families (Anka-cocks mated with Gushi-hens) and three reciprocal families (Gushi-cocks mated with Anka-hens) as described previously , semi-evisceration weight (SEW), evisceration weight (EW), liver weight (LW1), heart weight (HW), gizzard weight (GW), spleen weight (SW), pancreas weight (PW), head weight (HW1), claw weight (CW1), double pinion weight (DPW), breast muscle weight (BMW), leg muscle weight (LMW), leg weight (LW), fat bandwidth (FBW), skin fat thickness (SFT), and abdominal fat weight (AFW). Moreover, the percentage traits were also calculated. BMR, LMR, LR, and AFR were the ratios of BMW, LMW, LW, and AFW to EW, and the other ratios for each carcass trait were calculated by dividing their weight by BW12, for example, CWR = CWe/BW12. The measurement methods have been previously detailed by Han et al. (P < 0.05) were ranked by normal scores with the Tukey method, and then transformed data were used in the following genetic analyses.Phenotypes, including growth traits and carcass traits, were measured for all 860 chickens. Growth traits consisted of body weight (BW) and body size. BW was recorded individually every 2 weeks from birth to 12 weeks . Body size was measured at 4, 8, and 12 weeks and included shank length (SL), shank girth (SG), chest depth (CD), chest width (CW), breast bone length (BBL), pectus angle (PA), body slanting length (BSL) and pelvis breadth (PB). Carcass traits were collected after 860 Fn et al.  and Li en et al. . The pheIndividual genomic DNA was isolated from blood samples by the Qiagen DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. The DNA quality and concentration satisfied the requirements for library construction of double-digest genotyping-by-sequencing (ddGBS)   was implemented using GCTA  in the GCTA program.y is the phenotypic values for each trait; W is a matrix of covariates (fixed effects) that control the population structure (top two PCs), sex and genotyping batch effect; in particular, BW0 is also considered as a covariate in all BW traits; α is a vector of corresponding coefficients including the intercept; β is the SNP effect and x is a vector of SNP genotypes; u is a vector of random effects with a covariance structure that follows a normal distribution as u ~ N , where K is a known genetic relationship matrix; and e is a vector of random errors.The following MLM was used:P value threshold was calculated using Bonferroni correction with an effective number of independent tests. We calculated the number of genome-wide independent markers using PLINK  > 5.72) based on the 26,352 independent markers. Manhattan and Q-Q plots were derived from the GWAS results using the CMplot package (https://github.com/YinLiLin/R-CMplot) within the R software (http://www.r-project.org/).The genome-wide significance http://circos.ca/) software package  program . For example, the pairwise Pearson’s r of BW8, BW10 and BW12 was >0.85. In addition, different body size measurements, including SL, CW, SG, BBL, and BSL, showed stronger correlative relationships at the same stage . However, a relatively weak correlation was observed in both the BW and partial body size traits  between the early- and late-growth stages. Furthermore, a strong correlation occurred between the CWe trait and parts of the CWe, such as the BMW, LMW, HW1, LW, CW1, EW, and SEW  > 5.72)  Fig. .Fig. 3Th10(P) = 7.35; intergenic) explained 2.31% of the phenotypic variance. The genomic control inflation factor (λ) calculated for all growth traits ranged from 0.97 to 1.00, which indicated acceptably low false positives  = 14.16; intron) could explain 10.14% of phenotypic variance. The genomic control inflation factor (λ) calculated for all growth traits ranged from 0.95 to 1.00, which is similar to the value found for growth traits  < 4, two genomic regions (Chr1:167.5–170.5 Mb and Chr4: 70.9–81.7 Mb) were obtained within a total range of 3 Mb.The genome-wide significant SNPs were mainly distributed throughout Chr1, Chr4, and Chr27 ; iii. SNP set III, high-LD SNPs with SNP set I and SNP set II (r2 > 0.7) with the removal of one of any pair of adjacent SNPs <100 bp apart. The SNPs obtained from the three datasets are listed in Table We found that significant SNPs on Chr1 had effects on multiple traits, and these SNPs were located in relatively large QTLs, which might affect BW together. Therefore, we screened the SNPs with the following criteria and obtained different SNP data sets. i. SNP set I, significant SNPs associated with BW at the age of different weeks; ii. SNP set II, significant SNPs associated with traits strongly correlated with BW  neighboring the significant locus also remained. Finally, a total of 277 unique putative candidate genes were used for enrichment analysis (Table MLNR) gene was enriched.Annotation of each significant locus revealed putative candidate genes. Moreover, the genes located within the high-LD region . Chicken growth traits are a class of complex traits with important economic impacts. Analyzing the genetic basis not only served as a key link for chicken breeding programs but also enhanced our understanding of the genetic pattern of QTLs.A large number of GWAS have shown that most comprehensive traits and diseases are controlled by multiple genes, and the effect of a single gene is relatively small. For example, a joint analysis of 183,727 people identified 180 height-related loci, which explained a total of only 12% of the phenotypic variations   at Chr1 functions in the regulation of vitamin D metabolism by binding to the vitamin D receptor, further affecting the development and maintenance of mineral ion homeostasis and skeletal integrity  gene located at Chr1 catalyzes the process of phosphorylation to activate AMP-activated protein kinase and then regulates food intake  line to identify QTLs on Chr1, -2, -4, -7, -8, and -13 that affected BW, and Chr4 had the largest single additive effect  gene located at Chr4 has been reported many times to be associated with chicken growth traits  gene located at Chr27 has been demonstrated to be the major gene for growth traits in ducks (Zhou et al. Park et al. found that Chr27 harbored a highly significant QTL for shank weight but no significant effect on growth (Hee-Bok et al. Growth is a complex trait and combined process that is influenced by food intake, appetite, energy composition, nutrient distribution, physical activity, metabolic rate, etc. Therefore, the genetic basis of the growth trait might be affected by many loci, each of which only explained a small variation of the trait. The results of selective-sweep mapping suggested that >100 loci might contribute to the selection response (Johansson et al. This study provided systematic phenotypic measurements and genetic mapping of growth and carcass traits in 734 chickens from a Gushi-Anka F2 population based on ddGBS sequencing data. A total of 470 significant SNPs for growth and carcass traits were detected and mapped on Chr1-6, -9, -10, -16, -18, -23, and -27, including new loci and genes. Remarkable effects of QTLs on Chr1, -4, and -27 were detected. The significant SNPs associated with multiple traits were all located on Chr1, -4, and -27. Haplotype analysis of the QTL on Chr1 showed a mosaic inheritance pattern. Significant SNPs and pathway enrichment revealed that the MLNR, MED4, CAB39L, LDB2, and IGF2BP1 genes could be putative candidate genes for further study.Supplementary MaterialsSupplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7


These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A Biological membranes of mammalian cells are comprised mostly of proteins and lipids. Glycerophospholipids (phospholipids), along with sphingolipids and cholesterol, are the major lipid components. The basic structure of membrane phospholipids, with a glycerol backbone, polar headgroup, and two hydrophobic chains, provides a template for a diverse array of chemical species to support their many biological functions. These functions include formation of lipid bilayers that form hydrophobic barriers in cellular membranes, which encapsulate and compartmentalize cells as well as provide domains of molecular interactions . Phosphosn) -3 position and two fatty acids and linkages at the sn-1 and sn-2 positions of their glycerol-based structures (A). For headgroups, the simplest phospholipid class, phosphatidic acid (PA), has just a phosphate. During de novo phospholipid synthesis, PA is a precursor molecule for the production of other phospholipids with more complex headgroups: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL)  , 8. Chemectively . The memectively , 10.Figude novo phospholipid biosynthesis and phospholipid remodeling, as described in detail in The choices of headgroups, fatty acids, and linkages are all essential features that affect phospholipid function. For the fatty acids, carbon chain lengths and double-bond numbers/positions are important attributes, which may affect biophysical and biological properties of phospholipid layers , 12. TheB). Chemical diversity is endowed to phospholipid molecules in two distinct phases of their biosynthesis, the Kennedy pathway (de novo pathway)  B. Chemicpathway)  and the pathway)  Fig. 2)B). Chemipathway) .Figure 2B and via two main routes. In one route, PA is dephosphorylated by PA phosphatases  to produce diacylglycerol (DAG). DAG may be next metabolized to PC and PE, which may be further converted to PS by PS synthases  acyltransferases (GPATs) use G3P and acyl-CoA as substrates to produce a key intermediate in phospholipid synthesis, lysophosphatidic acid (LPA) B and 2. ynthases . DAG mayynthases . In the ynthases .1/2s and LPLATs d LPLATs , 21  and the membrane bound O-acyltransferase (MBOAT) families  1 and 2 , and several protein acyltransferases such as ghrelin O-acyltransferase (GOAT), porcupine O-acyltransferase (PORCN), hedgehog acyltransferase (HHAT), and HHAT-like  The numbers assigned in each LPLATx name are essentially in the order of discovery. (iii) The LPLATs for which we propose nomenclature belong to two families, AGPAT and MBOAT. Although there are a few reports of enzymes with potential LPLAT activities that belong to other protein families , the natural substrates utilized by LPLAT9 in PAF production. LPLAT9 possesses a hydrophobic cavity that faces the cytosol at one end and the membrane surface at the other, and acetyl-CoA and lyso-PAF could enter the cavity from the cytosol and the membrane, respectively, contacting with each other in the vicinity of the catalytic His146. AGPAT Motifs 1 to 3 constitute the cavity, whereas Motif 4 functions as a backing for the cavity. Besides the motifs, Arg166 and Arg195 could function in recognizing the phosphate groups in lyso-PAF and acetyl-CoA, respectively. Figure 8B, lower panel, shows a manually docked model of LPLAT12 with arachidonoyl-CoA and lyso-PC, the natural substrates utilized by LPLAT12 in arachidonoyl-containing-PC production. LPLAT12 possesses a hydrophobic cavity at the center that penetrates the membrane, and arachidonoyl-CoA and lyso-PC could bind to the cavity from the cytosol and the luminal side of the membrane, respectively, contacting with each other in the vicinity of the catalytic His374. MBOAT Motifs B and C, but not A or D, contribute to the construction of the cavity, which possesses a hydrophobic bulge at the center that might be suitable for accommodating the arachidonoyl chain. Recently, Zhang et al. reported on the structure of the LPLAT12 ortholog in chicken as determined by x-ray crystallography, cryo-EM, and sequence analysis  and MBOAT Motifs B-D in LPLAT12 (LPCAT3) are shown in Figure 8for PlsC  and DGATfor PlsC   is highly expressed in lipogenic tissues such as liver and adipose, where its levels are decreased by fasting and increased by insulin, indicating a metabolic function to regulate fat utilization and storage , 47. GPAholipids , 51. In holipids , 52.in vitro assays  shows selectivity to utilize C20:4-CoA as substrate o assays . Among so assays . This exo assays , 55. GPAo assays .GPAT3  is a microsomal GPAT, but was also suggested to possess LPAAT activities . During GPAT4  is abundantly expressed in various tissues. Its expression is high in the liver, BAT, and WAT, where GPAT4 was reported to be responsible for most GPAT activity . GPAT4-KO-acyltransferase (GNPAT) and increased synthesis of peroxisomal ether lipids  has been found to be an essential cofactor for GPAT4 and thereby regulate ER glycerolipid synthesis. CHP1 binding was required for GPAT4 activation, and myristoylation of CHP1 was required for the full interaction. Loss of CHP1 in mammalian cells led to severely reduced fatty acid incorporation and storage, but was partially compensated for by upregulation of glyceronephosphate r lipids .In this section, biochemical and biological characteristics of each LPLAT are summarized. The new proposed names for LPLATs are also introduced, which are in the format LPLATx, where x is a number . As descde novo phospholipid synthesis as well as TAG production (LPLAT1 (AGPAT1), also sometimes called LPAAT1, utilizes a variety of acyl-CoAs to produce PA during oduction , 66, 67 oduction . LPLAT1 oduction , 69. LPLoduction  (69). A oduction , suggestoduction .LPLAT2 (AGPAT2), also called LPAAT2, shares high homology with LPLAT1 and has similar biochemical properties to utilize a variety of acyl-CoAs to produce PA for phospholipid as well as TAG biosynthesis , 66, 67 LPLAT2 has an essential physiological role in adipose tissues, where it functions in TAG production and accumulation . In humain vitro assays (in vivo (LPLAT3 (AGPAT3), also sometimes called LPAAT3, utilizes several acyl-CoAs including docosahexaenoic acid -CoA as donor substrates in o assays , 81, 82 o assays . However, which may be partly compensated for by upregulation of other AGPATs in LPLAT4-KO tissues. Further analyses are required to understand the unique biological functions of LPLAT4.LPLAT4 (AGPAT4), also called LPAAT4, is expressed in many tissues including the brain, muscle, and WAT and has activity to incorporate unsaturated fatty acids into LPA to produce PA; with selectivity to utilize C22:6->C20:4->C18:2->C18:1->C16:0-CoAs as donor substrates , 88 Tab. LPLAT4  fission . Brains  fission . LPLAT4- fission . This wa fission . LPLAT4  fission . In skel fission . LPLAT4- fission . The stuIn vitro studies using cells overexpressing LPLAT5 (AGPAT5) revealed that it colocalized to mitochondria and showed both LPAAT and LPEAT activities with C18:1-CoA , also sometimes called LYCAT, ALCAT1, or AGPAT8, acylates diverse substrates including lyso-PI (LPI), lyso-PG (LPG), and LCL using a variety of acyl-CoAs , 99, 100 tissues . Indeed,sues  also showed similar abnormalities in seam cells  and LPGAT activities in mouse livers as well as decreased serum TAG and cholesterol levels, suggesting that LPLAT7 may function as an MGAT and play a significant role in hepatic TAG synthesis and secretion in db/db mice  localized to ER and had activity to incorporate C16:0, C18:0, and C18:1 into LPG  Table 4. Livers /db mice . However/db mice , 117. Po/db mice  (Table 5LPLAT8 (LPCAT1), also called AGPAT9, AGPAT10, or AYTL2, is expressed in the lung, retina, and other tissues where it generates C16:0-containing PC , 121 Ta. In lungrd11 mice causes rod and cone cell loss leading to retinal degeneration; and as in lung, DPPC is also reduced in retina in rd11 and LPLAT8-KO mice , also called AGPAT11 or AYTL1, incorporates C2:0 (acetic acid) into lyso-PAF to produce PAF , 129 Fi. LPLAT9 in vitro , 128. Thin vitro . LPLAT9 in vitro . (ii) Sein vitro . (iii) Lin vitro , 131. Thin vitro .in vitro and impaired immunogenic anticancer responses in vivo  are involved . NeuropaLPLAT10 (LPCAT4), also called LPEAT2, has activities to incorporate C16:0-CoA, C18:0-CoA, and C18:1-CoA into LPE, LPC, and LPG as well as alkyl-LPC and alkenyl-LPE, and suggested to have a role for biosynthesis of ethanolamine-containing phospholipids, especially in brain  Table 4. LPLAT10C. elegans, mboa-7 was discovered to selectively incorporate C20:4 into LPI, as does the closest mammalian homologue LPLAT11 (MBOAT7), also called LPI acyltransferase (LPIAT)1  , 143, al(LPIAT)1  (Table 4(LPIAT)1 . LPLAT11(LPIAT)1 . The mic(LPIAT)1 , 146. In(LPIAT)1 . The ass(LPIAT)1 , 146. In(LPIAT)1  , also called MBOAT5, can utilize a variety of substrates and shows selectivity to incorporate C18:2 and C20:4 into LPC, LPE, and LPS in o assays , 161  mice, LPLAT12-deficiency drastically increased tumor formation via disrupted cholesterol homeostasis  led to reduced hepatic TAG levels through suppression of lipogenesis, indicating that LPLAT12-regulated phospholipid compositions are important regulators of SREBP-1c activation and lipogenesis , also called LPCAT4, has both LPCAT and LPEAT activities in biochemical assays and selectivities to incorporate C18:1 into LPC and LPE , 159 Fi. LPLAT13enotypes .in vitro assays (LPLAT14 (MBOAT1), also called LPEAT1, exhibits LPEAT and LPS acyltransferase (LPSAT) activities and has selectivity to utilize C18:1-CoA as an acyl donor substrate o assays , 159  and LPLAT11 (MBOAT7), are expected to have positional specificities to incorporate fatty acids into the sn-2 position.The fatty acids in phospholipid molecules tend to show a nonsymmetrical distribution between sn-positional specificities of most LPLATs have been largely unknown, several reports within the past decade have examined the sn-positional specificities of a small subset of LPLATs. Imae et al. , LPLAT12 is a sn-2 position-selective enzyme (type 2), and LPLAT8 (LPCAT1) is an enzyme that has specificity to incorporate fatty acids towards both sn-1 and sn-2 positions (type 3)  . In the sn-1 and sn-2 specificities of several LPLATs, made possible by technological improvements (Here we have summarized the current progress on LPLATs of the AGPAT and MBOAT families. These enzymes function in the Kennedy pathway and the Lands cycle to regulate the fatty chain compositions of membrane phospholipids. Much progress in our knowledge of these enzymes has been made in the past decade. Gene-deficient mice for these enzymes have provided us important insights into mechanisms and biological significance of the generation of membrane phospholipid diversity. Another recent advance is determination of positional ovements , which wThe current nomenclature of LPLATs is complicated, with several cases of individual enzymes being assigned different names by different groups, and the same name being used for different enzymes in some instances. This overlapping nomenclature for some enzymes has caused confusion in the field, and as more studies come out, the inconsistent and overlapping naming creates even more of an impediment in accessing the expanding body of knowledge. This review puts forward a proposed enzyme nomenclature that is not based on the perceived lysophospholipid substrate specificities. It is hoped that in the near future a similar nomenclature might be agreed upon and broadly adopted that provides clear and specific names for each enzyme.Based on the current trajectory, the LPLAT field is still on the cusp of greatly expanding the known roles for LPLATs. Phospholipid analyses using gene-deficient mice of all of the enzymes, including inducible- and tissue-specific KOs, are expected to continue to accelerate the discovery of important roles for these enzymes in a variety of biological and physiological processes. These discoveries in parallel with ongoing technological advances in biomembrane analyses will greatly expand our knowledge of how/why membrane phospholipid compositions and diversity can impact human health and diseases.10.13039/501100013170ONO PHARMACEUTICAL CO, LTD.Department of Lipid Signaling, National Center for Global Health and Medicine is financially supported by


The peroxidase family of peroxiredoxins (PRDXs) plays a vital role in maintaining the intracellular balance of ROS. However, their function in head and neck squamous cell carcinoma (HNSCC) has not been investigated. We therefore explored the value of PRDXs in HNSCC. We found that the expression of PRDX1, PRDX4, and PRDX5 in HNSCC increased while the expression of PRDX2 decreased. Moreover, the high expression of PRDX4/5/6 indicated a poor prognosis. Lower expression of PRDX1/5 was linked to more immune cell infiltration, higher expression of immune-related molecules and a more likely response to anti-PD-1 treatment. Moreover, PRDX5 knockdown inhibited HNSCC cell proliferation, invasion and metastasis and it might promote apoptosis through its antioxidant property. Taken together, our study highlights the potential role of PRDXs in HNSCC. The function of PRDX5 in the development of HNSCC and the formation of the immune microenvironment makes it a promising potential therapeutic target. Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent tumor, annually accounting for ~ 830 000 new cases and 430 000 deaths worldwide . DespiteThe peroxiredoxin (PRDX) family of peroxidases comprises six members (PRDX1 to PRDX6) that constitute a broad range of cellular antioxidant defenders. Based on the number of cysteines, PRDXs are divided into three categories: typical 2-cysteine peroxiredoxins (PRDX1–PRDX4), atypical 2-cysteine peroxiredoxins PRDX5), and 1-cysteine peroxiredoxins (PRDX6). Although these proteins have different functions in antioxidant protection and cellular redox regulation, they share a common molecular mechanism. PRDXs catalyze the reduction of H2O2, peroxynitrite, and organic hydroperoxides to maintain the intracellular balance of reactive oxygen species (ROS) , 4. ROS , and 1-cAccording to recent reports, the PRDX family is dysregulated in multiple tumors. PRDX1 is up-regulated in some tumors such as pancreatic ductal adenocarcinoma, non-small cell lung cancer, cervical cancer and breast cancer, and promotes the growth and metastasis of cancer cells –9. PRDX2in vitro inhibited the proliferation, migration, and invasion of HNSCC and increased apoptosis, which might result from ROS elevation. In addition, patients with low expression of PRDX1/5 was more likely to respond to the PD-1 inhibitor.Thus, in this study, we focused on the biological roles of the PRDX family in HNSCC. In our results, PRDX5 and PRDX4 were upregulated in HNSCC patients and were associated with a shorter survival time. Patients that showed higher PRDX1/5 expression also had lower immune cell infiltration and lower expression of immune-related molecules. Knocking down PRDX5 https://portal.gdc.cancer.gov/). RNASeq data was transformed into transcripts per kilobase million (TPM) values.Level 3 RNASeq, miRNASeq data and the corresponding clinical information of the HNSCC datasets were obtained from The Cancer Genome Atlas database (TCGA) (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp) was used to evaluate the relationship between PRDXs and 14 functional states based on the GSE103322 data set, including angiogenesis, apoptosis, cell cycle, differentiation, DNA damage, DNA repair, EMT, hypoxia, inflammation, invasion, metastasis, proliferation, quiescence, and stemness.We divided TCGA HNSCC cohort into different groups (PRDXs-high and PRDXs-low) based on the “survminer” R package. To understand the biological pathways in which PRDXs could be involved, we performed Gene Set Variation Analysis (GSVA) using “GSVA” R packages. The gene sets of “hallmark” was downloaded from the “msigdbr” R package. An adjusted P-value of less than 0.01 was considered significant. In addition, CancerSEA . We quantified the immune cell infiltration using single-sample gene set enrichment analysis (ssGSEA) based on 23 types of immune cells signatures (We obtained tumor purity gnatures . The difhttps://mexpress.be/). The relationship between lncRNA and PRDXs was assessed using spearman correlation analysis. |Correlation coefficient| ≥ 0.3 and adjusted P-value < 0.05 were defined as significant.Methylation and copy number variation (CNV) of the PRDXs in HNSCC datasets was assessed and visualized based on MEXPRESS  targeting PRDX5 (si-PRDX5) and a negative control siRNA (si-NC) were designed and purchased from GenePharma . The sequence of si-PRDX5#1 was 5’- GGUGGCCUGUCUGAGUGUUdTdT-3’, and the sequence of si-PRDX5#2 was 5’- GCCUGGCACCCAAUAUCAUdTdT-3’. Lipofectamine 3000  was used to transfect si-PRDX5 and si-NC into HNSCC cells following the instructions of the manufacturer.Total RNA was isolated from NOK, HSC2, UM1, HN6, HSC4, and SCC4 cells by the RNA-quick purification kit  following the manufacturer’s instructions. A total of 2 µg RNA was synthesized from cDNA using HiScript III-RT SuperMix . The obtained cDNA product was used for real-time quantification PCR (RT-qPCR). The following primers were used: PRDX5, forward 5′-CTCCTGGCTGATCCCACT-3′, and reverse 5′- CACTATGCCATCCTGTACCAC-3′; GAPDH, forward 5′-CTCCTCCTGTTCGACAGTCAGC-3′, and reverse 5′ -CCCAATACGACCAAATCCGTT-3.Total proteins of HNSCC cell lines were extracted with RIPA lysis buffer and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a 0.22-µm PVDF membrane  and blocked with 5% non-fat skimmed milk at RT for 1 h. Next, the membranes were incubated with primary antibodies against PRDX5  and GAPDH  at 4°C overnight. After that, the membranes were incubated with species-specific secondary antibodies at RT for 1 hour. Finally, the bands were detected using the enhanced chemiluminescence system .HN6 and HSC4 cells with transfected siRNAs were seeded at 1500 cells/200 µl per well in 96-well plates. CCK-8 kit  was applied to assess cell viability at different time points according to the protocol of the manufacturer.HN6 and HSC4 cells were transfected with siRNAs, seeded in 6-well plates (500 cells per well), and cultured for 14 days. Then, colonies were fixed by methanol and stained with 0.5% crystal violet solution, and visible colonies were manually counted.HN6 and HSC4 cells transfected with siRNAs were grown in 6-well plates and cultured at 37 °C. When the cells grew to 90% confluence, a 200 µl tip was used to create a thin scratch. The supernatant cells were washed with PBS. Subsequently, the cells were subjected to a serum-free medium for starvation. The area of the scratch was recorded at 0 h and 24 h with an inverted microscope .Cell invasion assays were performed in transwell chambers  precoated with Matrigel . In detail, a total of 200 µl serum-free medium containing 8 × 105 cells were added in the upper chambers, and 600 µl medium containing 20% FBS was placed in the lower chambers. After 24 h of incubation, the invading cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 1 h, and counted under an inverted microscope .Apoptosis of HN6 and HSC4 cells was detected by Muse Annexin V & Dead Cell Assay Kit  according to the manufacture protocol. HN6 and HSC4 cells transfected with siRNAs were starved in serum-free medium and resuspended with Annexin V buffer. Then, the cells were incubated with Annexin V-FITC and PI in the dark. A flow cytometer (CytoFlex) was used to assess apoptotic cells.via a flow cytometer (CytoFlex).We employed MitoSOX Red reagent  to determinate mtROS according to the manufacturer’s instructions. HN6 and HSC4 cells transfected with siRNA were incubated with 5 µM MitoSox Red dye at 37°C for 10 minutes and then washed with PBS three times. The fluorescence signal of the labeled samples was analyzed First, we analyzed the expression levels of PRDXs (PRDX1-6) between HNSCC and normal controls in the TCGA database. Compared to the normal tissues, PRDX1, PRDX4 and PRDX5 were significantly upregulated, while PRDX2 was significantly downregulated in HNSCC tumors Table 1.GSVA analysis was performed to explore the possible roles of PRDXs in HNSCC patients. PRDX1, PRDX5 and PRDX6 were positively associated with DNA repair, MYC targets V1/V2, mTORC1 signaling, oxidative phosphorylation and fatty acid metabolism. Similarity, PRDX1, PRDX5 and PRDX6 were negatively associated with heme metabolism, KRAS signaling up, IL6_JAK_STAT3 signaling, complement, interferon gamma response and inflammatory response. PRDX2 and PRDX3 were positively associated with E2F targets, G2M checkpoint and spermatogenesis. PRDX3 and PRDX4 were negatively associated with heme metabolism, estrogen response, p53 pathway and cholesterol homeostasis  was used to assess the relationship between PRDXs and 14 functional state signatures in HNSCC tumor cells based on the CancerSEA database. PRDX1 was positively correlated with cell cycle, DNA repair and stemness while PRDX1 was negatively correlated with angiogenesis, apoptosis, differentiation, DNA damage, EMT, hypoxia, inflammation, invasion, metastasis, proliferation and quiescence. PRDX2 was positively correlated with angiogenesis, cell cycle, differentiation, DNA damage, DNA repair, EMT, hypoxia, invasion, metastasis, proliferation and quiescence, while it was negatively correlated with stemness. PRDX3 was positively correlated with apoptosis, cell cycle, differentiation, DNA damage, DNA repair, invasion, metastasis, proliferation and stemness, while it was negatively correlated with angiogenesis, inflammation and quiescence. PRDX4 was positively correlated with angiogenesis, cell cycle, DNA damage, DNA repair, EMT, hypoxia, invasion, metastasis and proliferation, while it was negatively correlated with inflammation and stemness. PRDX5 was positively correlated with angiogenesis, apoptosis, differentiation, EMT, hypoxia, inflammation, invasion and metastasis, while it was negatively correlated with cell cycle, DNA damage, DNA repair and stemness. PRDX6 was positively correlated with cell cycle, DNA damage and DNA repair, while it was negatively correlated with angiogenesis, apoptosis, differentiation, EMT, hypoxia, inflammation, invasion and metastasis, quiescence and stemness . PRDX2 showed a relatively strong correlation with methylation (from r = -0.186 to r =-0.610). PRDX3 was positively or negatively associated with methylation probes. PRDX5 was negatively correlated with 4 methylation probes (from r = -0.127 to r = -0.177). PRDX6 was also negatively associated with methylation. Only one methylation probe was positively associated with PRDX4. The expression of PRDXs was significantly associated with CNV, especially PRDX6 (r = 0.371) and PRDX5 (r = 0.366) , thereby recruiting T cells into the tumor . For anoltration . Therefoltration , 41. TheAlthough this study systematically analyzed the value of PRDXs and verified the function of PRDX5 in HNSCC, some limitations are acknowledged. The PRDXs expression and survival analysis is mainly based on the data retrieved from the TCGA database, and therefore multi-centered cohorts are required to confirm the results presented here. In addition, it is necessary to elucidate the in-depth mechanism by which PRDX5 promotes the aggressive phenotype of HNSCC.In summary, we found that PRDX1/2/4/5 were dysregulated in HNSCC and that higher expression of PRDX4/5/6 was associated with a shorter survival time. The possible function of PRDX5 in the development of HNSCC and the regulation of the immune microenvironment makes this protein a promising therapeutic target.The original contributions presented in the study are included in the article/in vitro experiments. RC, HZ, and WZ wrote and edited the manuscript. LW, XC, and XR collected and examined the data. JX and BC supervised the project. All authors read and approved the final manuscript.JX and BC designed the study. RC performed all the bioinformatics analysis described here. WZ performed This study was supported by the National Natural Science Foundation of China , Guangdong Financial fund for High-Caliber Hospital Construction (174-2018-XMZC-0001-03-0125/D-05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-Hydroxyethylpyrrolidone(HEP) gave the best results. Moreover, the methodology was successfullyapplied to the synthesis of an intermediate of the anticancer drugErlotinib, demonstrating the versatility of the new green protocol.The replacement oftoxic solvents with greener alternatives inHeck–Cassar–Sonogashira (HCS) cross-couplings was investigated.The fine-tuning of the HCS protocol allowed to achieve complete conversionsand high speed under mild conditions.  Since then, the Heck–Cassar–Sonogashira(HCS) reaction was successfully applied for industrial production.Several studies have investigated the influence of leaving groups,palladium ligands, cocatalyst, and bases.6The reaction was independently reported in 1975 by Sonogashira7 Solventsrepresent the main source of waste in chemical industrial processes,constituting, on average, 80–90% of the total process mass.8 Their selection is critical in Pd-catalyzed cross-couplings,because of the influence on the coordination sphere of the metals,the stability of the catalyst, the equilibrium, and the rate and selectivityof the reaction.9The greenness of industrial processes to preserve the environmentand to ensure health and safety of workers has evolved from an ethicapproach to an inescapable necessity.N,N-dimethylformamide(DMF),10 which is well-known as a highlyreprotoxic solvent, is classified as a substance of very high concern(SVHC), and is a potential source of N-dimethylnitrosamine.11 Other solvents also have been used, such astetrahydrofuran (THF), dimethylsulfoxide (DMSO), 1,4-dioxane, toluene,dimethoxyethane (DME), and amines, even if not representing real greeneralternatives.9 Alcohols and aqueous systems,12 ionic liquids,13 andbio-based solvents such as dimethylisosorbide,14 γ-valerolactone,15 andCyrene10 also were investigated.In the last decades,almost 40% of the published HCS reactionswere performed in N-methylpyrrolidone (NMP), which displays a similar polarityprofile. However, NMP has limitations, because of the potential developmentof toxic metabolites, such as oxidized derivatives and formaldehyde.16DMF has been successfully replaced in many processes by N-alkylpyrrolidonesmay offer novel opportunities,since their metabolites are less toxic than formaldehyde and relatedcompounds typically deriving from N-Me oxidationin DMF and NMP. Their lower toxicity allowed their use as surfactantsand their addition in cosmetic formulations.17Longer N-butylpyrrolidone (NBP) hasbeenalready successfully used in Heck and Suzuki cross-couplings,18 while less attention has been paid to pyrrolidoneswith longer alkyl chains (N-octylpyrrolidone (NOP), N-benzylpyrrolidone (NBnP), N-cyclohexylpyrrolidone(NCP)), and to N-hydroxyethylpyrrolidone (HEP). Inaddition, anisole and tert-butyl acetate (tBuOAc)have been included, since they are sustainable dipolar aprotic solvents.20Among them, 1a and phenylacetylene 2a, in the presence ofPd(PPh3)2Cl2 and CuI at 30 °Cto test the efficiency of new greener solvents, by screening severalparameters (see 21 A high-performance liquid chromatography–ultraviolet(HPLC-UV) signal at 210 nm was used to follow the transformation ofthe reagents to diphenylacetylene 3a.22 The reactions were stopped when no further evolution intime was observed. DMF and Cyrene experiments were performed as referencereactions and compared with literature data.10 Under the selected conditions, all of the solvents did not affordcomplete conversion  in place of the most commonly used TEA.Under these conditions, the reaction complete conversion was achievedwithin only 30 min, even in the presence of 1% copper co-catalyst, becauseof the complete migration of this solvent in water during the workup.This reaction was also performed on 10 mmol scale, with comparableresults, in order to verify HEP recovery. Distillation of the HEP/waterphase afforded the pyrrolidone in >90% yield. The E factor is comparableto the one achievable in DMF. However, HEP is a nontoxic solvent,23 manageable at high temperatures and easily removableby a simple workup as reported above. Furthermore, HEP can be potentiallyvery inexpensive, being an intermediate in the green synthesis of N-vinylpyrrolidone from biogenic acids.24One of the worst performing solvents, NOP, was usedto optimizethe reaction conditions in further experiments. An excess of 1a and 2a. Thus,all of the reactions were performed in HEP, using Pd(PPh3)2Cl2 (2 mol %) as a precatalyst, copperiodide (1 mmol %), and TMG (1.1 equiv) (see The reaction was extended to substituted aryl iodidesand acetylenessee 2. For eauiv) see 2. The re1b–1g) did not affect reactivity, since all testedreagents displayed complete conversions to 3b–3g at 30 °C in 30 min 2Cl2 had to be replaced by Pd(ACN)2Cl2/Xphos or Pd(DPPF)Cl2.To increase the reaction speed, the inexpensive Pd2Cl2/Xphos, with or withoutcopper  type 1 tyrosine kinase activity in manytypes of human cancers affecting lung, pancreas, ovary, kidney, stomach,liver, and breast tissue.Erlotinib hydrochloride is an oral antitumor drug28 requiresa Sonogashira reaction to convert 3-bromoaniline 4b to3-ethynylaniline 6b. Thus, the reaction between 4b and 2-methyl-3-butyn-2-ol 2h in HEP was studied.As reported in 2Cl2/Xphos catalytic system allowedto achieve complete conversion to the intermediate 5b only after 14 h without CuI Cl2 under copper-free HC conditions, whichallowed complete conversion to be attained within 3 h  in the HCScross-coupling between aryl halides and substituted acetylenes.In summary, several green solventshave been tested to replacetoxic DMF and N-hydroxyethyl pyrrolidone (HEP) has been shownto be the most suitable candidate, allowing one to find mild conditionsfor poorly reactive alkynes and aryl bromides. The versatility ofthe solvent is particularly important when complex molecules are synthesizedvia multistep procedures. The excellent results obtained in the synthesisof an intermediate of the drug Erlotinib encourage in the applicationof HEP on a large scale.


Mesothelioma is an aggressive cancer that is associated with exposure to asbestos. Although asbestos is banned in several countries, including the UK, an epidemic of mesothelioma is predicted to affect middle-income countries during this century owing to their heavy consumption of asbestos. The prognosis for patients with mesothelioma is poor, reflecting a failure of conventional chemotherapy that has ultimately resulted from an inadequate understanding of its biology. However, recent work has revolutionised the study of mesothelioma, identifying genetic and pathophysiological vulnerabilities, including the loss of tumour suppressors, epigenetic dysregulation and susceptibility to nutrient stress. We discuss how this knowledge, combined with advances in immunotherapy, is enabling the development of novel targeted therapies. Mesothelioma is a malignancy primarily of the thoracic and abdominal linings that affects 2600 individuals in the UK annually with a median survival of about 1-year post-diagnosis . The pleura itself is only 40 μm thick and consists of five layers: a monolayer of mesothelial cells on a basal lamina; a superficial elastic layer; a collagen-rich layer containing vessels, nerves and immune cells; and a deep fibroelastic layer. The deep layer is tightly adherent to the underlying structures e.g. muscle, rib or lung parenchyma. Kampmeier foci on the mediastinal pleura contain aggregates of macrophages and lymphocytes, which are involved in immune surveillance and phagocytosis of irritants and pathogens.Mesothelial cells can exist in flat or cuboidal forms. Cell–cell contacts towards their apical surfaces regulate pleural permeability and maintain cell polarity; N-cadherin rather than epithelial E-cadherin is found in mesothelial adherens junctions. Mesothelin is a 40-kDa glycoprotein expressed on mesothelial cells, which mediates cell adhesion partially through its interaction with mucin CA125/MUC16 . Its purPleural fluid is secreted predominantly from the apical zones of the parietal pleura by filtration from capillaries. Numerous microvilli provide a large surface area for secretion and absorption. In addition, primary cilia detect friction and inflammatory cytokines, and so are important in mesothelial repair. When these roles in lubrication and inflammation are perturbed, fibrosis and adhesions can occur.Targeting intrinsic features of cancer, such as genetic instability, unchecked growth, aberrant energetics, enhanced angiogenesis, and immune evasion, suggest a variety of therapeutic options in mesothelioma. We therefore begin by assessing this biology.CDKN2A), BRCA1-associated protein 1 (BAP1) and neurofibromin 2 (NF2) [Mesothelioma has a modest mutational burden compared with other cancers –16. Neve 2 (NF2) –19 [Fig. 2 (NF2) , 21.Fig.BAP1 predispose to malignant mesothelioma [BAP1 have a significantly better prognosis compared with those with sporadic disease, although the mechanism of this is unknown [BAP1 enhances the susceptibility of fibroblasts and mesothelial cells to ionising or UV irradiation, and to asbestos [BAP1 gene product is a deubiquitinating enzyme that plays a key role in the nucleus in the cell cycle, cell death and the DNA damage response [The importance of BAP1 genetics in the development and prognosis of mesothelioma has been extensively reviewed elsewhere –24. In hthelioma , and togthelioma . Remarka unknown , 27. BAP unknown , and losasbestos , which casbestos , 31. Theresponse , 23; whiresponse . BAP1 isresponse , as the response . One sucNF2 encodes Merlin, which negatively regulates receptor-dependent mitogenic signalling, and downstream phosphatidylinositol 3-kinase (PI3K)–AKT activity, and activates Hippo pathway [NF2;INK4a/ARF or NF2;p53 [DCAF1, to stabilise large tumour suppressor kinase 1/2 (LATS1/2), which in turn inhibits the transcriptional co-activators YAP and TAZ, two downstream effectors of the Hippo pathway [LATS1 and LATS2, have been observed in many mesothelioma specimens with aberrant YAP activation [LATS2 and NF2 in mesothelioma cell lines triggers the loss of cell–cell contact inhibition, dysregulation of Hippo and mammalian target of rapamycin (mTOR) signalling, and correlates with higher sensitivity to inhibitors of the PI3K–AKT–mTOR pathway [ pathway . Althoug pathway , additio NF2;p53 . Merlin  pathway . Mutatiotivation , 21. Fur pathway . Targeti pathway  either b pathway . Interes pathway . This mi pathway .CDKN2A encodes two cell-cycle regulators, p16INK4a and p14ARF [INK4a inhibits cyclin-dependent kinase (CDK)4 and CDK6-dependent phosphorylation of retinoblastoma protein (RB), whereas p14ARF prevents p53 degradation by Mdm2 [TP53 occur in less than 10% of mesotheliomas [CDKN2A is deleted in approximately 45% of mesothelioma cases [d p14ARF , 41. p16heliomas , 21 but ma cases , palbocima cases . TreatmePTEN or TP53 promotes the development of non-epithelioid mesotheliomas, with activation of PI3K and MEK–ERK/MAPK [The majority of studies have focused on the more common epithelioid mesothelioma, with relatively few reports pertaining to the rarer, but more aggressive, sarcomatoid subtype. A report in 2020 showed that loss of ERK/MAPK . AccordiERK/MAPK , pointinERK/MAPK –46. For ERK/MAPK , 44, whiERK/MAPK . AnotherCDK2NA, APC, CCND2 and RASSF1) promoters and upregulation of DNA methyltransferases (DNMTs) have been reported [SETDB1 and SETD2) are often found in patient samples [Post-translational modification of histones and methylation of DNA are often altered in cancers  and, in reported , while m samples , 21, 49. samples , 51. A p samples  in 13 pa samples . Unfortu samples . Newer H samples . In vitr samples , indicat samples .Loss of tumour suppressive microRNAs that control cell growth, migration and apoptosis is often observed in mesothelioma . SeveralFocal adhesion kinase (FAK) is a tyrosine kinase that regulates cancer cell survival, proliferation, migration and invasiveness . In vitrc-MET, a receptor tyrosine kinase important in cell proliferation and motility, is also overexpressed in mesothelioma . In vitrMost solid cancers promote angiogenesis to support their growth . AngiogeMesothelial cells produce high quantities of cell-surface glycoproteins, most likely to help lubricate the pleural cavities, and therefore depend on the presence of a functional endoplasmic reticulum (ER) in which to fold these proteins. Increased protein misfolding, which might well occur in response to increased demands for protein secretion, is known to cause ER stress signalling, which has emerged as a key adaptive mechanism supporting cancer progression and resistance to treatment . The expNutrient stress can also affect mesothelioma growth. For example, inhibition of a key component of the glycolytic pathway, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), results in anti-tumour activity in mesothelioma, with death occurring at least in part via the induction of ER stress . MesotheREG) cells [+ lymphocytes correlates with better patient prognosis [+ and low CD4+, predict poor prognosis [Tumour-infiltrating lymphocytes (TILs), macrophages and natural killer (NK) cells are detectable in mesothelioma tissue , 102, whG) cells . Whereasrognosis , 104, hirognosis . Interesrognosis . In pleurognosis . Such knRare cases of spontaneous regression of mesothelioma have been attributed to immune responses , suggestThe tumour-killing immune response can be inhibited by cancer cells that express regulators of immune checkpoints such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin mucin-3 (TIM-3) and PD-L1 . PD-L1, CTLA-4 appears not to be a useful target in mesothelioma. In an early trial, the anti-CTLA-4 monoclonal antibody (mAb) tremelimumab showed a limited response rate , whereasCompared to other malignancies, mesothelioma is not highly immunogenic, which might explain its poor response to existing immune checkpoint inhibitors. Nevertheless, a number of proteins expressed by mesothelioma cells could serve as antigens for alternative immunotherapies, such as vaccines and CAR T cell therapy.In 1982, immunotherapy for mesothelioma using the Bacillus Calmette–Guérin (BCG) vaccine was described in 30 patients, and resulted in apparent increased survival in a group of patients with low tumour burden. . The impListeria monocytogenes engineered to express human mesothelin, stimulated a tumour-specific CD8+ T cell response in 60% of subjects with advanced cancer, including two patients with mesothelioma [Pseudomonas exotoxin A fused to a high-affinity anti-mesothelin antibody, showed limited efficacy in phase I trials, possibly due to the development of neutralising antibodies, which occurred in most patients after a single treatment cycle [Mesothelin is a 40-kDa cell surface glycoprotein expressed by all epithelioid, but not by sarcomatoid or the spindle component of biphasic mesotheliomas . High lethelioma . An earlthelioma . Howeverthelioma . SS1P, ant cycle . A subsent cycle . Anothernt cycle .+ and CD8+ T cell responses [As overexpression of the Wilms’ tumour 1 (WT1) transcription factor occurs in several malignancies, including mesothelioma , WT1 pepesponses . A favouesponses .REG cells [Following the success of the approach in murine models , dendritEG cells . Only onEG cells . A partiEG cells . A phaseEG cells .The immune system can also be engaged by generating chimeric antigen receptor (CAR) T cells. In this approach, homologous T cells are collected from the individual, genetically engineered to express a tumour-specific antigen receptor and, following ex vivo expansion, re-administered to the patient. The CAR consists of an antibody single-chain variable fragment (scFvs) fused to a transmembrane domain, followed by an intracellular co-stimulatory domain  and an intracellular T cell receptor CD3ζ chain . FollowiAntibody–drug conjugates (ADCs) use recombinant monoclonal antibodies that recognise tumour antigens to deliver cytotoxic payloads. Initially, as proof of concept, transferrin was used to deliver intracellular doxorubicin , but a nAnother ADC with potent anti-mesothelioma activity, αMSLN-MMAE, is a humanised anti-mesothelin mAb conjugated to the microtubule-disrupting drug monomethyl auristatin A (MMAE) with a lysosomal-protease-cleavable valine–citrulline linker . A phase+ mesothelioma (NCT03007030).In vitro, epithelioid mesothelioma appears sensitive to brentuximab vedotin (BV), a recombinant chimeric mAb generated against CD30 and conjugated to MMAE via a protease-sensitive linker , 179. SiTripterygium wilfordii that shows a wide spectrum of anti-tumour activities [A panel of ADCs that target trophoblast glycoprotein  conjugated to a tubulin polymerisation inhibitor, yielded encouraging results in mesothelioma cells cultures expressing high levels of 5TA . Anothertivities . It showtivities .+ and CD8+ T cells, as well as NK cells [Oncolytic viruses offer another promising therapeutic approach for several malignancies, including mesothelioma , 188, thNK cells . OncolytNK cells , 191.Thanks to its localised nature, relative lack of metastasis and its physical accessibility, mesothelioma is an attractive candidate for viral therapy. The intrapleural or intraperitoneal administration of double-stranded RNA was reported in 1976 to prevent tumour growth  and the INK4a deletion [CDKN2A. Deletion of ICP34.5, a viral homologue of GADD34, markedly reduces the neurovirulence of the construct but inhibits viral protein synthesis [GADD34 gene into the HSV genome [Another promising oncolytic virus for mesothelioma therapy is a replication-competent HSV in which the viral genes encoding ICP6 and ICP34.5 have been deleted. The lack of ICP6 confers virus selectivity towards mitotic cells or cells with a p16deletion , suggestynthesis , which cynthesis , 206; alV genome . The latV genome .IFNα2b induced an anti-tumour immune response [Clinical trials investigating oncolytic viral therapy for mesothelioma are summarised in Table . The resresponse , which, response . When thresponse . The outAlthough studies of mesothelioma biology have revolutionised our understanding of this cancer, the prognosis for newly diagnosed patients nevertheless remains poor. The current epidemic of mesothelioma that is affecting industrialised countries will soon peak, but the number of cases is predicted to tail well into this century . UnfortuTargeted therapies and immunotherapies that show promise in early trials frequently later fail in phase III trials. This might plausibly reflect a failure to pre-select those patients most likely to benefit from a particular treatment. Improved personalisation of therapies, through stratification of individuals by tumour type, antigen expression, or even genotype, might help overcome this . There i


MYC is a transcription factor in the nuclei that modulates cell growth and development, and regulates immune response in an antitumor direction by mediating programmed death ligand 1 (PD-L1) and promoting CRC tumor recurrence after adjuvant chemotherapy. However, the molecular mechanism through which c-MYC maintains stemness and confers treatment resistance still remains elusive in CRC. In addition, recent reports demonstrated that CRC solid colon tumors expresses C-X-C motif chemokine ligand 8 (CXCL8). Expression of CXCL8 in CRC was reported to activate the expression of PD-L1 immune checkpoint through c-MYC, this ultimately induces chemoresistance in CRC. Accumulating studies have also demonstrated increased expression of CXCL8, matrix metalloproteinase 7 (MMP7), tissue inhibitor of metalloproteinase 1 (TIMP1), and epithelial-to-mesenchymal transition (EMT) components, in CRC tumors suggesting their potential collaboration to promote EMT and CSCs. TIMP1 is MMP-independent and regulates cell development and apoptosis in various cancer cell types, including CRC. Recent studies showed that TIMP1 cleaves CXCL8 on its chemoattractant, thereby influencing its mechanistic response to therapy. This therefore suggests crosstalk among the c-MYC/CXCL8/TIMP1 oncogenic signatures. In this study, we explored computer simulations through bioinformatics to identify and validate that the MYC/CXCL8/TIMP1 oncogenic signatures are overexpressed in CRC, Moreover, our docking results exhibited putative binding affinities of the above-mentioned oncogenes, with our novel small molecule, RV59, Finally, we demonstrated the anticancer activities of RV59 against NCI human CRC cancer cell lines both as single-dose and dose-dependent treatments, and also demonstrated the MYC/CXCL8/TIMP1 signaling pathway as a potential RV59 drug target. Tumor recurrence from cancer stem cells (CSCs) and metastasis often occur post-treatment in colorectal cancer (CRC), leading to chemoresistance and resistance to targeted therapy.  MYC is a transcription factor in nuclei that modulates cell growth and development, and MYC overexpression promotes CRC development and progression [c-MYC is a member of the MYC family, which is commonly expressed in CRC, and regulates cell proliferation, apoptosis, transformation, and therapeutic resistance. In CRC, c-MYC was demonstrated to regulate gene expression which plays an important role as regulator of epithelial stem cells in colon tissue [Wnt/β-catenin, which is reported to play a major role as promoter of CSCs, serves as a transcriptional factor for c-MYC in the nucleus, which ultimately regulates major functions of CSCs. Therefore, c-MYC serves as one of the core stem cell markers [Colorectal cancer (CRC) is the 3rd most common cancer ,2, with gression . c-MYC in tissue ,15. In p markers ,17.c-MYC were demonstrated to regulate antitumor immune responses, by mediating programmed death ligand 1 (PD-L1) and cluster of differentiation 47 (CD47) [c-MYC activates mitogen-activated protein kinase (MAPK) and rat sarcoma (RAS) signaling, which eventually alters the response to oxaliplatin or vorinostat treatment in breast cancer and CRC patients [c-MYC was also reported to be one of the major stem cell markers in CRC. However, the molecular mechanism through which c-MYC maintains stemness and confers treatment resistance still remains elusive in CRC [CXCL8), also known as interleukin (IL)-8 [CXCL8 functions as an inflammatory cytokine secreted by immune cells, such as macrophages within the tumor microenvironment (TME) [CXCL8 expression in CRC was reported to activate the expression of the immune checkpoint PD-L1 through c-MYC [CXCL8 was revealed to play a significant role by promoting tumor formation, progression, and invasion via activating phosphoinositide 3-kinase (PI3K) signaling, which subsequently phosphorylates protein kinase B (PKB) also known as (AKT) and MAPK in breast cancer and CRC [CXCL8 mediates tumor metastasis, stemness, and ultimately induces chemoresistance in CRC [Additionally, high expression levels of 7 (CD47) , as well7 (CD47) . Severalpatients ,21. In ae in CRC ,22. Thise in CRC ,24. Recen (IL)-8 , In addint (TME) ,27. CXCLgh c-MYC . Moreove and CRC ,30. In ae in CRC ,32.CXCL8, MMP7, tissue inhibitor of metalloproteinase 1 (TIMP1), and components of the epithelial-to-mesenchymal transition (EMT) were found to be highly expressed in CRC tumors [CXCL8/MMP7/TIMP7 oncogenes may collaborate and promote the EMT and colon stem cells [TIMP1 is matrix metalloproteinase (MMP)-independent and regulates cell development and apoptosis in various cancer cell types, including CRC [TIMP1 cleaves to CXCL8 on its chemoattractant, thereby influencing its mechanisms and responses to therapy [TIMP1 was also reported to participate in various cell functions including proliferation and survival, leading to reduced sensitivity to chemotherapy in colon cancer [TIPM1 was shown to be associated with poor clinical outcomes in CRC patients compared to normal samples [c-MYC/CXCL8/TIMP1 oncogenic signatures. In the current study, we used computation studies through a bioinformatics analysis to identify and validate expressions of the c-MYC/CXCL8/TIMP1 signaling pathway in CRC, and used in silico molecular docking to evaluate potential interactions of the RV59 with MYC/CXCL8/TIMP1 signaling. RV59 is a small molecule, a derivative of EGFR inhibitor osimertinib derivative [Furthermore, study evidence from a microarray analysis also outlined significant differences in several oncogenes between tumor tissues and normal tissues. Interestingly, C tumors . This suem cells ,35. TIMPding CRC . Recent  therapy . TIMP1 wn cancer ,38,39. I samples . This thrivative ,42, whicrivative .http://way2drug.com/PassOnline/) web resources to predict the spectrum of interactions for known protein kinase inhibitors [50), which is the IC50 as an endpoint, and National Safety Code (NSC) number (763967) as a delimiter [To predict potential drug targets of RV59, we used computer-based Prediction of Biological Activity Spectra (PASS) (hibitors . Additioelimiter .https://www.ncbi.nlm.nih.gov/geo/). The GEO database, a free public genomic database, stores array data and sequence data. GSE74602 included 10 CRC cancer samples and 10 matched adjacent normal samples [In total, three gene expression profiles  were extracted from the Gene Expression Omnibus , an open-access public online tool for analysis of The Cancer Genome Atlas (TCGA) [MYC/CXCL8/TIMP1 in CRC samples (red) were compared to adjacent normal samples (blue), with p < 0.05 indicating statistical significance. Furthermore, we used GECO, a gene expression correlation analytical tool, which distinguishes two expression datasets into positive and negative correlations [p < 0.05 as statistically significant.Expression levels of the s (TCGA) . Expresselations , with pohttps://string-db.org/) to construct a PPI clustering network [https://genemania.org/) and cytoscape software (version 3.8.2) were used to build gene interactions and PPI networks respectively, and from analysis of the results, the interactive networks were based on gene co-expression, co-localization, genetic interactions, and various pathways involved within the network. The database for annotation, visualization, and integrated discovery (DAVID), (https://david.ncifcrf.gov/.jsp), was used to analyze enriched GO including biological processes and molecular functions involved, with the criterion set to p < 0.05.Protein interactions were analyzed using the STRING tool  [Interpretation of gene expression network was analyzed using the Network Analyst 3.0 tool (yst.ca/) , a compryst.ca/) .MYC/CXCL8/TIMP1 expressions and tumor infiltration levels were analyzed with the Tumor Immune Estimation Resource (TIMER) (https://cistrome.shinyapps.io/timer/), an online computational tool used to analyze the nature of tumor immune interactions across a variety of cancers [MYC, CXCL8, and TIMP1 with a set of gene markers of immune infiltration cells including CD8+ T cells and macrophages (with p < 0.05). Furthermore, we applied the SCNA statistical module, a sub-tool of TIMER to identify the abundances of tumor infiltrates including CD8+ T cells, macrophages, and dendritic cells (DCs) in colon cancer. The infiltration level was compared to the normal level using a two-sided Wilcoxon rank-sum test.Correlations between  cancers . Herein,MYC/CXCL8/TIMP1 oncogenes and drug sensitivity of the genomics of drug sensitivity in cancer (GDSC) top 30 drugs in pan-cancer, we used the Gene Set Cancer Analysis (GSCA), a web-based tool used to analyze differentially expressed genes (DEGs) and correlation to drug sensitivity [To determine the correlation between sitivity . All theMYC/CXCL8/TIMP1 oncogenes. Crystal structures of MYC (PDB: 6G6K), CXCL8 (PDB:5D14), and TIMP1 (1UEA), were downloaded from the Protein Data Bank (PDB). The 3D structure of RV59 was constructed with the Avogadro molecular visualization tool [14H2ON2O3), repaxicin (CID:9838712), with a Mw of (283.39 g/mol and molecular formula (C14H21NO3) and ilomastat (CID: 132519), with a Mw of (388.5 g/mol and molecular formula (C20H28N4O4), were all retrieved from PubChem as SDF files. For further processing, we used PyMol software (https://pymol.org/2/) to visualize the ligands and convert them into PDB file format, and these files were subsequently converted into PDBQT format using the computer-based software, autodock (http://autodock.scripps.edu/resources/adt). For visualization and interpretation, docking results were analyzed using Discovery Studio [In silico molecular docking is a technique used to predict protein-ligand interactions, and this includes detection of the position and orientation of the ligand . In ordeion tool , while ty Studio .MYC/CXCL8/TIMP1 expressions in CRC cancer types. The statistical significance of differentially expressed genes (DEGs) was evaluated using the Wilcoxon test. * p < 0.05 was accepted as being statistically significant.Pearson’s correlations were used to assess correlations of 50 as an endpoint and the NSC number as a delimiter [MYC, CXCL8, and TIMP1 were among the genes displayed by the prediction tools , and lipophilicity . Moreover, pharmacokinetics (PKs), drug-likeness, and medicinal chemical properties of RV59 , which was recently synthesized in our laboratory. Using swissADME, a free web tool to evaluate pharmacokinetics, drug-likeness, and medicinal chemical friendliness of small molecules , we asse of RV59  indicateDEGs from the microarray dataset were shown by identifying expressed genes between colon cancer samples and normal samples tallied from separate studies. Results showed that 536, 144, and 607 DEGs were respectively obtained from the GSE41328, GSE44861, and GSE74602 datasets. Eighty-five genes overlapped in these datasets as demonstrated in a Venn diagram A. Red anMYC/CXCL8/TIMP1 oncogenes were analyzed using UALCAN , a comprehensive open-access public online tool for analyzing cancer data [MYC/CXCL8/TIMP1 oncogenes in CRC primary tumor samples (red) were compared to adjacent normal samples (blue), with p < 0.05 considered statistically significant  to construct a PPI clustering network [https://genemania.org/) and cytoscape software were used to build the PPI network. From analysis of the results, interactive networks were based on gene co-expression, co-localization, genetic interactions, and various pathways involved within the network. In short, we identified interactions of CXCL8 with MYC, TIMP1, MMP9 and ACKR; of MYC with CXCL8, TIMP1, MMP9, CDK4, CCND1, RIOX2, BCAT and DDX18; and of TIMP1 with MYC, CXCL8, MMP9, CCNA2, JUND, and STAT3 (https://david.ncifcrf.gov/.jsp) was used to analyze the enriched GO including involved biological processes and biological pathways, with the criterion set to p < 0.05 , where markers were adjusted by purity. As expected, results showed correlations of immune cell markers in colorectal adenocarcinoma (COAD), specifically CD8+ T cells and M2 macrophages , to be less sensitive to the drugs. Interestingly, high expression levels of MYC, TIMP1 and CXCL8 gene signatures, were shown to be resistance to Bx-912 (PDK-1 inhibitor) [HDAC and MYC inhibitor) [HDAC inhibitor) [To determine the drug sensitivity of response . The corhibitor) , navitochibitor) , vorinoshibitor) ,62 and thibitor)  among otMYC, −7.7 kcal/mol for CXCL8, and −6.9 kcal/mol for TIMP1. In addition, a visualization analysis showed that docking of the small molecule (ligand) displayed shorter binding distances with the proteins (receptors) of 2.49 Å for MYC, of 2.03 Å for CXCL8, and of 2.51, 3.14, 3.4, and 3.29 Å for TIMP1 (MYC (vorinostat), CXCL8 (reparixin), and TIMP1 (ilomastat). Interestingly, the standard inhibitors exhibited lower binding energies for MYC  and CXCL8 , with the exception of TIM1 , which showed a slightly higher binding energy compared to the RV59 compound , and 50% lethal concentration (LC50) values. Results showed potential anti-proliferative effects in a dose-dependent manner  chemotherapy. Among those 20 compounds, RV59 was more effective, overcame cNrf2-mediated chemoresistance, and suppressed tumor growth in colon cancer cells [Advanced chemotherapy and targeted therapies still offer limited prolonged overall survival in CRC patients. One of the main causes of poor prognoses in patients is resistance to these therapeutic interventions . Moreovemertinib . Our preer cells . Howeverer cells .MYC/CXCL8/TIMP1 as target genes for RV59; in addition, an analysis of the results also predicted that all probability active (Pa) values were greater that all predicted probability inactive (Pi) values, indicating the inhibitory or antagonistic activities of RV59 on MYC and CXCL8, among other identified activities. In the early stages of drug discovery and development, the disposition of a compound is assessed in terms of its absorption, distribution, metabolism, and excretion (ADME), with the final goal of identifying potential effectiveness of a medicine for patients [2), and lipophilicity (XLOGP3 = 2.76), and all the values were within recommended values as described in MYC/CXCL8/TIMP1 oncogenic signatures in CRC primary tumors and compared them to adjacent normal samples. The genes also exhibited positive correlations among each other with p < 0.05 indicating statistical significance.Herein, we used a bioinformatics simulation study to further expand on that previous study; however, the present study focused more on identifying and validating oncogenes associated with chemoresistance and alteration of oxaliplatin or doxorubicin treatment responses in advanced CRC. Studies showed that accumulation of tumor recurrence from cancer stem cells (CSCs), and metastasis often occur post-treatment in CRC, which leads to therapeutic resistance ,71. Usinpatients . After aMYC/CXCL8/TIMP1 oncogenes at the protein level. Interestingly, the PPI network analysis from two independent databases showed interactions of all of the proteins with each other, as well as enrichment in GO terms, including biological processes and biological pathways involved in CRC, with p < 0.05 indicating statistical significance  by pembrolizumab enhanced regulation of cytotoxic T-cell tumoricidal activities, which ultimately leads to increased expression levels of tumor-enhanced CXCL8, which subsequently induces infiltration of tumor-associated macrophages (M2) in the immune system [TIMP1 gene derived from tumor cells creates a metastatic niche, to which circulating tumor cells cling and promote CRC metastasis [MYC/CXCL8/TIMP1 oncogenic signatures to chemoresistance and resistance to targeted treatment. To identify associations of MYC/CXCL8/TIMP1 gene expressions with selected immune cells in CRC, we applied a correlation analysis between the above-mentioned oncogenes with immune infiltration cells (CD8+ and macrophages), where markers were adjusted by purity. As expected, MYC/CXCL8/TIMP1 gene expression levels displayed associations with tumor purity and were positively correlated with infiltrating levels of CD8+ T cells and macrophages (p < 0.05), and were correlated with the abundances of tumor infiltrates including CD8+ T cells, macrophages and DCs in CRC , CXCL8 , and TIMP1 , and short binding distances with these protein (receptors): MYC (2.49 Å), CXCL8 (2.03 Å), and TIMP1 . These results were compared to standard inhibitors of MYC (vorinostat), CXCL8 (reparixin), and TIMP1 (ilomastat). Interestingly, the inhibitors exhibited lower binding energies for MYC  and CXCL8 , with the exception of TIMP1  compared to RV59. Moreover, RV59 showed anticancer activities against NCI human colon cancer cell lines with single-dose treatment of 10 μM, toward the COLO 205, HCC-2998, and HCT-15 cell lines, and anti-proliferative activities toward the HCT-116, HT29, KM12, and SW-620 cell lines, in dose-dependent manners. measured Results of measuring the GI50, TGI, and LC50 levels showed potential anti-proliferative effects in dose-dependent manners (50 results ranged 0.18–1.85 μM on colon cancer cell lines, with HCT-15 cells being the most responsive at 0.18 μM, followed by HCT116 at 0.28 μM, SW-620 at 0.29 μM, HT-29 at 0.47 μM, KW12 at 0.84 μM, and COLO 205 at 1.15 μM, with HCC-2998 cells at 1.85 μM showing the least responsiveness compared to the aforementioned cell lines. This suggests that RV59 exhibits drug-like characteristics, and is a potential oral drug candidate. In summary, the findings from this study revealed the anticancer activities of RV59 in CRC, and highlight additional avenues for RV59 as a potential treatment for CRC chemotherapy and resistance to targeted therapy, particularly by targeting the MYC/CXCL8/TIMP1 signaling pathway. The compound is currently being assessed for its therapeutic potential effectiveness in CRC and breast cancer both in vitro and in vivo in our laboratory.We further predicted protein-ligand interactions using molecular docking between RV59 and the  manners B,C. MoreMYC/CXCL8/TIMP1 signaling pathway, which is responsible for resistance to both chemotherapies and targeted therapies, to be a potential RV59 drug target. Moreover, the in silico molecular docking study exhibited putative binding affinities of RV59 with the above-mentioned oncogenes, which were even higher than the standard inhibitors of these genes. Currently in our lab, there are ongoing cell and animal experiments to evaluate the therapeutic effectiveness of RV59 in CRC and breast cancer.In conclusion, we revealed the anticancer activities of RV59 against NCI human colon cancer cell lines both as a single dose and dose-dependent treatment, and also demonstrated the