Protein aggregation is classically considered the main cause of neuronal death in neurodegenerative diseases (NDDs). However, increasing evidence suggests that alteration of RNA metabolism is a key factor in the etiopathogenesis of these complex disorders. Non-coding RNAs are the major contributor to the human transcriptome and are particularly abundant in the central nervous system, where they have been proposed to be involved in the onset and development of NDDs. Interestingly, some ncRNAs  share a common functionality in their ability to regulate gene expression by modulating miRNAs in a phenomenon known as the competing endogenous RNA mechanism. Moreover, ncRNAs are found in body fluids where their presence and concentration could serve as potential non-invasive biomarkers of NDDs. In this review, we summarize the ceRNA networks described in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis and spinocerebellar ataxia type 7, and discuss their potential as biomarkers of these NDDs. Although numerous studies have been carried out, further research is needed to validate these complex interactions between RNAs and the alterations in RNA editing that could provide specific ceRNET profiles for neurodegenerative disorders, paving the way to a better understanding of these diseases. Neurodegenerative diseases (NDDs) are of increasing relevance in public health due to the aging of the global population. These common and complex disorders are characterized by a progressive and selective loss of neurons from specific regions of the central nervous system (CNS), the most prevalent NDDs being Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Although protein aggregation is a common hallmark for these disorders, there is growing evidence that alterations in RNA metabolism contribute to the etiopathogenesis of NDDs ,2,3. DefncRNAs can be classified into two groups according to their length: small ncRNAs (<200 nucleotides) and long ncRNAs (>200 nucleotides) . Among sOn the other hand, a considerable number of pseudogenes can be transcribed to ncRNAs, even though they have historically been regarded as inactive gene sequences ,14. In fIn 2011, Pier Paolo Pandolfi’s group proposed the so-called ceRNA hypothesis , which sIn a simplified manner, when two RNA molecules share the same MRE they potentially compete for the same pool of miRNAs. Thus, when the expression of a ceRNA is upregulated, it will bind and titrate more miRNAs , leaving fewer miRNA molecules available for binding the mRNA with shared MRE. Hence, this corresponding mRNA will become derepressed. In reverse, when the ceRNA levels are reduced as a consequence of a biological disturbance, the corresponding mRNA will be downregulated due to hyperrepression . Without doubt, the reality is more complex and a miRNA can bind more than one mRNA (50% of miRNAs are predicted to target 1–400 mRNAs and some of them up to 1000) . LikewisThe potential of ceRNAs as biomarkers, the major focus of this review, is augmented by the fact that ncRNAs have been found in body fluids like blood or urine free or inside extracellular vesicles including exosomes, which would allow obtaining new biomarkers in a non-invasive way. On this basis, emerging research on the role of ncRNAs in various diseases has arisen ,18,19. IOver the last years, the ceRNA hypothesis has been corroborated by a large number of experiments. However, investigation of ceRNA mechanisms and their interaction networks has been mainly carried out in cancer research ,21,22,23We searched the PubMed database for articles in English. The main search terms included neurodegenerative diseases, Alzheimer’s disease, AD, Parkinson’s disease, PD, multiple sclerosis, MS, amyotrophic lateral sclerosis, ALS or spinocerebellar ataxia type 7, combined with competing endogenous RNA, ceRNA, long non-coding RNA, lncRNA, circular RNA, circRNA, pseudogene or miRNA. Since this is a relatively new research topic, no filter for the years was applied. We hand-searched the retrieved articles and selected the most relevant articles based on a subjective evaluation of their quality and relevance. Additional articles on specific topics were searched if needed.In the context of NDDs, most ceRNA research has been done in AD, probably due to its growing prevalence in the aged population of most developed countries . Such isAt molecular level, dementia associated with AD is characterized by proteinopathy with extracellular deposition of β-amyloid (Aβ) plaques in the brain and the presence of TAU neurofibrillary tangles (NFTs) in neurons. Consequences of AD at cellular level include deregulation of redox homeostasis and low-grade chronic inflammation . BACE1 post-transcriptional regulation, one of the most relevant being the lncRNA BACE1-AS [BACE1 in a ceRNA regulation system that involves several miRNAs including miR-29, miR-107 and miR-124 [BACE1, thus preventing BACE1 mRNA from degradation. This mechanism complements the one previously reported by which BACE1-AS increases the stability of BACE1 mRNA through the formation of RNA duplexes [Atg12 and having hence a neuroprotective effect [As mentioned above, the deposition of Aβ plaques in the brain is one of the hallmarks of AD. β-secretase 1 (BACE1), one of the enzymes involved in the Aβ formation, is elevated in brains of AD patients ,122. TheBACE1-AS , an antiBACE1-AS . Also, BBACE1-AS . Furtherduplexes . BACE1-Aduplexes , being tIn addition, BACE1-AS is a ceRNA for miR-132-3p , which iBACE1 mRNA levels in AD cell and mouse models [BACE1 regulation in AD being mediated by several ceRNA networks.Besides BACE1-AS, other lncRNAs such as XIST (through miR-124), NEAT1 (which bind miR-124 and miR-107) and SOX21-AS1 (targeting miR-107) may regulate e models ,29,30,31BACE1 expression and the levels of other genes involved in AD [CDK5R1 levels [CDK5R1 codes for the main activator of cyclin-dependent kinase 5 (CDK5), p35, essential for brain development and functioning so that its deregulation could be implicated in AD onset and progression [CDK5  is regulated by miR-125b, which in turn is reversely regulated through MALAT1 [Other members of the miR-15/107 family can also regulate ed in AD . In this1 levels . CDK5R1 gression . Interesh MALAT1 . Furtherh MALAT1 .SIRT1 [ROCK1 is upregulated in AD cellular and mouse models and silencing of the lncRNA TUG1 depresses apoptosis of hippocampal neurons (like XIST1 knockdown) by elevating miR-15a and repressing ROCK1 expression. ROCK1 is a ubiquitous serine/threonine kinase whose reduction has been reported to diminish Aβ levels by enhancing APP protein degradation in AD [Besides miR-124, the lncRNA XIST targets another miRNA and could participate in more than one ceRNET in AD. Wang et al.  showed tSIRT1 ,133. CurSIRT1 ,136,137.SIRT1  reportedon in AD .KREMEN1 levels [ZNF217 [ZNF217 (zinc finger gene 217) has been described as a potential oncogene [ZNF217 [Aβ peptides have also been related to other lncRNAs that have been proposed to act as ceRNA in AD: the lncRNAs SNHG1, lncRNA-ATB, LINC00094, MIAT1 and Rpph1. Small nucleolar RNA host gene 1 (SNHG1) is a recently described lncRNA involved in the development of multiple human tumors, as well as in other types of diseases such as AD or PD ,143,144.1 levels  and as a [ZNF217 . KREMEN1 [ZNF217 ,146, wheoncogene . Notably [ZNF217 . NeverthSH3GL2 mRNA [SH3GL2 codes Endophilin-1, an endocytosis protein markedly increased in the AD brain and involved in Aβ induced postsynaptic dysfunction [SH3GL2 mRNA [LncRNA LINC00094  has been reported to regulate blood-brain barrier (BBB) permeability in AD microenvironment by sponging miR-224-5p and miR-497-5p, both of which target GL2 mRNA . SH3GL2 function . Taken tGL2 mRNA , it seemVEGF axis, acting as a vascular dysfunction regulator [LncRNA MIAT  is aberrantly expressed under neurovascular dysfunction , a condiegulator ,151,152.egulator  and obsePKM2 [Wnt1 [CD42 [In contrast to the lncRNAs discussed above, Rpph1 (ribonuclease P RNA component H1) seems to exert a neuroprotective compensation mechanism in AD pathology through three different ceRNA axes: Rpph1/miR-326/PKM2 , Rpph1/mM2 [Wnt1  and Rppht1 [CD42 . In partt1 [CD42 ,42, as wt1 [CD42 . Rpph1 wt1 [CD42 , where iKlf4 (upregulated at the early stage of the disease) and Akap5 (downregulated at late-stage), respectively, and LNC_000033, which acts as a ceRNA of 5 miRNAs that regulate the expression of Synpo. Klf4 and Akap5 are two genes related to Aβ-induced neuroinflammation, and synaptic plasticity and memory, respectively. As for Synpo, it codes for synaptopodin, an essential protein for dendritic spine plasticity of the developing hippocampus that was found upregulated at early stage [In continuation to this work, the same laboratory studied the RNA expression profile in cortex samples from the same mouse model at different ages to determine the distinct lncRNA-associated ceRNA network (LncACeNET) that could be participating in the progression of the disease depending on the stage . In genely stage .Similarly, LncACeNETs have also been constructed based on human samples ,98. WangMAPT and TTBK1 by sponging miR-181c-5p. As mentioned above, p35 is the main activator of CDK5, a kinase implicated in AD onset and progression [As CTB-89H12.4, two other lncRNAs have recently been implicated in AD pathogenesis through tau phosphorylation and neuroinflammation. First, Yan et al.  demonstrgression . Interesgression ,153,154.gression  showed tCDR1 mRNA [UBE2A that is essential for Aβ clearance [NF-κB p65 subunit mRNA levels were not affected by ciRS-7 here, other studies have shown that the expression of p65 is downregulated by miR-7 [NF-κB(p65) could represent another potential axis in AD and it might be influenced by APP, since it may reduce the levels of ciRS-7 [LncRNAs are not the only ncRNAs proposed as AD-associated ceRNAs, as these also include circRNAs. One of the best-studied circRNAs is CDR1as, an antisense circular transcript of the Cerebellar Degeneration-Related protein 1 (CDR1), highly expressed in the brain . This ciDR1 mRNA  and to aDR1 mRNA . Intereslearance . This walearance . Neverthlearance . Althougby miR-7 ,49. Therf ciRS-7 .PTGS2, a pro-inflammatory gene reversely regulated by miR-103 [Sirt1, since it has been proposed to act as a sponge for this miRNA. As previously mentioned, Sirt1 regulates Aβ production in AD through ROCK1 or ADAM10 [Sirt1 and ADAM10 levels, thus mediating synaptic function and APP processing in AD [AKAP5 and DRD1, which are known to interact with PSD-95 [AKAP5 is a gene related to synaptic plasticity and memory and is part of a LncACeNET in AD [Recently, other circRNAs have been found altered in AD, but, unlike ciRS-7, they are less well known. Among these, circ_0000950 promotes neuronal apoptosis, enhances inflammatory cytokine levels and suppresses neurite outgrowth in two cellular models of AD by directly sponging miR-103 and increasing the mRNA expression of r ADAM10 ,136,137.r ADAM10 . This cong in AD . In lineng in AD  demonstrng in AD . Although PSD-95 . As prevET in AD . As for ET in AD .iodothyronine deiodinase Dio2 mRNA expression, which is reduced in AD patients and is related to the myelination process [SLY. SLY encodes Silymarin, a natural Aβ aggregation inhibitor with a large potential for the treatment of AD [The rest of the circRNA-associated ceRNA networks (cirCeNET) described in AD have been identified by transcriptome profiling studies in different animal models and patient samples. In the brain of the senescence-accelerated mouse model (SAMP8), Zhang et al.  construc process ,161. In  process  identifint of AD ,163,164.Aqp3, where Aqp3 codes for an aquaporin expressed in astrocytes and neurons, but its role in AD remains scarcely investigated [Gsdmd. GSDMD is a key executive protein of pyroptosis, a highly inflammatory form of programmed cell death that has been reported to be an AD therapeutic target [Sorbs2, a gene that influences memory and dendritic development and its variants consistently were associated with delayed onset in AD [Cntnap2, involved in the development of neural systems critical for learning and cross-modal integration [Creb mRNA. CREB is an important transcriptional factor in the regulation of brain-derived neurotrophic factor (BDNF) and with the CREB-BDNF signaling pathway modulating cognitive status and Aβ toxicity in AD [Aanat, a critical enzyme for melatonin synthesis [Other studies carried out in animal models analyzed specific brain areas such as the hippocampus, cortex or pineal gland. In the hippocampus of Aβ1-42-induced AD rat model , Wang etstigated . In hippstigated  establisstigated . Seven cc target . Thus, tc target . In the c target  construcc target . Four miet in AD ,170. Sevegration . Mmu_cirty in AD . In the ty in AD  construcynthesis .PSEN2, KIAA1586/hsa-miR-101/UBE2A and KIAA1586/hsa-miR-15a, hsa-miR-29b/BACE1. Accordingly, other studies have demonstrated the relationship between miR-29b and BACE1 in AD [APP [In AD patients, three studies have provided circRNA expression profiling in the brain, and two in body fluids. Using different brain regions, Zhang et al.  construcE1 in AD ,175, and AD [APP ,176.HOMER1, a gene involved in Aβ processing whose dysregulation may underlie the early phase of memory loss that occurs in AD [PSEN1 and PSEN2. Likewise, circCORO1C was predicted to contain two binding sites for miR-105 that target APP and SNCA. In the brain cortex of patients, Dube et al.  identifirs in AD ,178,179.SLC8A1, a gene that codes for Na+/Ca2+ Exchange Protein 1 (NCX1) which could exert a neuroprotective role in AD [EPHA4, an emerging Aβ oligomer receptor that could be involved in the synaptic spine alterations found in AD [EPHA4 mRNA and confirm this ceRNA network.Similar to the last study, Lo et al.  investigle in AD . Both cind in AD . HoweverBDNF [retinoic acid receptor alpha (RARA) involved in the regulation of GSK-3β activity [SIRT1 since miR-138-5p could also bind to SIRT1 and mediate APP processing in AD (see above) [In cerebrospinal fluid (CSF) from patients, Li et al.  construcBDNF . In turnactivity . Anothere above) . This ste above) .SLC8A1 (involved in neuroprotection) and hsa_circ_061346/hsa-miR-103a-2-5p/HOMER1 (involved in Aβ processing). Interestingly, this study also found that the differentially expressed circRNAs partially overlapped in this network analysis. In this sense, 13 circRNAs contain MRE sequences for hsa-miR-455-3p, which has been confirmed to bind at the 3’UTR of the APP gene and regulates its expression, exerting a protective effect on AD [In peripheral blood mononuclear cells (PBMCs) of patients, Li et al.  construcct on AD .SNCA gene [PD is the second most frequent neurodegenerative disease after AD, and the most prevalent movement disorder, affecting approximately 1% of the population aged over 60 . The etiNCA gene .GBA gene and its 96% homology pseudogene GBAP1 by miR-22-3p binding [GBA encodes a lysosomal glucocerebrosidase and its mutations represent the major genetic predisposing factor for PD [GBA has been closely related to key processes in this disease, including α-synuclein aggregation, lysosomal and autophagy dysfunction and endoplasmic reticulum stress [The first ceRNA network described in PD involved the regulation of  binding . GBA encr for PD . Moreovem stress . HoweverPTEN expression [+-induced cytotoxicity. It is worth noting that this signaling pathway has been also associated with the HAGLROS/miR-100/ATG10 LncACeNET [GSKβ3 gene, establishing a new ceRNA axis implicated in neuron cytotoxicity and reactive oxygen species production [NLRP3 axis [The lncRNA small nucleolar RNA host gene SNHG1 has been found upregulated in in vitro models of PD from neurons and microglia, as well as murine models ,56,57,58pression . PTEN ispression . This ispression  and thatncACeNET . AdditioncACeNET ,194. miRoduction . FurtherATG10 [RAB3IP axis could provide a new pathomechanism and therapeutic target in the disease. To unravel the role of HOTAIR in vivo, PD mice were injected with HOTAIR shRNA, resulting in protection against the initiation and development of PD, including cognitive impairment and bradykinesia. At molecular level, these mice had lower levels of RAB3IP and increased miR-126-5p expression compared to the PD control group. Moreover, at cellular level, shRNA-HOTAIR increased the number of TH-positive cells, reduced α-synuclein-positive cells and protected neurons from apoptosis [LncRNA HOTAIR is overexpressed in both in vitro and in vivo models of PD ,61,195. ATG10 , a gene ATG10  that codATG10 . HOTAIR ATG10 ,198. In RAB3IP is also a part of another ceRNA axis, in this case together with the lncRNA NEAT1 and miR-212-5p [RAB3IP being upregulated and miR-212-5p downregulated. Importantly, NEAT1 is increased in peripheral blood cells of PD patients [ARHGAP26 [R-212-5p . This axpatients . Its knopatients , supportpatients . NEAT1 aARHGAP26  and NEATARHGAP26  axes.MAPK1 overexpression. Likewise, MALAT1 also acts through a ceRNA mechanism involving miR-205-5p but to regulate LRRK2 expression, contributing to cell apoptosis in in vitro and in vivo models of PD [LncRNAs AL049437, MALAT1, SNHG14, lincRNA-p21, GAS5 and BDNF-AS, upregulated in mouse and in vitro models of PD, have also shown to exert a detrimental role and contribute to the progression of the disease ,67,70. Als of PD .LRRK2 [MAPK1 and LRRK2 genes.Taken together, these data may point towards a common regulatory network where all MALAT1, AL049437 and HOTAIR, which also regulated LRRK2 , sponge DAPK1 [DAPK1 downregulation, alleviating cell apoptosis in vitro and in vivo and improving behavioral changes in PD mice. Alternatively, other mRNA targets of miR-124-3p are likely to play a role in PD. Those include MAP3K3, RELA/p65 [Bim [SQSTM1, MAPK11 [STAT3 [ANXA5 [PRKCD (PKC-δ) family feedback loop [TRPM2 axis [NLRP3 expression via competitive sponging of miR-223-3p and miR-125b-5p, enhancing in vivo and in vitro microglial inflammatory response [Additionally, Liu et al. proposed a second ceRNA mechanism, in which MALAT1 would serve as a molecular sponge of miR-124, although the mRNA downstream this edge was not investigated . Later sDAPK1 , which eDAPK1 ,204. In RELA/p65 , Bim [20p65 [Bim , SQSTM1,, MAPK11 , STAT3 [1 [STAT3 , and ANX3 [ANXA5 , among o3 [ANXA5 ,212,213.3 [ANXA5 . Similar3 [ANXA5 ,71, and 3 [ANXA5 ,70,71. Lack loop , and plaPM2 axis . Finallyresponse  and promresponse , respectHPRT1 [PIK3R3 axis [PIK3R3 a gene that codes for a regulatory subunit of PI3K associated with increased susceptibility to PD [As opposed to the above-mentioned ceRNA networks, lncRNAs can also have a protective role in PD. Such is the case of the lncRNA Mirt2 or lncRNA H19. Mirt2 prevents TNFα-triggered inflammation via the repression of miR-101 , whereasHPRT1 , and con3R3 axis , being Pty to PD .IRF1 and RIMKLA , NAV1 (related to RPS14P3) and SACS and SDC2 (G046036), all thought to be related to neurodegenerative processes although their role in PD still awaits clarification. One year later, Chi et al. established a PD-associated network from differentially expressed RNAs in PD patients’ blood versus controls [PTBP1, FBXL7, SRSF1, PTBP2, UBE2Q2 and RBBP6) related to mRNA metabolism, mitochondrial function and injury, DNA damage and protein polyubiquitination. Interestingly, PTBP1 was previously reported in PD [Besides experimentally validated ceRNA networks, in recent years, bioinformatic predictions have uncovered new disease-associated ceRNAs and their principal mechanisms of action. In 2017, Lin and colleagues  establiscontrols . This necontrols  identified in PD ,216.NLRP3 mRNA could compete for it [SNCA mRNA and repress α-synuclein protein levels [SNCA axis has been proposed as a possible ceRNET in PD [SNCA gene itself  acts as a ceRNA of miR-7 and upregulates SNCA in PD. Furthermore, pramipexol (PPX), a common treatment for PD, exerts suppressive effects on circSNCA expression. In agreement, Sang and coworkers showed that the inhibition of circSNCA and SNCA reduce apoptosis and promote autophagy, thus attenuating the progression of PD [SNCA), or even ciRS-7, circSNCA and SNHG1 may be part of the same ceRNET (miR-7/SNCA and NLRP3) exerting a cooperative regulation. As previously mentioned, miR-7 is part of a PD-related ceRNET where the lncRNA SNHG1 and e for it . Howevern levels . Interesn levels . Since Cn levels ,47 , a bZIP transcription factor involved in PD by regulating α-synuclein levels [M60.4, ZK470.2, igeg-2 and idhg-1, the ortholog of mitochondrial isocitrate hydrogenase (NAD+). On the other hand, Feng et al. [BDNF, Bcl-2 and PGC-1a, all of which are neuroprotective factors involved in many NDDs including AD and PD [In contrast to circSNCA, the circRNAs circzip-2 and circDLGAP4 have been found altered in PD with a protective role. On one hand, Kumar et al.  observedn levels ,217. Thrg et al.  describeD and PD ,219,220.Recently, two studies have provided circRNA expression profiling in PD brain. Jia et al.  identifiSLC8A1 gene that codes for NCX1, which could be involved in AD (see above) and also PD [BMI1, SIRT1 and AXIN1) was reported in PD brains. Therefore, a new ceRNET  was described that could be involved in PD pathology through oxidative stress [Hanan et al.  explored also PD . circSLCe stress . MS is a chronic autoimmune disease, characterized by an immune-cell-mediated attack to the white matter in the CNS causing inflammation, demyelination and axonal loss ,225. MS IL17 mRNA and protein levels, as well as mRNA of RORϒt, a lineage defining transcription factor required for Th17 cell differentiation and function [SOCS5 in EAE mice were associated with an increased number of Th17 cells and markers of inflammation  in the spinal cord of this model. These inflammation markers were reduced with the administration of exosomes from M2 microglia, which are enriched in PVT1; and this effect was reversed by the administration of M2 exosomes with shRNA for PVT1. Hence, it was proposed that PVT1 could ultimately inhibit the proinflammatory response of Th17 cells through a JAKs/STAT3-mediated pathway. Lately, a large number of studies support the importance of lncRNAs role in the differentiation, function and misproportion of immune cells , as wellfunction . These afunction . This, tfunction . On the function  and in tfunction . Wu et afunction , a protefunction . SOCSs pfunction , regulatfunction . MoreoveNFkB1(p50) ceRNET described by Yue et al. in 2019 [The above results indicate that the lncRNA-ceRNA networks influence the inflammatory response in MS. This idea is also supported by the TUG1/miR-9-5p/ in 2019 . TUG1, o in 2019  and last in 2019 . ImportaBesides inflammation, demyelination is another important MS hallmark, a process that could also be influenced by lncRNAs acting as miRNAs sponges. Sulfasalazine, a drug that promotes remyelination and improves the outcome of MS patients ,236, exeIL7R, SP140) and contribute to back-splicing of approximately 50 circRNAs [Finally, lncRNA MALAT1, which is also dysregulated in MS, can cause alternative splicing abnormalities of MS-associated genes  from the roptosis , have beroptosis . Severalroptosis ,243. It s levels . ASIC1a  disease ,247,248. miR-149 . Another miR-149  found twdiseases ,250. Thrdiseases ,94. HoweFUS and TARDBP) are involved in transcription and RNA metabolism [ALS is the third most common progressive neurodegenerative disease, affecting 150,000 people in the world . It is ctabolism  and, nottabolism ,255,256.FUS and TARDBP genes, respectively. These specialized RNAs are especially enriched in the lncRNA NEAT1_2, which has been identified predominantly expressed in spinal motor neurons in early phases of ALS pathological process and was shown to directly bind FUS and TDP-43 [In agreement, a potentially pathological paraspeckle enrichment was observed in motor neurons of ALS patients’ brains . Parasped TDP-43 ,258. Thed TDP-43  by sequed TDP-43 , remainsd TDP-43 .FUS mutation [Whole transcriptome RNA-seq analyses have revealed differential expression of lncRNAs both in blood PBMCs and iPSC-derived motoneurons from ALS patients ,261,262,mutation .NEFL [NEFL), where hsa_circ_0023919 could act as a miR-9 sponge and regulate the metabolism of intermediate filaments (NEFL) observed in ALS [PTEN, a direct target of miR-647 [PTEN has been seen involved in AD- and PD-associated ceRNA networks. Since PTEN has been reported as a potential therapeutic target in motor neuron diseases, including ALS or SMA  [PTEN mRNA could also be part of ALS-linked ceRNETs.Similar to lncRNAs, little is known about circRNA in ALS. However, three circRNAs have shown some potential as diagnostic biomarkers . DolinarNEFL , which ed in ALS . In contd in ALS . However miR-647 . As mentatrophy) ,269,270,It is important to note that, although there are no experimentally confirmed ceRNAs associated with ALS, ceRNA mechanisms can regulate myogenesis , an alteSCA7 gene. This results in formation of protein aggregates and decreased protein activity [Spinocerebellar Ataxia Type 7 (SCA7) is a rare inherited neurodegenerative disease caused by polyglutamine repeat expansion in Ataxin 7 (ATXN7), a product of the activity  that conactivity . ATXN7L3 gene , has been shown to regulate the expression of the ATXN7 gene in a miR-124-dependent manner [ATXN7 mRNA levels increased in both patient-derived fibroblasts and SCA7 mouse model brain, while miR-124 levels decreased. Furthermore, in mice, this increase was more prominent in tissues relevant to SCA7, such as the retina and cerebellum. Surprisingly, reduction of lnc-SCA7 levels led to a depletion of both mature and precursor miR-124 levels, generating a novel negative feedback loop involving ATXN7 and miR-124. In SCA7, crosstalk between ncRNAs have been shown to contribute to cell-specific neurodegeneration. Specifically, lnc-SCA7, a highly conserved lncRNA derived from the retrotransposition of the t manner . Tan et RNA editing is an important mechanism of post-transcriptional processing that can modify RNA molecules by altering its sequences through insertion, deletion, or conversion of a nucleotide ,282. RecThe most common type of RNA editing is the conversion of adenosine to inosine (A-to-I), in which enzymes encoded by the adenosine deaminase acting on RNA (ADAR) gene family catalyze the deamination of adenosine (A) nucleotides to inosines (I) . CriticaInterestingly, A-to-I editing has been reported specifically reduced in SALS motor neurons due to the progressive downregulation of ADAR2 ,291. BasOther NDDs, such as AD and PD, also present alterations in RNA editing patterns ,293,294.The vast majority of NDDs can be definitively diagnosed only after death or in advanced stage, and their previous diagnosis is based on ruling out other possible causes for the symptoms. For most NDDs, there is no cure or treatment capable of reversing the damage due to neuronal death. Therefore, it is critical to find new biomarkers that would facilitate an early diagnosis, prognosis and efficient monitoring of therapeutic interventions.In the search for new biomarkers, non-coding RNAs have been proposed as promising tools for diagnosis and prognosis. Many ncRNAs often arise from genes that cause NDDs or are somehow involved in the development of one of these disorders (like BACE1-AS or circSNCA). Thus, ceRNETs established by these ncRNAs could well be, at least in some cases, disease and even stage-specific. However, as reported in this review, ncRNAs are commonly misregulated in several NDDs . This isIn this way, by analyzing various elements of the altered ceRNETs, it may be possible to differentiate one NDD from another even if there were common components. Ideally, working with several correlatable molecular targets at the same time (lncRNAs/circRNAs/pseudogenes-miRNA-mRNAs) increases the sensitivity and reliability of ceRNETs as biomarkers. It should be noted that ceRNETs construction also contributes to the identification of new molecular mechanisms of gene regulation that may lead to a better understanding of the etiopathogenesis of the diverse NDDs, as well as to reveal new therapeutic targets and obtain relevant information about the pathological processes of the disease.In this sense, ceRNETs may also reflect the editing efficiencies of ADAR, a post-transcriptional phenomenon dysregulated in several NDDs. RNA editing can affect the levels and the efficiency of RNA interaction networks, so its alterations could provide a specific RNA fingerprint that helps in the diagnosis or prognosis of NDDs. Finally, the described crosstalk between the RNA molecules in certain ceRNETs is relatively conserved between species, paving the way for translation of data obtained from animal models into clinical practice ,298. Among the main advantages of ceRNETs for biomarker research, the fact that these ncRNAs are easily accessible is noteworthy, since they are extremely stable in circulation and may be detected in exosomes. Such is the case for circRNA CDR1as/ciRS-7 and lncRNA MALAT1, found in exosomes. Interestingly, levels of ciRS-7 in these vesicles depend on the intracellular abundance of the miRNA that it sponges (miR-7) . FurtherBecause ceRNA interaction networks are multifactorial, they may represent an advantage in studies of these complex neurodegenerative disorders, one being at the level of biomarkers (combined RNA biomarkers panels) and another at the level of therapeutic targets (modulate the levels of multiple disease-associated RNAs at once by just targeting one).Nevertheless, it must be taken into account that there is still much to do, since these networks are very complex and their interactions must be experimentally defined . In thisAlthough the full extent of ceRNA networks still needs to be still determined, the competition of ncRNA and mRNAs for miRNAs constitutes a key point of gene regulation that could underlie some pathological aspects of neurodegenerative diseases, favoring at the end the identification of specific pathological mechanisms for each disease.


HEV is the most causative agent of acute viral hepatitis globally. HEV causes acute, chronic, and extrahepatic manifestations. Chronic HEV infection develops in immunocompromised patients such as organ transplant patients, HIV-infected patients, and leukemic patients. The source of chronic HEV infection is not known. Also, the source of extrahepatic manifestations associated with HEV infection is still unclear. Hepatotropic viruses such as HCV and HBV replicate in peripheral blood mononuclear cells (PBMCs) and these cells become a source of chronic reactivation of the infections in allograft organ transplant patients. Herein, we reported that PBMCs and bone marrow-derived macrophages (BMDMs), isolated from healthy donors (n = 3), are susceptible to HEV in vitro. Human monocytes (HMOs), human macrophages (HMACs), and human BMDMs were challenged with HEV-1 and HEV-3 viruses. HEV RNA was measured by qPCR, the marker of the intermediate replicative form (ds-RNA) was assessed by immunofluorescence, and HEV capsid protein was assessed by flow cytometry and ELISA. HEV infection was successfully established in primary HMOs, HMACs, and human BMDMs, but not in the corresponding cells of murine origin. Intermediate replicative form (ds RNA) was detected in HMOs and HMACs challenged with HEV. The HEV load was increased over time, and the HEV capsid protein was detected intracellularly in the HEV-infected cells and accumulated extracellularly over time, confirming that HEV completes the life cycle inside these cells. The HEV particles produced from the infected BMDMs were infectious to naive HMOs in vitro. The HEV viral load was comparable in HEV-1- and HEV-3-infected cells, but HEV-1 induced more inflammatory responses. In conclusion, HMOs, HMACs, and human BMDMs are permissive to HEV infection and these cells could be the source of chronic and recurrent infection, especially in immunocompromised patients. Replication of HEV in human BMDMs could be related to hematological disorders associated with extrahepatic manifestations. Orthohepevirus genus of the Hepeviridae family, and there are eight known HEV genotypes [HEV infection is endemic in developing and developed countries ,2. HEV ienotypes . HEV-3 aenotypes ,5,6. Infenotypes . HEV-5 aenotypes ,9. HEV-7enotypes ; an infeenotypes .HEV is a small icosahedral positive-sense single-strand RNA virus that includes three open reading frames (ORF). ORF1, located at the 5′ end of the genome, represents two-thirds of the viral genome and encodes nonstructural enzymes that are responsible for viral replication such as RNA-dependent RNA polymerase, helicase, and cysteine protease. ORF2, located at the 3′ end of the genome, encodes a structural capsid protein, and ORF3, which overlaps with ORF1 and ORF2, encodes a small phosphoprotein that plays a role in viral morphogenesis .HEV infection was first recognized as an acute self-limiting disease, but these infections can evolve to chronicity in immunocompromised patients such as organ transplant patients, HIV-infected patients, and patients with hematological disorders ,14,15. ESeveral studies have reported the replication of hepatotropic viruses such as HAV, HBV, and HCV in peripheral blood mononuclear cells (PBMCs). PBMCs could serve as a reservoir for these hepatotropic viruses and sources for chronic infection. ,20,21. IHerein, we investigated the replication of HEV in human-derived monocytes (HMOs) and/or macrophages (HMACs) and human bone marrow-derived macrophages (BMDMs), isolated from healthy donors, in vitro. To our knowledge, our study is the first report that shows the replication of HEV in HMOs, HMACs, and human BMDMsBone marrow aspirates were obtained from the end of femurs removed from patients (n = 3) undergoing orthopedic hip reconstruction operations. Blood samples were collected from the same donors. All donors were screened accroding to the protocol of Assiut University Hospitals and they tested negative for viral hepatitis markers  at the time of sample collection as described previously ,26,27. ABalb/c (n = 3) male mice (8–12 weeks) were obtained from Animal Facility, Faculty of Medicine, Assiut University, Egypt. Blood and bone marrow were collected from the mice after euthanasia. All experiments were done and approved in accordance with by the Institutional Review Board (IRB) at the Faculty of Medicine, Assiut University, Egypt. (IRB no 17200190)2. RAW 264.7 were maintained in Dulbecco’s modified Eagle’s media (DMEM)  under the same condition.Immortalized human monocytes cells (THP-1) and mouse macrophage cells (RAW 264.7) were kindly provided by the VACSERA-Cell Culture Unit, Cairo. THP-1 were maintained in Roswell Park Memorial Institute (RPMI-1640) medium  containing 10% fetal bovine serum (FBS)  and kept at 37 °C and 5% COHEV inoculums were isolated from the stool of acute HEV-infected patients admitted for diagnostic purposes to Assiut University hospitals as described previously .Mouse monocytes were isolated from whole blood by immunomagnetic negative selection using the EasySep™ Mouse Monocyte Isolation Kit  according to the manufacturer’s instructions. The purity of the isolated cells was analyzed by flow cytometry after staining with PE anti-mouse CD14  and FITC anti-mouse CD11b antibodies .2 c. After 6–8 days, the cells strongly adhered to the plastic dish. The adherent differentiated cells were then scraped and assessed for macrophage marker (F4/80) before plating them for HEV infection.For isolation of cells from the bone marrow, the femurs were removed and stripped of muscle, exposed bone was washed in 70% EtOH, and a small piece at both extremities of the femur head was cut using scissors. Bone marrow cells were flushed with cold phosphate buffered saline . Then, the cell suspension was filtered through a cell strainer (70 μm) and centrifuged. To differentiate blood monocytes and bone marrow-derived monocytes into macrophages, the cell pellets were dissolved in RPMI medium containing 1% penicillin/streptomycin and macrophage-colony stimulating factor  and were incubated at 37 °C and 5% CO2 in the presence Granulocyte-Macrophage Colony-Stimulating Factor  to generate human macrophages (HMACs) within 6–8 days [Human peripheral blood was taken from the antecubital vein of healthy volunteers (n = 3), and the buffy coat was separated using Ficoll-Hypaque density gradient centrifugation. Human monocytes were obtained from human blood by negative selection of freshly isolated PBMCs using a Pan Monocyte Isolation Kit, human , with a typical purity of 90–98% as assessed by flow cytometry after staining with PE anti-human CD14  and FITC anti-human CD16 antibodies . Monocytes were cultured in RPMI media  containing 1% penicillin/streptomycin, supplemented with 10% FBS , and cultured at 37 °C and 5% CO6–8 days . Followi4 cells/well of HMOs and HMACs were seeded in 24-well plates. A filtered 10% w/v fecal preparation was added to the cells at a dose of 106 IU/well and incubated for 6 h. Then, the inoculum was removed and replaced with fresh culture medium. Collections of supernatants were done at 0, 1, 3, 5, 7, 10, and 12-days post-infection. Supernatants from each time point were stored at −80 °C, and processing of all samples was done at the same time. As proof of concept for HEV replication, HMOs and HMACs were treated with RBV  at final conc. 50 µM and challenged with HEV inoculum as described previously [Infection of HMOs and HMACs with HEV inoculums was assessed as described previously . Brieflyeviously ,29,30,31eviously . BrieflyQuantification of HEV RNA was done as described previously ,33,34,35Detection of HEV ORF2 Ag in the infected cells was done by flow cytometry as described previously . BrieflyPlus assay  according to the manufacturer’s instructions, with slight modifications in the procedure of the cut-off (C.O.) calculation as described previously [Detection of HEV ORF2 Ag in cell culture supernatants was performed using the HEV-Ag ELISAeviously .Supernatants, collected from uninfected and HEV-infected HMACs at day 12 pi were tested for IL-12, IL-6, IL1-β, MCP-1, TNF-α, and IFN-γ cytokines by ELISA kits  according to the manufacturer’s instructions. The level of these cytokines was compared in HEV-infected cells versus the uninfected cells with the same cell number. As a control group, HMACs were challenged with Ultraviolet (UV)-inactivated HEV inoculums and assayed for the same cytokines. UV inactivation was performed in an UV transilluminator for 30 mint-test. p < 0.05 was considered significant.Statistical analyses were performed using GraphPad Prism software 6  using an unpaired Primary murine monocytes (MMOs) were isolated from the blood of Balb/c mice (n = 3) as described in the previous section. Analysis of the isolated cells by flow cytometry showed that the cells expressed the monocyte (MO) marker  A. MMOs w3 IU/mL in the supernatant of infected cells by day 12 pi (3 IU/mL by day 12 p.i in the supernatants of the infected cells (Primary human monocytes (HMOs) were isolated from the blood of healthy volunteers (n = 3) as described in the previous section. Analysis of the isolated cells showed that the cells expressed the MO marker (CD14+). Differentiation of HMOs into human macrophages (HMACs) was done in vitro, and the differentiated cells expressed the MAC marker (CD68) A; the HMay 12 pi B. Similaed cells C. Generaed cells  ContraryTo examine the expression of the HEV capsid protein in HMOs and HMACs, we assessed HEV ORF2 Ag expression in HEV-infected HMOs and HMACs on day 12 pi by flow cytometry. Uninfected HMOs and HMACs served as a negative control. We detected intracellular HEV ORF2 Ag inside the infected cells A; the pe4 IU/mL by day 15 pi , and the cells were differentiated in vitro into macrophages (BMDMs) A. We chaay 15 pi B. The viay 15 pi A. HEV RNay 15 pi C.Then, we wanted to assess the level of proinflammatory cytokines released due to HEV infection. Supernatants from HMACs challenged with HEV-1 and HEV-3 were collected on day 12 pi, and the levels of IL1-β, MCP-1, TNF-α, IFN-γ, IL-6, and IL-12 cytokines were measured by ELISA. We found that the level of the proinflammatory cytokines were higher in HEV-infected HMACs than in uninfected ones. The levels of IL1-β, MCP-1, TNF-α, and IFN-γ were significantly higher in HEV-1-infected HMACs than in HEV-3-infected HMACs. There was no statistical difference in the level of IL-12 and IL-6 between HEV-1- and HEV-3-infected cells . As a coHEV causes acute, chronic, and extrahepatic manifestations. Chronic HEV infection develops in immunocompromised patients such as HIV-infected patients, leukemia patients, and patients with organ transplants . The eti+/+/SCID and FRG) were not susceptible to HEV-1 and HEV-3 infection [In this study, we assessed the replication of HEV in murine and human myeloid cells. Although mice are considered a query model for HEV ,41, we tnfection ,33,36. Unfection ,33,34. Hnfection ,33,34,36Then, we determined whether human PBMCs are permissive to HEV Infection. We infected primary HMOs and HMACs with HEV inoculums and then we assessed HEV markers in the challenged cells. We found that HEV RNA and HEV ORF2 Ag increased over time in HMOs and HMACs challenged with HEV inoculums. Similar findings were recorded using human BMDMs challenged with HEV. In addition, the HEV capsid protein was detected intracellularly alongside dsRNA which is an intermediate of viral genome replication. Several studies used dsRNA as a marker of viral replication, especially positive-sense RNA viruses such as HCV , the SinIn this study, we did not record a significant difference in the replication of HEV-1 and HEV-3 in HMOs, HMACs, and BMDMs. Although HEV-1 is more virulent than HEV-3 in infected patients and in vivo animal model ,49, HEV-In this study, HEV could not replicate in the human monocyte cell line (THP1), which originated from a patient with acute leukemia. While HEV replicates in primary HMOs and HMACs. HEV-1 replicates efficiently in primary human hepatocytes, while its replication in the hepatoma-derived cell line is reduced or inhibited ,53,54. TOur results suggest that HEV replicates inside HMOs and HMACs and these cells could be a reservoir and a source of HEV infection for other organs. Although the viral load released in the culture supernatant was low, the presence of dsRNA and the detection of intracellular and extracellular HEV capsid proteins confirmd the complete replication of HEV in these cells. In a swine model, both positive (+)-strand HEV RNA and negative (−)-strand HEV RNA were detected in the lymph node of pigs inoculated with swine-derived HEV-3 and human-derived HEV-3, suggesting that a lymph node is a site for extrahepatic HEV replication . Similarα were unchanged. They also reported that the serum IL-1β level was higher in acute HEV patients than in the controls [In this study, we found the levels of proinflammatory cytokines such as MCP-1, IL-6, TNF-α, IL-β, IFN-γ, and IL-12 were higher in HEV-infected HMACs than in control cells. IL-12 and IFN-γ also play a role in the activation of adaptive immune responses, especially T-helper 1. Similarly, Saravanabalaji et al. isolated PBMCs from acute HEV patients, fulminant hepatitis failure (FHF) patients, and controls, and they stimulated these PBMCs with the recombinant ORF2 peptide (rORF2p) of HEV-1. They found that IL-12 and IFN-γ were higher in acute HEV patients than in controls, while TNF-α and IL-2 levels were similar in both groups . IFN-γ, controls . Collectcontrols . Likewiscontrols ,62.Our study is the first report that shows evidence of HEV replication in PBMCs in vitro. The fact that HEV persists and multiplies in HMOs, HMACs, and BMDMs is an important finding in understanding HEV pathogenesis. PBMCs could be a reservoir for HEV and the source of chronic, recurrent infection, as well as extrahepatic manifestations. HEV infection of BMDMs could be related to hematological disorders associated with HEV infection.


Arabidopsis, which is a complex process controlled by multiple quantitative trait loci of various effect sizes. Two types of factors are distinguished that contribute to natural regenerative variation: master regulators that are conserved in all experimental systems  and conditional regulators whose relative role depends on the explant and the incubation settings. We further elaborate on epigenetic variation and protocol variables that likely contribute to differential explant responsivity within species and conclude that in vitro shoot organogenesis occurs at the intersection between (epi) genetics, endogenous hormone levels, and environmental influences.Plant regeneration is essential for survival upon wounding and is, hence, considered to be a strong natural selective trait. The capacity of plant tissues to regenerate in vitro, however, varies substantially between and within species and depends on the applied incubation conditions. Insight into the genetic factors underlying this variation may help to improve numerous biotechnological applications that exploit in vitro regeneration. Here, we review the state of the art on the molecular framework of de novo shoot organogenesis from root explants in  Arabidopsis thaliana. Based on that knowledge, we attempt to bridge the gap between reports on organogenic variability between and within different species and natural variation in several regulatory layers of regeneration.As sessile organisms, plants face numerous environmental stresses, and, accordingly, they have retained extended developmental plasticity compared to animals . This abArabidopsis thaliana (WUSCHEL-RELATED HOMEOBOX (WOX) 11 and 12, WOX5&7, and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) 16 and 29 . T,2. TArabthaliana  1,11]. . Arabido6 and 29 ,16,17. S6 and 29 ,10,18. A6 and 29 ,20,21,22TIR1/AFB1-5 coreceptor complex binds Aux/IAA (INDOLE-3-ACETIC ACID INDUCIBLE) repressor proteins and targets them for proteasomal degradation, which releases TOPLESS (TPL)-mediated repression of AUXIN RESPONSE FACTORs (ARFs) that induce auxin-responsive gene expression. Cytokinins, on the other hand, are synthesized from adenosine triphosphate (ATP) or diphosphate (ADP) in two successive reactions, respectively catalyzed by ISOPENTENYLTRANSFERASE (IPT) and LONELEY GUY (LOG) enzymes [i to ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINs (AHPs). The latter act as shuttle proteins, conveying the signal into the nucleus, where they activate two types of ARABIDOPSIS RESPONSE REGULATORS (ARRs): type-B ARRs that act as transcriptional activators of the cytokinin response, and type-A ARRs that form a negative feedback loop on the signal [Auxin and cytokinin play vital, but often antagonistic roles in de novo shoot formation and mutations in the homeostatic or signaling pathway of either hormone are known to impair regeneration ,23. Auxi enzymes . Active  enzymes . Cytokine signal . Auxin ae signal ,26. Ethye signal ,23.2+ influxes, plasma membrane depolarization, a burst of reactive oxygen species (ROS), disruption of cellular communication, and jasmonic acid (JA) accumulation, although it is not well understood how these are translated into waves of extensive transcriptional changes observed after wounding [WIND1, ERF113/RAP2.6L, and LBD16) tend to be rapidly induced by cutting, whereas genes with H3K36me3 and H3K27me3 are less responsive to wounding. Notably, it has been suggested that wound-induced calli differ from calli formed on CIM because the former do not express root markers and their induction is not affected in solitary root (slr) mutants [Intact seedlings subjected to the CIM–SIM procedure regenerate lateral roots instead of shoots, indicating the importance of wound stress for de novo SAM formation . Moreovewounding ,28. Centwounding ,30. Recewounding . Moreovewounding . Other Awounding . Genes w mutants ,33,34.ALF4 by very-long-chain fatty acids confines the capacity of pericycle cells to form callus [When explants are placed on CIM, the high auxin concentration triggers cell division to initiate the formation of a proliferating cell mass or callus ,35. Whilm callus .WOX5, SHORT ROOT (SHR), SCARECROW (SCR), PLT1&2, PIN1, QUIESCENT CENTER 25 (QC25), ROOT CLAVATA HOMOLOG 1 (RCH1), and GLABRA2 (GL2). Accordingly, callus formation on CIM proceeds through a similar developmental program as lateral root initiation [LBD expression by removing repressive H3K9me3 marks [ARF7&19 and LBD genes [E2 PROMOTER BINDING FACTOR a (E2Fa), which associates with DIMERIZATION PARTNERs (DPs) to stimulate DNA replication genes [KRPs are further silenced via H4R3 dimethylation and alternative splicing of RELATED TO KPC1 (RKP) by PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) [In-vitro-induced callus resembles root primordia on a morphological and transcriptional level, even when derived from aerial organs ,39,40. Iitiation ,20,34. Aitiation ,42. JUMOe3 marks , while tBD genes . In turnon genes . Auxin a (PRMT5) . A serie (PRMT5) , which i (PRMT5) ,47. Upon (PRMT5) . These A (PRMT5) .WUSCHEL (WUS) on SIM requires several days of CIM preincubation [WOX5, PLT1&2, SCR, and SHR, in a subset of callus cells confers pluripotency, but how these are activated is not completely understood [WOX5&14, PLT1&2, and SCR loci to potentiate their transcription on CIM [WOX5&11, WUS, and SHOOT MERISTEMLESS (STM), undergo dynamic changes in PRC2-mediated H3K27me3 during organogenesis, but the machinery underlying histone demethylation is still poorly characterized [ARR12, WUS, and CLAVATA3 (CLV3)) on SIM [CMT3 and DOMAINS REARRANGED METHYLTRANSFERASE (DMR) 1 and 2 allows to bypass CIM preincubation for WUS expression and shoot regeneration.On CIM, calli also acquire competence to respond to shoot inductive cues as activation of key shoot meristem regulators like cubation . Recentlderstood ,19,50. Oderstood . The latderstood . Notablyderstood . Furtherderstood ,35. For n on CIM ,54. Manycterized ,55. Howe) on SIM . Besides) on SIM . AccordiWOX5-expressing stem cells, for transition [It has been proposed that de novo establishment of the shoot apical meristem on SIM reflects transdifferentiation from root to shoot identity instead of a true de- and redifferentiation process ,39,58. Aansition .CUC1&2 are upregulated by PLT3, PLT5, and PLT7 during CIM preincubation, but also by ESR1&2 in response to cytokinin and wound-induced WIND1 [CUC genes to the peripheral zone, where they induce LIGHT SENSISTIVE HYPOCOTYLS (LSH) 3 and 4, which suppress differentiation in the organ boundary [wus mutants fail to regenerate [ARGONAUTE 10 (AGO10) was found to enhance pro-SAM formation by releasing miR165&166 to degrade HD-ZIP III mRNAs [WUS transcription, in agreement with other reports showing that phb phv rev and inducible miR165/166 lines fail to regenerate [STM and RAP2.6L [IPT5 by ARF3 and inhibition of STM-mediated IPT7 activation maintains auxin signaling at the peripheral and apical zone of the primordium [WUS is only induced after 2–3 days on SIM because it harbors the repressive H3K27me3 mark maintained by PRC2 components CLF and SWN, removal of which depends on cytokinin-controlled cell cycle progression and possibly involves active demethylation by EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6) [WUS locus also undergoes dynamic changes in the levels of other repressive histone modifications  and activating marks , which is modulated by LDL3 and JMJ14 (H3K4 demethylases), KRYPTONITE  and HAG1 [WUS via DNA methylation, which is gradually lost on SIM [MET1 through CYCD3-mediated activation of E2Fa to prevent early WUS transcription, after which MET1 expression migrates to the outer layers of the callus and the resulting dilution of DNA methylation enables WUS activation by ARRs [WUS is confined to the center of the dome-shaped promeristem when CUC1&2 activity shifts to the peripheral zone and STM is expressed throughout the primordium [Upon transfer to SIM, overlapping signaling domains of auxin and cytokinin diverge into mutually exclusive regions, thereby partitioning cellular identity through complementary expression of CUC2 and WUS ,22,25,60ed WIND1 ,51,61. Ted WIND1 ,11,19. Sboundary . On the generate ,26. Thisgenerate ,64,65. O RAP2.6L ,68, and  RAP2.6L ,70,71. C6 (REF6) . The WUSand HAG1 ,72. Moret on SIM . It was  by ARRs . WUS is  by ARRs . UltimatCLV3, in turn blocking WUS expression in the central zone and the rib meristem through CLV1&2 and CORYNE (CRN) [CLV3 is upregulated after WUS expression is established in the center of the shoot promeristem [ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) [ARABIDOPSIS THALIANA HOMEOBOX GENE 1 (ATH1), KNOTTED1-LIKE HOMEOBOX GENE 4 (KNAT4), SAWTOOTH (SAW) 1 and 2, TCP10, and YABBY5 (YAB5) by H3K4me3 deposition [A functional SAM consists of an organizing center (OC) between a lower rib meristem and a central zone with slowly dividing stem cells at the apex, surrounded by a peripheral zone undergoing rapid proliferation ,76. WhilNE (CRN) ,75. Thismeristem . In thismeristem . PIN1 an (ATML1) ,77,78. Rposition ,79.Arabidopsis thaliana accessions, and various strategies have been applied to map such differences at the genetic level (er was linked to one common region (85–89 cM) on chromosome 1 underlying callus formation and shoot regeneration from leaf explants, whereas several areas on chromosomes 4  and 5  specifically contributed to shoot regeneration from roots or leaves [SHOOT REDIFFERENTIATION DEFECTIVE 1 , although it also overlaps with RECEPTOR-LIKE PROTEIN KINASE 1 , which was later discovered by Motte et al. [er RILs also revealed quantitative trait loci (QTLs) on chromosomes 1, 4, and 5, but at different positions  [er variant of the major QTL on chromosome 5, while fixing the beneficial Ler alleles at the minor loci and contrasting gene expression patterns in both genotype pools uncovered 845 differentially expressed genes, including ARR18, AGL6, and AT4G36590 (2 MADS-box TFs), AT5G50820 (a NO APICAL MERISTEM family member related to CUC1&2), HOMEOBOX-LEUCINE ZIPPER PROTEIN 17 (HB17), and 3 subtilases . On the other hand, analysis of Ler x Cvi RILs respectively identified 8, 5, and 4 loci for shoot, root, and callus formation from leaf or root explants [SRD3, ROOT REDIFFERENTIATION DEFECTIVE 4 (RRD4), CYTOKININ HYPERSENSITIVE 1 (CKH1), INCREASED ORGAN REGENERATION 1 (IRE1), or AUXIN RESISTANT 1 (AXR1) [Several studies have reported substantial variation in the regenerative potential of natural r leaves . Combinee et al. ,83,84. Cn region –89 cM onRPK1 was found to be expressed in dividing pericycle cells during CIM preincubation and loss-of-function impaired de novo shoot formation [WOX5 expression [WUS expression to control SAM maintenance, and, although this function is not conserved in RPK1 [rpk1 mutants are capable of expressing shoot markers and occasionally regenerate ectopic shoot meristems [DCC1 (encoding a thiol-disulfide oxidoreductase family protein with a conserved DxxCxxC motif) were strongly linked to regenerative variation among 48 ecotypes [DCC1 or CA2 caused increased ROS levels by reducing the activity of complex I, in turn hampering shoot regeneration by downregulating essential genes for callus and shoot promeristem formation  and lowering the expression of auxin biosynthesis and signaling genes , and SMALL AUXIN UPREGULATED 51 (SAUR51)), potentially via redox modification of PHV and TCP15 [DCC1 abolished the interaction with CA2 and, accordingly, the superior haplotype was associated with lower ROS levels. It is important to note, however, that while DCC1 may be correlated with variation in shoot regeneration, it might not reflect a rate-limiting factor in natural populations as it was not discovered by mapping .More recently, linkage mapping with RILs between Nok-3 and Ga-0 revealed five major QTLs responsible for the difference in de novo shoot organogenesis between these lines . One of ormation . This lepression ,87,88. N in RPK1 , the two in RPK1 ,86, indieristems . Motte eeristems ,81. Anotecotypes . DCC1 lond TCP15 . Moreoveer x Col, or Ler x Cvi RILs, highlighting the complex polygenic nature of regeneration . Moreover, our GWAS also exposed several novel QTGs that might act as master regulators, including AT3G09925 , SUPERMAN (SUP), EMBRYO SAC DEVELOPMENT ARREST (EDA40), and DNA-BINDING WITH ONE FINGER 4.4 (DOF4.4). Finally, the genetic architecture of in planta shoot growth was shown to be complex as well, comprising around 112 QTLs grouped in hotspots on chromosomes 1, 2 and 5, of which ~10% were considered major QTLs ), and the rest were loci with intermediate or minor effects that occasionally interacted with drought stress [Notably, a poor overlap is observed between QTLs obtained by linkage mapping using Nok-3 x Ga-0, Lneration  [81,82,8nditions . In agreAgrobacterium-mediated transformation of in vitro cultures, and, although efficient protocols have been established for callus induction and regeneration from mature seeds, many commercial cultivars  are recalcitrant to this system [Arabidopsis, early endeavors to map variation in several callus induction traits by following the segregation of amplified/restriction fragment length polymorphism (AFLP/RFLP) markers after crossing strongly and poorly regenerating  varieties uncovered a myriad of QTLs across the 12 chromosomes, some of which could be used for marker-assisted selection, but failed to identify specific genes (PROMOTER OF SHOOT REGENERATION 1 (PSR1) [PSR1 is a putative ferredoxin-nitrite reductase with lower expression and activity in poorly regenerating Koshihikari plants. Another study monitored simple sequence repeat (SSR) markers in chromosome segment substitution lines (CSSLs) between Nipponbare and Zhenshan 97B to identify 29 QTLs for 4 callus traits under 2 protocols, which were grouped in hotspots on chromosomes 1, 3, and 10 [Arabidopsis [Arabidopsis: CROWN ROOTLESS 1 , OsBBM1 (an ortholog of BBM), and OsSET1 . While the latter was downregulated during in vitro culture, expression levels of the former two increased, consistent with the repressive and promotive roles of their respective counterparts in Arabidopsis. Fourteen additional candidates were put forward based on p-values, annotations, and expression patterns, including a putative thioredoxin, two AP2-domain-containing proteins, and OsIAA10, RNAi knockdown of which impeded callus induction and auxin responsivity [BROWNING OF CALLUS 1 (BOC1) from wild rice was found to reduce callus browning in indica cultivars by decreasing cell death and senescence in response to oxidative stress [The creation of transgenic rice plants for breeding and functional genome research relies on s system ,94. Likeic genes  [93,95,91 (PSR1) . Cloning, and 10 . More re, and 10 . The disbidopsis . Accordibidopsis . Among tonsivity . Lastly,e stress .ALDOLASE 1 (ALD1), ZmWOX2A, and ZmWOX5B. Furthermore, a multilocus GWAS for five embryonic callus traits using 43427 SNPs in 144 inbred maize lines identified 63 common quantitative trait nucleotides (QTNs), 15 of which were retained in multiple environments [WOX2, showing elevated expression in a strong regenerator, which is consistent with the role of WUS in Arabidopsis organogenic variation [WUS2- or BBM-expressing constructs regenerate more seedlings [WUS2 and BBM [STM, PICKLE (PKL), LEC, AGL24, and CUCs.QTL mapping of regeneration traits has been attempted in several other cereal crops such as maize ,105,106,ariation  and the eedlings . In wheaeedlings ,109,114.eedlings . Higher  and BBM . Finally and BBM . Four ofLycopersicon peruvianum with cultivated L. esculentum plants using morphological and RFLP markers pinpointed a QTL near the middle of chromosome 3, designated Rg-1, and proposed to act together with one or two other loci in the control of shoot regeneration from root explants [Rg-2 allele from L. chilense was mapped to the same region and linked to an acid invertase gene [L. esculentum and L. pennellii identified 6 QTLs on chromosomes 1, 3, 4, 7 and 8, underlying shoot regeneration from leaf disks and mapping of prior candidates Rg-2 and LESK1 (a serine/threonine kinase upregulated during shoot induction), suggested that the QTL on chromosome 3 might be another allele of Rg-2, which was dubbed Rg-3 [LESK1 was not located in a QTL but other regions contained serine/threonine kinases, histidine kinases, AP2/ERF TFs, cyclins, and MADS-box genes. Notably, only the two main QTLs on chromosomes 1 and 7 were found for all three investigated traits, and only the Rg-3 locus overlapped with previous findings based on root explants, confirming the occurrence of conditional regeneration determinants in tomato. On the other hand, Rg-1 appears to be a master regulator as it increases both shoot and root formation, rescuing the dual effects caused by the knockout of the DELLA protein PROCERA (PRO) and the specific phenotypes of auxin-resistant diageotropica (dgt) and lateral suppresser (ls) mutants, while overexpression of MOUSE EARS (ME) only increased shoot induction on SIM [Arabidopsis J3 . This gene was upregulated in highly regenerable genotypes, and overexpression enhanced the performance of the recalcitrant RIL parent [PRC2 subunits and flavin-binding monooxygenases with a role in auxin biosynthesis and de novo root formation , and RPK1. Sunflower, on the other hand, is an important crop for oil production, and because mass clonal propagation using cultured explants would facilitate genetic engineering, a RIL population has been developed to screen for molecular markers linked to shoot regeneration from cotyledon explants and somatic embryogenesis from epidermal layers [LBD16, LEC1&2, WUS, TSD1, and CLF , while the observation that eQTLs and epi-eQTLs  underlying gene expression) were often found at transcription start sites indicates that the general silencing effect of methylation is mediated by altered TF binding [CUC2 binding sites were specifically enriched at eQTLs). Notably, epiRILs (RILs with similar DNA sequences but diverging methylomes) show comparable phenotypic inheritance as RILs or natural accessions [er strains, which has been coupled to the spreading of repressive heterochromatin marks  from nearby TE insertions [Because many key histone modifiers and DNA methylation enzymes, as well as their target marks and genes, are differentially regulated during incubation on CIM and SIM, epigenetics constitute an important regulatory layer of de novo shoot organogenesis ,53,130. esponses ,132,133.esponses ,135. Wheesponses , the extesponses . On the esponses ,139. GWA binding . This alcessions , confirmcessions . These ecessions . Hence, cessions . In addisertions . Althougsertions ,142. TraIRE genes [Arabidopsis leaves [PLT2 underlying lower repair potential at the proximal root end [miR156 and SPL9 that is responsible for progressively declining shoot regeneration in older plants [Explant age, physiological state, and source tissue  determine the efficiency of regeneration, concordant with different medium requirements for callus, root, and shoot formation from root, hypocotyl, and leaf explants of diverse ecotypes ,84,143. RE genes ,146. ForRE genes . Accordis leaves ,74. Intrs leaves . While ms leaves . Variabls leaves . Furthers leaves . Severalroot end , an intrr plants .Arabidopsis strains [rho with an absolute value greater than 0.3 and an FDR below 10%. Contrary to previous reports, we detected significant positive correlations between shoot numbers, shoot primordia, undefined structures, and green callus area under two regeneration protocols, differing in the age of source plants, applied light spectrum, and external cytokinin concentration [To determine whether the capacity for shoot formation from root explants is linked to other characteristics of the parent plant, we performed a correlation meta-analysis between 16 regeneration traits in 170  strains  and 383  strains ,153. In ntration . Root-lintration , suggestntration . Finallyntration , as wellntration , suggestArabidopsis, and this response was different in 4 accessions [Arabidopsis, indicating the existence of an optimum for every system [Arabidopsis differed between ecotypes [OsPIN1, and LATE EMBRYOGENESIS ABUNDANT 1 (OsLEA1) genes, implicating interaction between auxin, ABA, and carbohydrate metabolism for osmotic stress regulation in callus. Notably, constitutive osmotic stress in plastids of Arabidopsis MscS-like mechanosensitive ion channel (msl2 msl3) mutants leads to callus development at the shoot apex, involving perception of elevated cytokinin levels by AHK2, upregulation of WUS, and downregulation of ARR7&15 [Lathyrus, on the other hand, polyethylene glycol (PEG)-induced osmotic stress reduced the multiplication rate and vigor of regenerated shoots [Arabidopsis and Thellungiella [Arabidopsis, leaf or root explants respectively prefer sucrose or glucose [It is likely that part of the regenerative variation among explants from the same individual or subspecies can be attributed to fluctuations in environmental conditions, such as light, temperature, osmotic stress, and medium composition. For instance, it was found that variable light exposure of cotyledon explants during the first 5 days after excision affects shoot regeneration in cessions . More spcessions . Notablycessions . Low ligcessions , and appcessions . Curiouscessions , whereascessions . The usecessions . Similary system ,158. Exty system . Optimalecotypes , and theecotypes . The lat ARR7&15 . A second shoots , and NaCungiella . Finallyungiella ,164,168. glucose , cytokin glucose , activat glucose , and sup glucose .Arabidopsis and which ones are conserved, such as WUSCHEL-RELATED HOMEOBOX genes, AP2/ERF transcription factors like BBM, and receptor-like protein kinases. Knowledge of which genes are rate-limiting in a particular system can be used to overcome recalcitrance or improve the regeneration efficiency through genetic engineering. In order to avoid deleterious side-effects, the best strategies for ectopic gene activation involve the use of tissue-specific or inducible promoters , transient expression , or excision of genes at a later stage  [CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-LIKE 4 (CPL4) was already shown to boost de novo shoot organogenesis from root explants in Arabidopsis [Arabidopsis accessions, will help to shed light on this matter by enabling accurate association of genetic, epigenetic, and transcriptional effects underlying the differential organogenic potential between and within species. Ultimately, this will contribute to the development of selectable markers to screen for highly regenerable cultivars, while a better understanding of how rate-limiting factors interact with the environment could empower targeted design of protocols for biotechnology applications in crops. Machine learning algorithms provide a complementary prospect for fine-tuning protocol development, as they allow to predict optimal requirements in terms of incubation conditions, plant growth regulators, explant source, and genotype, without the need for large-scale, time-consuming, and costly experimental trials. The potential of this approach is illustrated by recent reports on the application of artificial neural networks to model and augment somatic embryogenesis and shoot proliferation in Chrysanthemum and shoot regeneration in wheat by altering medium composition [Swertia paniculata and Bryophyllum [Taken together, regeneration is a highly complex trait controlled by multiple intertwined regulatory layers that are all subject to variation, making it difficult to predict in vitro culture responses. Although significant progress has been made in elucidating the molecular framework and many potential QTLs have been identified, only a subset was mapped to specific genes. These can be divided into conditional factors and master regulators, posing the question of which established meristem determinants have specific roles in binases) . Adding binases) ,174,175.bidopsis . Next, fposition ,178,179.ophyllum ,181. Advophyllum . Finally


Sialic acids, a subset of nine carbon acidic sugars, often exist as the terminal sugars of glycans on either glycoproteins or glycolipids on the cell surface. Sialic acids play important roles in many physiological and pathological processes via carbohydrate-protein interactions, including cell–cell communication, bacterial and viral infections. In particular, hypersialylation in tumors, as well as their roles in tumor growth and metastasis, have been widely described. Recent studies have indicated that the aberrant sialylation is a vital way for tumor cells to escape immune surveillance and keep malignance. In this article, we outline the present state of knowledge on the metabolic pathway of human sialic acids, the function of hypersialylation in tumors, as well as the recent labeling and analytical techniques for sialic acids. It is expected to offer a brief introduction of sialic acid metabolism and provide advanced analytical strategies in sialic acid studies. N-acetylneuraminic acid, a major product released by mild acid hydrolysis of glycolipids in the brain or salivary mucins [N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac has an acetyl group on the fifth carbon atom (C5) while Neu5Gc has a glycolyl group instead. Interestingly, humans lack Neu5Gc caused by the mutation of the cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) gene that codes the enzyme transforming CMP-Neu5Ac to CMP-Neu5Gc [The term “sialic acid” first appeared in 1952 to describe P-Neu5Gc ,4,5. HowP-Neu5Gc . In thisN-acetylneuraminic acid synthetase (CMAS) to form CMP-Neu5Ac [In general, sialic acids normally exist as the terminal sugars in the periphery of oligosaccharides via different glycosidic linkages  . PolysiaP-Neu5Ac . N-acetylneuraminate lyase (NAL) to ManNAc and pyruvate [After activation, CMP-Neu5Ac is transferred to the glycoconjugates in the Golgi apparatus by a family of linkage-specific sialyltransferases. About 20 sialyltransferases have been cloned and characterized, including ST3GAL1-5, ST6GAL1, 2 and ST6GALNAC1-6, and ST8SIA1-6, which link Neu5Ac via its second carbon (C2) to the C3, C6 positions of other carbohydrates or the C8, C9 positions of another sialic acids, generating α2,3-, α2,6-, α2,8, or α2,9-linked sialic acids, respectively . Finallypyruvate . Interespyruvate .In the following, we summarize the functions of sialic acids during tumor progression, especially in immune escape, tumor proliferation and metastasis, tumor angiogenesis and apoptosis resistance. Moreover, we also introduce the prevalent analytical methods, including the sialic acid quantification and labeling strategies by bio-affinity, the chemical reaction, and metabolic labeling.CMAS gene, a key node in sialic acid metabolism, inhibited the synthesis of the activated form of sialic acid and decreased the formation of lung metastases in vivo [Sialic acids on tumor cell surfaces are aberrantly expressed during tumor transformation and malignant progression. In general, hypersialylation is frequently observed in tumor tissues compared to corresponding normal tissue . The tot in vivo . On the Growing evidence suggests that sialic acids control the immune homeostasis and weaken immune activation in order to avoid or limit the damage of sialylated cells . Sialic Expression of sialylglycans are positively correlated with aggressiveness and metastasis in many cancers. Altered expression of sialylglycans is associated with epithelial-mesenchymal transition (EMT), an essential step for tumor progression and metastasis . TransfoN-glycans with terminal α2,6-sialylation on the receptors of blood vessels such as VEGFR2 are required for the VEGF engagement and proangiogenic activation of endothelial cells [ST6GAL1−/− mice prevented PECAM-VEGFR2 interaction on the endothelial surface, inducing endothelial cell apoptosis and inhibiting angiogenesis [Angiogenesis is the formation of new blood vessels from pre-existing ones, which is accurately controlled during embryonic development and wound repair. Around the microenvironment where tumor cells grow rapidly, new blood vessels are needed to meet the oxygen and nutrient requirements. Angiogenesis is stimulated by the angiogenic growth factors (AGFs), which are released by inflammatory cells or tumor cells. Among them, the most important AGF is the vascular endothelial growth factor (VEGF) family that includes VEGF-A, B, C, D, E, and placental growth factor . VEGF afal cells ,82. α2,6ogenesis .Sialic acids affect cell apoptosis mainly through two pathways, the Fas receptor-Fas ligand (FasR-FasL) apoptotic pathway and anoikis. FasR-FasL interaction, induced through activated T cells and mediated by caspase activation, is important for homeostasis of cells in the immune system and for immune-privileged site maintenance in the human body . SialylaSince sialic acids have important functions in pathology and physiology, accurate, simple and rapid methods for sialic acid quantification and analysis have been developed, as summarized in The high sensitivity of sialic acids in serum or plasma as a tumor marker has been reported in various cancerous conditions . QuantifSambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL) are commonly used to recognize the α2,6-linked and α2,3-linked sialic acid residues, respectively [Over the past decade, several bio-affinity-based approaches for directly detecting sialic acids and sialylglycans have been developed, including lectins, antibodies, and recombinant sialic acid-binding proteins . Lectinsectively ,117,118,ectively , lectin ectively ,121,122,ectively , fluoresectively ,124. Usiectively . Detectiectively . Thus, aectively . Alternaectively . Those m4 treatment followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) and subsequent LC-MS/MS analysis. The TMT tag improved the ionization efficiency of complex gangliosides and could simultaneously quantify up to six samples, as well as the identification of glycan and lipid compositions in a single injection [N-glycans, triplex mdSUGAR tags were developed for quantitative glycomics. mdSUGAR tags could be easily linked to the reducing end of glycans and the additional aldehyde group introduced by mild periodate oxidation, which extend the mass difference and lower the requirement for resolving power [Since the side chain at C7, C8, C9 of sialic acid is specifically sensitive to periodate oxidation under mild conditions, periodate oxidation has been designed to generate aldehyde groups from the sialic acids. This reaction selectively oxidizes the glycerol side chain of sialic acid to form a 7-aldehyde sialic acid derivative, which further can be linked to hydrazine beads or fluorescence probes and used for detecting the sialic acids on living cell surface ,127 and njection . For N-gng power . In brieN-acetylglucosamine (UDP-GlcNAc) enzymatic conversion. The exogenous sialic acid precursors, including ManNAc, sialic acid, and CMP-sialic acid, can be taken up and metabolized by mammalian cells, resulting in the high expression of sialic acids in cell surface sialoglycoconjugates. Due to the broader substrate tolerance of enzymes involved in sialylglycans biosynthesis, it is possible to use the sugar analogues with functional groups  for sialic acid metabolic labeling [N-azido-acetylmannosamine (ManNAz) can be used in living cell surface glycans labeling. However, the absence of an efficient transporter and hydrophilic property limit ManNAz entering cells. Instead, the hydrophobic per-O-acetylated ManNAz (Ac4ManNAz) was used for sialic acid metabolic labeling [4ManNAz can easily cross the cell membrane. After Ac4ManNAz have entered the cell, the extra acetyl groups are deacetylated by cytosolic esterases, and glycan biosynthetic enzymes can utilize these deprotected sugar derivatives [4ManNAz has been widely used in the cell surface sialic acid labeling, such as the sialoglycoprotein expression upon megakaryocytic differentiation [4ManNAz metabolic labeling strategy, sialylated N-glycans on a select group of small noncoding RNAs (glycoRNAs), including Y RNAs, have been observed in mammalian cells [O-acetylated monosaccharides can spontaneously react with numerous cysteines in proteomes with non-enzymatic catalysis, resulting in abnormal S-glycosylation [4ManNAz. Despite the defect, metabolic labeling has been widely applied to cell engineering, chemical tumor targeting, and tumor therapy [As shown in labeling ,132. Thelabeling . Ac4ManNivatives . Therefontiation , imagingntiation , single ntiation  or selecntiation  metabolintiation . Amazingan cells , which man cells . It is wsylation . In cons therapy ,144,145.N-acetylgalactosamine . Usually, lectins are used to recognize sialic acids with specific linkages. However, non-specific binding of lectin SNA and MAL makes the results unreliable. Considering the different spatial conformations, α2,3-linked sialic acid can form a stable lactone to the neighbor galactose, whereas α2,6-linked sialic acid cannot. This character makes it possible to differentiate the specific linkage of sialic acid on glycans through molecular weight. When sialylglycans were derivatized by dimethylamine, α2,3-linked sialic acid formed a stable amidation structure, which showed a mass shift of −0.984 Da, whereas α2,6-linked sialic acid formed a stable dimethyl amide and remained stable in ammonium hydroxide, and the dimethylamidation of α2,6-linked sialic changed glycans mass by +27.047 Da. The different mass shifts of α2,3- and α2,6-linked sialic acid derivatives can be distinguished by MALDI-TOF MS [Sialic acids are frequently the terminal residues of glycan chains and are typically connected either by a α2,3- or α2,6-glycosidic bond to galactose  and/or I-TOF MS . Using tI-TOF MS ,148, chaI-TOF MS ,150, andI-TOF MS  have beeY glycans enhanced the antigen presentation to dendritic cells, which might act as a “vaccine” for tumor treatment [In this review, we summarized the possible functions of sialic acids in tumor development and progression. Sialic acids commonly locate at the terminal of glycans on the cell surface. The negative charge and hydrophilic property give the sialic acid coated cells unique characters. Hypersialylation is usually found in tumor tissues and is supposed to be a potential hallmark of cancer. Sialic acids enhance tumor progression at multiple levels by facilitating escape from immunological surveillance, angiogenesis, the formation of metastasis, and resistance to apoptosis and therapy. For this reason, the therapeutic approaches to modulate sialic acids and their receptors can be of high potency in tumor therapy ,153,154.reatment . The polreatment . Moreovereatment . 4ManNAz is mostly used as the azido group can be easily linked to probes or beads through click chemistry reaction. Sialic acid metabolic labeling combined with high resolution LC-MS offers simple detection in sialic acid modification sites of a certain protein [Fortunately, owing to novel technologies and methods, increasing discoveries of sialic acid functions in physiology and pathology are being reported. In clinical studies, sialylation in human serum or saliva can be accurately detected using quantification methods. Notably, specific proteins, such as IgA1 with sialylation, were proved to relate to certain tumor occurrences . Enhance protein . As a re