PtrMYB55 and PtrMYB121, were isolated and their functions were elucidated. Tissue expression analysis revealed that PtrMYB55 and PtrMYB121 had a similar expression pattern, which had the highest expression in stems. Their expression continuously increased with the growth of poplar, and the expression of PtrMYB121 was significantly upregulated in the process. The full length of PtrMYB121 was 1395 bp, and encoded protein contained 464 amino acids including conserved R2 and R3 MYB domains. We overexpressed PtrMYB121 in Arabidopsis thaliana, and the transgenic lines had the wider xylem as compared with wild-type Arabidopsis. The contents of cellulose and lignin were obviously higher than those in wild-type materials, but there was no significant change in hemicellulose. Quantitative real-time PCR demonstrated that the key enzyme genes regulating the synthesis of lignin and cellulose were significantly upregulated in the transgenic lines. Furthermore, the effector-reporter experiment confirmed that PtrMYB121 bound directly to the promoters of genes relating to the synthesis of lignin and cellulose. These results suggest that PtrMYB121 may positively regulate the formation of secondary cell wall by promoting the synthesis of lignin and cellulose.MYB transcription factors have a wide range of functions in plant growth, hormone signaling, salt, and drought tolerances. In this study, two homologous transcription factors,  The most abundant biological resource produced by woody plants is wood, which is widely used in traditional industries including lignum, papermaking, and pulping. Wood is also one of the important materials used in the production of bioethanol . FormatiUp to now, biosynthesis of the main SCW components has been clearly studied. Cellulose is synthesized by cellulose synthase (CesA) complexes, which can be divided into the following two types: type I which is mainly involved in the formation of primary walls and type II which is involved in fiber, vessels, and xylem parenchyma synthesis of SCW. Bioinformatics analysis has revealed that there were 10 CesA genes in Arabidopsis , 18 CesAAs compared with biosynthesis, transcriptional regulation of SCW has not been completely understood. On the basis of increasing data regarding whole genome sequences from different plants, a great number of transcription factors (TFs) that regulate SCW formation have been identified, such as the NAC, MYB, and WRKY families. Previous reports have indicated that there was a transcriptional regulatory network among these families for regulating biosynthesis of SCW . Within AtMYB46 and AtMYB83 [AtMYB61 was founded to be a multifunctional TF in Arabidopsis. It specifically regulates lignin biosynthesis [AtMYB61 also links the transcription regulation of multiple aspects of resource allocation [Another important gene family that regulates SCW biosynthesis is R2R3-MYB TFs, which belong to the second-level switch genes. Of these members, MYB46 and MYB83 in Arabidopsis are the key genes ,22, and  AtMYB83 ,25. Meanynthesis ,27, affeynthesis , regulatynthesis , and alsynthesis . Furtherlocation .Populus tricocharpa (P. tricocharpa), but less than 10% have been isolated and characterized for function analysis [Poplar is one of the most widely cultivated fast-growing trees around the world and important to the wood industry. As a woody model plant, the whole genome of several poplar species ,32,33 haanalysis . TherefoPtrMYB121 from P. tricocharpa, and its function was investigated by tissue expression analysis, overexpression in Arabidopsis thaliana , qRT-PCR, and transcriptional activation assays. The results indicate that PtrMYB121 can activate the expression of structural genes involved in the biosynthesis of lignin and cellulose and promote the accumulation of lignin and cellulose.In this study, we isolated a MYB TF AtMYB61 was used to blast and multiple alignment. Two pairs of R2R3-MYB homologous genes (PtrMYB55/121 and PtoMYB170/216) of AtMYB61 were isolated  of three-month-old P. trichocarpa seedlings was extracted for qRT-PCR analysis. The qRT-PCR analysis showed that PtrMYB55 and PtrMYB121 had similar expression patterns, and transcript abundance of PtrMYB55 and PtrMYB121 could be detected in all organs and tissues  in wild-type A. thaliana. Then, we obtained 11 hygromycin-resistant A. thaliana lines and analyzed the function of PtrMYB121 in the T2 homozygous generation. A part (783bp) of the CDS sequence of PtrMYB121 was amplified by PCR using gene-specific primers , L4 (11.3-fold), and L6 (9.6-fold) had relative higher expression levels of PtrMYB121 as compared with other lines  and fference . The thrPtrMYB121-overexpressing transgenic Arabidopsis, we further made the anatomical cross-sections of inflorescence stems and took pictures with an optical microscope system. Then, Image J software was used for calculating the cell layer number and radial width of xylem. Both WT (WT-1) and PtrMYB121-overexpressing Arabidopsis (L1) contained three independent samples, and every sample had 30 slices for analysis. The cell layers of xylem increased 89.73–120.84% in PtrMYB121-overexpressing Arabidopsis  method was used to estimate the total lignin content , while tT plants , while tT plants . Howeverng lines .PtrMYB121-mediated SCW formation, especially the accumulation of lignin and cellulose, we used qRT-PCR analysis to analyze the expression profiles of SCW biosynthetic genes. From the results of qRT-PCR analysis, we found that all of the detected lignin synthetic genes  had a significant increase in the PtrMYB121-overexpressing transgenic lines. Meanwhile, the expression of 4CL2, CCoAOMT1, COMT, and CAD5 was upregulated five- to 20-fold in transgenic lines  9 and IRX14, and they had no difference in WT and PtrMYB121-overexpressing transgenic lines  and cellulose (CesA7 and CesA8) biosynthetic genes. They all had secondary wall MYB-responsive elements ACC(A/T)A(A/C)(T/C) [4CL2, CAD5 and CesA7), four (CCOAOMT1 and COMT1), and five (CesA8)  significantly enhanced the relative LUC activity as compared with the mock  and cellulose biosynthetic genes (CesA7 and CesA8).To confirm that /C)(T/C)  in the p (CesA8) . Then lueporters A. Co-expthe mock B. The re4CL, C3H, CAD, C4H, CCOAOMT, COMT, F5H, PAL, CesA7, CesA8, IRX9, and IRX14 [AtMYB58, AtMYB63, and AtMYB85 can specifically activate the expression of lignin biosynthetic genes [AtMYB61 plays crucial roles in lignin biosynthesis, trichome formation, and seed coat pigmentation [AtMYB4, AtMYB32, and AtMYB7 have been proved to play negative regulatory roles in the biosynthesis of lignin and other phenylpropanoid compounds [SbMyb60 not only impacts phenylpropanoid biosynthesis, but also alters SCW composition in Sorghum bicolor [OsMYB103L plays an important role in GA-mediated SCW synthesis, and functions as a master switch of downstream TFs [BdSWAM1 is a positive regulator of SCW thickening in Brachypodium distachyon and interacts with cellulose and lignin gene promoters [SCW formation is a very complex process that requires coordinated regulation of a series of structural genes and TFs in vascular plants . Researcnd IRX14 ,42,43,44ic genes ,46. Meanentation ,28,29,30ompounds . R2R3-MYompounds ,49,50. I bicolor . In riceream TFs . BdSWAM1romoters .PtrMYB2/3, PtrMYB20/21, and PtrMYB74 are the MYB master genes that simultaneously regulate the synthesis of cellulose, lignin, and hemicellulose [PtrMYB152 and PtoMYB170/216 specifically promote the accumulation of lignin [PtoMYB92, the homologous gene of AtMYB85, promotes the accumulation of lignin, but inhibits the hemicellulose synthesis [PtoMYB156 and PdMYB221 were also found to inhibit the accumulation of cellulose, lignin, and hemicellulose [PpDof5 in hybrid poplars promoted the accumulation of lignin in stems and basal leaves [ARK1 gene from the ARBORKNOX family reduced the lignin content by downregulating lignin synthesis-related genes [PdOLP1, an osmotin-like protein gene in poplar, caused a reduction in the radial width and cell layer number in xylem and phloem zones, and downregulated expression of genes involved in lignin biosynthesis, and in the fibers and vessels of xylem cell walls [As perennial plants, the SCW formation of woody plants is complicated, and its transcriptional regulation mechanism is largely unknown. There are at least 194 R2R3-MYB TFs in the poplar genome, and only a few of them have been demonstrated to regulate SCW formation. Meanwhile, ellulose ,54; overf lignin ,36. PtoMynthesis . In addiellulose . Recentll leaves . The ARKed genes . KNAT 2/ed genes . The ovell walls .PtrMYB121 from P. trichocarpa and demonstrated that PtrMYB121 positively regulated the biosynthesis of lignin and cellulose. Phylogenetic analysis showed that PtrMYB55/121 and PtoMYB170/216 from P. tomentosa were homologous genes of Arabidopsis AtMYB55 and AtMYB61  and N. benthamiana were used in this study. Seeds of A. thaliana and N. benthamiana were sterilized with 70% ethanol (30 s) and 1% sodium hypochlorite (10 min), and then germinated on plates with MS medium  supplemented with 8 g/L agar. Fourteen days later, the seedings were transferred into soil and grown in a greenhouse. The culture conditions of P. trichocarpa and N. benthamiana were 16/8 h light/dark cycle, 23–25 °C, 10,000 Lux light, and relative humidity of about 40%. The culture conditions of Arabidopsis were 16/8 h light/dark cycle, 22–23 °C, 5000 Lux light, and relative humidity of about 60% [The seedlings of bout 60% .https://www.ncbi.nlm.nih.gov). Then, the sequences were used to construct the phylogenetic tree by neighbor-joining (NJ) method in p-distance model using MEGA8.0 software  [https://www.megasoftware.net/), and multiple sequence alignment was carried out with DNAMAN software [The amino acid sequences of R2R3-MYB TFs were obtained from the GenBank of NCBI   , following the manufacturer’s instructions. Then, the reverse-transcription PCR was performed with the PrimeScript RT reagent Kit (QIAGEN) to synthesize the first-strand cDNA. The cDNA was stored at −20 °C and used as the template for gene cloning and qRT-PCR. The qRT-PCR analysis was performed as previous studies [Total RNA was extracted from various tissues  of  studies .PtrMYB121 was amplified by PCR reaction with gene-specific primers listed in PtrMYB121 binary vector was introduced into the Agrobacterium tumefaciens strains GV3101. The reaction system (25 μL) contained 12.5 μL Prime STAR DNA polymerase , 0.25 μM forward primer, 0.25 μM reverse primer, 100 ng DNA template from stems of P. trichocarpa, and supplemented with ddH2O. The procedure was as follows: 94 °C 300 s; 94 °C 45 s, 58 °C 60 s, and 72 °C 90 s for 30 cycles; 72 °C 600 s.The full-length CDS (without the termination codon) of PtrMYB121 binary vector was transformed into 30-day-old Arabidopsis plants by floral dip method [The 35S: p method . TransgeW/V) TBO for 30 s immediately, and the temporary slides were observed with an optical microscope. Among them, the location of blue pigmentation represented the xylem.TBO staining was used to observe the lignin deposition. The bases of inflorescence stems of 40-day-old Arabidopsis were sliced, and paraffin sections (15 mm) were made with an ultra-thin semiautomatic microtome , as previous studies . Hence, Paraffin sections were also used for the observation of lignin autofluorescence, and the temporary slides were used for the detection of fluorescence signal using a confocal laser scanning microscopy  at 408 nm laser.The components of SCW were measured, as previously described . The acePtrMYB121 was amplified by PCR amplification with gene-specific primers  were independently cloned  of  primers  and ligay cloned  and ligat-test. In all experiments, significant differences of the data were evaluated by one-way ANOVA. * p < 0.05 and ** p < 0.01.All experimental data were obtained from three biological replicates, and the statistical analysis were performed with Student’s The GenBank accession numbers of other genes used in this study are as follows: PtrMYB121 (XP_002302702.1), AtMYB46 (AT5G12870.1), AtMYB55 (NM_116398.4), AtMYB58 (AF062893.1), AtMYB61 (AF062896.1), AtMYB63 (AT1G79180), AtMYB83 (AT3G08500.1), AtMYB103 (AT1G63910.1), EgMYB1 (AJ576024), EgMYB2 (AJ576023.1), HvMYB3 (X70881.1), OsMYB46 (JN634085), PtMYB1 (AY356372.1), PtMYB4 (AY356371.1), PtMYB8 (DQ399057.1), PtoMYB74 (KX887329.1), PtoMYB92 (KP710214.1), PtoMYB170 (KY114929.1), PtoMYB216 (JQ801749.1), PtrMYB2 (KF148677.1), PtrMYB3 (KF148675.1), PtrMYB20 (KF148676.1), PtrMYB55 (XM_002302666.3), PtrMYB192 (XM_002310643.2), PvMYB4a (JF299185), ZmMYB31 (NM_001112479), ZmMYB42 (NM_001112539)


Their impact on human health, especially through inhalation, has been studied for more than two decades. TiO2 is known for its strong affinity towards phosphates, and consequently interaction with cellular phosphates may be one of the mechanisms driving its toxicity. In the present study, we used a phosphoproteomics approach to document the interaction of TiO2-NP with phosphoproteins from A549 human pulmonary alveolar epithelial cells. Cells were exposed to 21 nm anatase/rutile TiO2-NPs, then their phosphopeptides were extracted and analyzed using shotgun proteomics. By comparing the phosphoprotein content, phosphorylation status and phosphorylation sites of exposed cells with that of control cells, our results show that by affecting the phosphoproteome, TiO2-NPs affect cellular processes such as apoptosis, linked with cell cycle and the DNA damage response, TP53 being central to these pathways. Other pathways including inflammation and molecular transport are also affected. These molecular mechanisms of TiO2-NP toxicity have been reported previously, our study shows for the first time that they may derive from phosphoproteome modulation, which could be one of their upstream regulators.TiO The analysis conditions were as described by Dedieu et al. . Full-scudo-MS3)  was acti2 and samples from control cells, based on spectral counts after standard normalisation. PatternLab software was used for normalization and statistical significance assessment [p < 0.05 and fold-change > 1.5. Gene ontology and pathway analyses were performed using Database for Annotation, Visualization and Integrated Discovery (David) [® Pathway Analysis . Enriched pathways were considered statistically significant when p < 0.05.Peak lists were generated with the MASCOT DAEMON software  using the extract_msn.exe data import filter from the Xcalibur FT package (version 2.0.7) . The filtering options were, as previously : minimumsessment . The cut (David) ,44 and I2-NPs, then rinsed three times with PBS containing 2 mM of EDTA. Then they were fixed for 30 min in 70% ice-cold ethanol and diluted in PBS-EDTA (2 mM). This fixative solution was removed and cells were suspended in 25 µg/mL propidium iodide prepared in PBS-EDTA (2 mM) to which was added 25 µg/mL RNase A. Samples were analyzed by flow cytometry using a FACS Calibur . This experiment was repeated three times independently, with four replicates per experiment.Cells were exposed to 100 µg/mL of TiO2-NPs for 24 h were washed three times with phosphate saline buffer, fixed with 2.5% glutaraldehyde and post-fixed with OsO4. They were then dehydrated by immersion in solutions of ethanol with increasing concentration and embedded in Epon. Ultrathin sections were prepared by ultramicrotomy and counterstained with uranyl acetate and lead citrate. These samples were observed using a CM12 Philips electron microscope, operating at 80 kV.Cells exposed to 100 µg/mL TiO2-NPs to A549 cells, i.e., less than 25% of cell mortality in exposed cells according to the MTT assay. Moreover, we previously showed that this exposure condition leads to a significant elevation of intracellular reactive oxygen species level, suggesting oxidative stress. We also showed that it causes significant DNA damage, as assessed via the comet assay and quantification of 8-oxo-dGuo using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS), as well as elevation of DNA repair capacities in exposed cells [Cell exposure conditions were chosen based on results that we previously obtained, showing mild cytotoxicity of TiOed cells ,14. The ed cells .2-NPs were analyzed after enrichment by high-resolution tandem mass spectrometry [Phosphoproteomes of control cells and cells exposed to TiOtrometry . As show2-NPs, respectively, corresponding respectively to 1310 and 1283 unique peptides  showed different phosphorylation level rather than a strong modification of phosphorylation level of a few P-sites . As reported in 2-exposed cells, 89 proteins (17.5%) showed different phosphorylation levels in cells exposed to TiO2-NPs as compared to control cells according to at least one of the three criteria (Category 1). Twenty-six proteins showed different phosphorylation levels according to all three criteria (Category 2). Most of these phosphoproteins showed increased phosphorylation levels in cells exposed to TiO2-NPs, compared to control cells (We compared the protein phosphorylation level in control cells and in cells exposed to TiOee 2-NPs . When usol cells .2-exposed cells. These 139 P-sites belonged to 111 phosphoproteins (Category 3). Contrary to what was found when analyzing protein phosphorylation level, 70% of P-sites were less phosphorylated in TiO2-NP-exposed cells than in control cells. Therefore, the overall protein phosphorylation level is not affected in cells exposed to TiO2-NPs. Considering the strong affinity of TiO2 towards phosphate groups, and the intense accumulation of these TiO2-NPs in A549 cells (see [2-NPs are referred to as TiO2-Degussa), a much more intense dysregulation of the overall protein phosphorylation levels was expected. Our results suggest that the impact of TiO2-NPs on the cellular phosphoproteome is much more finely tuned. This could be explained by the cellular distribution of TiO2-NPs in A549 cells, which shows preferential location in endosomes and/or lysosomes [2 with phosphoproteins.When using the same approach on individual P-sites, out of the 1606 detected P-sites, 139 showed different phosphorylation levels in control and TiOlls  was analyzed via protein ontology, using David and Ingenuity® Pathway Analysis , followed by data mining using the Uniprot protein annotation. The whole human proteome was used as reference group. Since both David and IPA analyses, based on the lists of individual P-sites, phosphocounts or phosphopeptides, highlighted the same cellular functions and processes, we present here the results obtained using David and the analysis performed on the list of phosphopeptides, showing a false discovery rate (FDR) lower than 20%  and P-sites are reported in 2-NPs [2-NPs [2-NPs as in the present study leads to significant accumulation of TiO2-NPs in autophagic vesicles and increased LC3II/LC3I ratio, suggesting alteration of the autophagic process [2-NP-exposed cells, particularly via impairment of the DNA repair as previously demonstrated in A549 cells exposed to the same TiO2-NPs and in the same conditions as in the present study [2-NPs for 24 h shows a mild but statistically significant reduction of the proportion of cells in the G1 phase, as compared to unexposed cells  and “negative regulation of extrinsic apoptotic signaling pathway” FDR 18%) attracted our attention, since apoptosis has already been described as being one of the cellular processes induced by TiO8% attrac2-NPs  and ACIN2-NPs . BAG3 ex2-NPs . TPX2 pa2-NPs . Some ofs [2-NPs ,49,50. Fs [2-NPs . HTT is s [2-NPs  and it is [2-NPs . SQSTM1,s [2-NPs , while Ss [2-NPs  and RB1 s [2-NPs . In linent study ,14 and vnt study ,18,19,20nt study . Moreovent study . SON is nt study . In addint study , while Rnt study . Moreovent study ,62. Its ed cells . Moreovell cycle . Moreove2-NPs as in the present study, up to two months, leads to increased phosphorylation and acetylation of TP53 [2-NPs. Induction of DNA damage and/or apoptosis is known to be related to activation of the TP53 pathway [Our analysis highlights the central role of TP53 in these processes, as illustrated by a network identified using IPA . This ne of TP53 . This su pathway ,67,68,692-NPs is generally reported as cell accumulation in S or G2-M phases [Impaired cell cycle progression induced by TiOM phases ,18,19,20M phases . The pre2-NPs on cellular development, movement and morphology . Some of these proteins play roles in cell migration, motility, and adhesion. For instance, TLN2 is a component of focal adhesion plates and is involved in cell adhesion in synapses, ZYX is a stabilizer of focal adhesions in muscle cells and synapses , and CTTrphology .2-NPs showed strong accumulation of NPs inside cells, and localization in cytoplasmic vesicles  RNA binding” FDR 0.03, “alternative splicing” FDR 0.25, “RNA-binding” FDR 19.65), particularly to splicing of pre-mRNA and mRNA . SON is a cofactor for transcript splicing, especially for transcripts related to cell cycle and DNA repair , while Snslation . The prenditions .2-NPs [2-NPs dysregulate the expression of genes or proteins related to ion transport [Finally, several proteins are involved in molecular transport , in and out of the nucleus in particular. NUP214 is a nucleoporin involved in nuclear export  and SEC62-NPs  or impai2-NPs ,14. Thesransport ,84, tranransport ,95, and ransport ,96. 421 and CARHSP141, which are dephosphorylated by PTP1B and PP2A, respectively [1151, ENSA67, ERF526, FLNA1084, KIF4A801, MISP397 and RB1826 have been shown to regulate the cell cycle [1151, CTTN421, FLNA1084, HSPB115 and VIM56 have been reported to be involved in cytoskeleton organization [We then analyzed the literature relative to the function of the 139 dysregulated P-sites, which would explain some of the impacted biological activities. The biological function of only 14 P-sites is known . Some kiectively ,97. Agaill cycle ,103,104,nization ,108,109.115 and CARHSP141) [56 and NDRG1330) [2-induced cellular stress [Other functions are identified through this approach, i.e., the oxidative stress response HSPBRHSP141) ,111,112 DRG1330) ,113. Bot41 ,111,12-NPs reveals that these nanoparticles affect DNA damage response, by means of dysregulation of the cell cycle and DNA repair, the process of autophagy, the cytoskeleton dynamics and structure, the RNA dynamics, as well as intracellular transport. It highlights the central role of p53 in some of these processes. Although these effects of TiO2-NPs have already been reported using other techniques, here we suggest that there is a link between the dysregulation of phophoprotein phosphorylation status and these cellular events, using an exposure condition that does not induce any overt cytotoxicity. It should be highlighted that the A549 cell line is derived from an alveolar carcinoma. Therefore, it would be important to repeat this experiment on a non-cancerous cell line. This would demonstrate that the observed effects are not specific to cancer cells and that they are representative of the impact caused by inhalation exposure of healthy individuals to TiO2-NP.Analysis of the phosphoproteome of A549 cells exposed to TiO


APOE AD risk genes, based on whether these variants were associated with altered mRNA transcript levels, as well as brain amyloidosis, tauopathy, and neurodegeneration.Genome-wide association studies have identified more than 30 Alzheimer’s disease (AD) risk genes, although the detailed mechanism through which all these genes are associated with AD pathogenesis remains unknown. We comprehensively evaluate the roles of the variants in top 30 non-Human brain gene expression data were obtained from the UK Brain Expression Consortium (UKBEC), while other data used in our study were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. We examined the association of AD risk allele carrier status with the levels of gene expression in blood and brain regions and tested the association with brain amyloidosis, tauopathy, and neurodegeneration at baseline, using a multivariable linear regression model. Next, we analyzed the longitudinal effects of these variants on the change rates of pathology using a mixed effect model.ADAM10, IGHV1-68, and SLC24A4/RIN3) were associated with brain amyloidosis, 7 variants  with brain tauopathy, and 8 variants  with brain neurodegeneration. Variants in ADAMTS1, BZRAP1-AS1, CELF1, CD2AP, and SLC24A4/RIN3 participated in more than one cerebral pathological process.Altogether, 27 variants were detected to be associated with the altered expression of 21 nearby genes in blood and brain regions. Eleven variants (especially novel variants in Genetic variants might play functional roles and suggest potential mechanisms in AD pathogenesis, which opens doors to uncover novel targets for AD treatment.The online version contains supplementary material available at 10.1186/s13195-020-00755-7. APOE is still the strongest genetic risk factor for LOAD, responsible for a 3- to 15-fold increase in risk  biomarkers, particularly elevated CSF phosphorylated tau (pTau) or cortical tau-PET ligand binding; and neurodegeneration [N] biomarkers, particularly elevated CSF total tau (tTau), diminished 18F-fluorodeoxyglucose (FDG)-PET, or brain structural atrophy on MRI.Although most of the non- effects , 13. In  effects . Recentl effects  that proAPOE AD risk genes from current large-scale GWAS studies of the transcript expression levels, and the pathological processes of brain amyloidosis, tauopathy, and neurodegeneration, using the baseline and follow-up data from AD-related CSF, PET, and MRI measures. Understanding the mechanisms by which these variants contribute to AD risk will lead to a better understanding of the disease-associated mechanisms and help uncover novel therapeutic avenues.Many of the early identified AD variants have been associated with expression levels of their nearby genes and implicated in brain amyloidosis and neurodegeneration , 16, 17.http://adni.loni.usc.edu). ADNI was launched in 2003 as a public-private partnership, led by Principal Investigator Michael W. Weiner, MD, VA Medical Center and University of California-San Francisco. Our analyses included all individuals with diagnosed AD, mild cognitive impairment (MCI), and normal cognition (NC), with clinical information, GWAS data, and gene expression data from peripheral blood (Affymetrix Human Genome U219 Array platform), or longitudinal AD-related CSF, PET, and MRI data. Furthermore, we selected only non-Hispanic white individuals to avoid population stratification effects which can lead to spurious findings. In total, 1183 individuals at baseline were included in our study , and included 10 brain regions from 134 neuropathologically normal individuals of European descent. The 10 brain regions were the cerebellar cortex (CRBL), frontal cortex (FCTX), hippocampus (HIPP), medulla (MEDU), occipital cortex (OCTX), putamen (PUTM), substantia nigra (SNIG), temporal cortex (TCTX), thalamus (THAL), and intralobular white matter (WHMT). In the Braineac dataset, Affymetrix GeneChip Human exon 1.0ST arrays were used to measure the gene expression in transcript levels. Detailed information on these methods is described in the Braineac database . Genomicκ) statistics. When choosing between 2 variants with significant overlap (high r2 and high κ), we retained the variant with the smallest amount of missing data. Our final number of non-APOE AD variants was thus reduced to 42  participants were genotyped according to the manufacturer’s protocol. We focused on well-established AD risk genes identified in AD GWASs available to date  using the multiplex xMAP Luminex platform  with Innogenetics  immunoassay kit-based reagents. All CSF biomarker assays were performed in duplicate and averaged.CSF Aβhttp://adni.loni.usc.edu/datasamples/pet/. The AV45-PET (amyloid-PET) and AV1415-PET (tau-PET) standardized uptake value ratios (SUVRs) were formed by normalizing composite multi-region target regions of interest (ROIs) to the cerebellar crus gray matter. The amyloid-PET target meta-ROI included the frontal, anterior cingulate, precuneus, and parietal cortex [A detailed description of PET image acquisition and processing can be found at l cortex . The taul cortex .The cerebral metabolic rate for glucose (CMRgl) data on FDG-PET was downloaded from the ADNI dataset. Mean FDG uptake was averaged from 5 meta-ROIs including the right and left angular gyri, right and left inferior temporal regions, and bilateral posterior cingulate. PET images were spatially normalized in Statistical Parametric Mapping (SPM) to the MNI PET template. We intensity-normalized each meta-ROI mean by dividing it by the pons/vermis reference region mean.https://surfer.nmr.mgh.harvard.edu). HV was adjusted for eICV using the following equation: Adjusted HV (HVa) = Raw HV – b (eICV – Mean eICV), where b is the regression coefficient when HV is regressed against eICV.Hippocampal volume (HV) and estimated intracranial volume (eICV) were performed from T1-weighted MRI acquired with a Siemens Trio 3.0 T or 1.5 T scanner. Regional volume estimates were processed using the Freesurfer software  from baseline, AD risk allele carrier status, and interaction between time from baseline and AD risk allele carrier status. The model included random slope and intercept terms for each participant with age, gender, educational level, APOEε4 genotype, and diagnosis as covariates. All statistical analyses were conducted using R statistical software. We defined associations with false discovery rate (FDR)-adjusted P values < 0.05 as statistically significant using the Benjamini-Hochberg procedure.Clinical and demographic characteristics for each variant were compared using CR1, ECHDC3, MS4A6A, and NME8 were associated with decreased expressions, while the other 10 variants in BIN1, EPHA1, HLA-DRB1, PTK2B, SLC24A4/RIN3, and ZCWPW1 associated with increased expressions.The associations between AD risk allele carrier status and gene expression in the blood are shown in Additional file CLU and rs6701713 in CR1 were associated with increased expressions of CLU and CR1. The minor alleles of rs2526378 in BZRAP1-AS1 and rs12590654 in SLC24A4/RIN3 were also associated with increased expression especially in TCTX/FCTX and FCTX, respectively, while the minor allele of rs3865444 in CD33 was associated with decreased expression in TCTX  and amyloid-PET levels (FDR-adjusted P = 0.001) at baseline , but these changes over time were not clear from the longitudinal analysis  and tau-PET levels (FDR-adjusted P = 0.045) at baseline . The minor allele carrier of PTK2B rs28834970 was associated with the rapid growth rate of CSF pTau levels  these variants are associated with altered expression levels and (2) the variants are associated with brain amyloidosis, tauopathy, and neurodegeneration.Although large GWAS has recently identified novel variants that are associated with altered AD risk, we have a relatively poor understanding of their functional impact. In this study, we comprehensively analyze possible functional effects of the variants in top 30 non-ABCA7, ADAM10, ADAMTS1, BZRAP1-AS1, CASS4, CD2AP, CD33, CELF1, CLU, HS3ST1, and PICALM were associated with the altered gene expressions in different brain regions alone, and variants in BIN1, ECHDC3, EPHA1, HLA-DRB1, NME8, and PTK2B were associated with the altered gene expressions only in the blood, while only 6 variants in CR1, MS4A6A, SLC24A4/RIN3, and ZCWPW1 affected these 4 nearby gene expressions both in the blood and in the brain, which perhaps become promising key biomarkers for AD diagnosis. Previous studies have shown that the overlap of genomic variants influencing transcript expression levels in both the human brain and blood is relatively low [CR1 rs6701713 was associated with decreased expression in blood but increased expression in specific brain regions. The direction of association of the same variant in different brain regions was also different  or WHMT  cleavage) [ADAMTS1 (within 665 kb of APP on chromosome 21) [IGHV1-68, BZRAP1-AS1, and SLC24A4/RIN3 with brain amyloidosis.Based on the baseline and longitudinal follow-up data, we were able to confirm the previously reported associations of genetic variants in escribed , 20, 31.leavage) , 33 and some 21)  were alsBIN1 rs744373 was associated with altered tau-PET and CSF pTau levels [BIN1 are also significantly associated with tau biomarkers. Similarly, we confirm the previously reported association of INPP5D, CD2AP, and PTK2B with brain tauopathy [SLC24A4/RIN3 and tau pathology is a new discovery. SLC24A4 CpG methylation sites were found associated with Aβ burden and tau pathology previously [SLC24A4 also appeared to take part in lipid metabolism [Previous studies have shown that u levels , 35. Ourauopathy , 36, 37.eviously  and SLC2tabolism  and braitabolism .CD2AP, CD33, CELF1, and PICALM on the changes in brain metabolism or atrophy over time, supporting the reported associations of these genes with brain neurodegeneration [ADAMTS1, ECHDC3, BZRAP1-AS1, and HS3ST1 with neurodegeneration biomarkers at baseline are novel findings of this research. Aligned with this, HS3ST1 was reported as significantly associated with working memory in probable-MCI patients [As most of the earlier studies were cross-sectional analyses, a stage-specific association might occur for genetic variants that influence the course of neurodegeneration . Using lneration , 40–42. patients .ADAMTS1, BZRAP1-AS1, and CELF1 affect the mechanisms involved both in brain amyloidosis and neurodegeneration, CD2AP in brain tauopathy and neurodegeneration, and SLC24A4/RIN3 in brain amyloidosis and tauopathy  is a subunit of the benzodiazepine receptor complex in mitochondria and a marker of neuroinflammation. Previous studies have demonstrated that the TSPO ligand can reverse Aβ accumulation and behavioral impairment in transgenic mice [CELF1 variant has been shown to affect cognition and CSF Aβ42 levels by modifying expression [CELF1, aret, also shows mediation of tau toxicity [According to our results, variants in AD brain , 44 and AD brain . BZRAP1 nic mice . Moreovepression , 47. Andtoxicity . Much mo42 is therefore best considered a biomarker of a pathologic state that is associated with amyloid plaque formation and not a measure of amyloid plaque load as amyloid-PET is. Similarly, CSF pTau is best considered a biomarker of a pathologic state that is associated with PHF tau formation and not a measure of pathologic tau deposits as tau-PET is [It is worth noting that, in our study, we place CSF and PET imaging biomarkers all into analysis; that is because the fundamental difference between the two should be recognized. CSF biomarkers are measures of the concentrations of proteins in CSF from the lumbar sac that reflect the rates of both production (protein expression or release/secretion from neurons or other brain cells) and clearance  at a given point in time. While imaging measures, on the other hand, represent the magnitude of the neuropathologic load or damage accumulated over time. Low CSF Aβu-PET is . In addiu-PET is , suggestGenerally, the ultimate goal of understanding the genetic architecture of AD is to enhance the understanding of disease mechanisms. Based on the above A/T/N classification system, our identification of many risk genes suggests their shared function in brain amyloidosis, tauopathy, or neurodegeneration, which might provide interesting targets for future functional follow-up and biological interpretation.Our current study has limitations. Firstly, the subjects with tau-PET data, especially the longitudinal tau-PET data, are limited. Secondly, the follow-up CSF data are limited because of the invasiveness of the procedure to acquire samples. With the rapid development and wide application of PET technology, future studies will validate our findings in a large, independent, longitudinal cohort with a greater number of individuals and time points and with a longer follow-up time to provide more statistically powerful results.APOE AD risk genes associated with transcript expression levels and involved in the pathological processes of brain amyloidosis, tauopathy, and neurodegeneration. This evidence increases the possibility that genetic variants might play functional roles and suggest potential mechanisms in AD pathogenesis. Further studies are needed to fully understand their roles in AD process, and our research opens doors to the investigation of novel targets for AD treatment.In summary, our current study provides new insights into the variants in the top 30 non-Additional file 1.


Calcium homeostasis plays an essential role in maintaining excitation–contraction coupling (ECC) in cardiomyocytes, including calcium release, recapture, and storage. Disruption of calcium homeostasis may affect heart function, leading to the development of various heart diseases. Myocardial ischemia/reperfusion (MI/R) injury may occur after revascularization, which is a treatment used in coronary heart disease. MI/R injury is a complex pathological process, and the main cause of increased mortality and disability after treatment of coronary heart disease. However, current methods and drugs for treating MI/R injury are very scarce, not ideal, and have limitations. Studies have shown that MI/R injury can cause calcium overload that can further aggravate MI/R injury. Therefore, we reviewed the effects of critical calcium pathway regulators on MI/R injury and drew an intuitive diagram of the calcium homeostasis pathway. We also summarized and analyzed calcium pathway-related or MI/R drugs under research or marketing by searching Therapeutic Target and PubMed Databases. The data analysis showed that six drugs and corresponding targets are used to treat MI/R injury and involved in calcium signaling pathways. We emphasize the relevance of further detailed investigation of MI/R injury and calcium homeostasis and the therapeutic role of calcium homeostasis in MI/R injury, which bridges basic research and clinical applications of MI/R injury. Myocardial ischemia/reperfusion (MI/R) is an inevitable process in the treatment of cardiovascular diseases such as acute myocardial infarction, thrombolysis, coronary angioplasty, and cardiac arrest . MI/R in2+ is an important cytoplasmic signaling molecule in most cellular responses and is mainly distributed in extracellular and intracellular organelles, such as sarcoplasmic reticulum (SR) and mitochondria. Calcium plays a vital role in excitation–contraction coupling (ECC) of heart tissue. When cardiac action potential occurs, extracellular Ca2+ enters the cell through L-type Ca2+ channels (LTCC), and intracellular Ca2+ activates ryanodine receptor 2 (RyR2) and more Ca2+ is released from the sarcoplasmic reticulum releases  plasmic reticulum Ca2+-ATPase (SERCA) recaptures intracellular Ca2+ back to SR. On the other hand, sodium/calcium exchanger (NCX) expels Ca2+ from the cells and the dissociation of Ca2+ from myofilament protein relaxes the myocardial cells  interacts directly with LTCC (Cav1.2); LTCC is regulated by G protein, adenylate cyclase, PKA, PP1, and PP2A, which affects channel activity. Some studies have shown that loss or deficiency of LTCC function can lead to short QT intervals and sudden cardiac death , and Cav2 and Cav3, which are non-LTCCs. Cav1.2 and Cav1.3 are co-expressed in multiple tissues  and play similar regulatory roles. Cav1.1 and Cav1.4 are expressed only in skeletal muscle and retina, respectively . LTCC chribution . At myocac death . Cav1.2 ac death . Therefo2+ sensitivity, and SR Ca2+ content , and phosphatases are involved in RyR2 phosphorylation. PKA-induced RyR2 phosphorylation (Ser-2808) promotes FKBP12.6 and RyR2 dissociation, thereby opening RyR2 and promoting calcium-activated calcium release (The FK506 binding proteins (FKBP) are expressed in cardiomyocytes and act as essential RyR2 regulators. FKBP12 and FKBP12.6 subtypes correspond to low and high RyR2 affinity, respectively. Reportedly, FKBP12.6 and not FKBP12 can inhibit RyR2 activity. FKBP12.6 and RyR2 association keeps RyR2 closed, preventing calcium release from the SR, thereby, ensuring heart relaxation . PKA, Ca release . This prRyR2 phosphorylation caused by CaMKII (Ser-2814) is more critical than that by PKA. Studies have shown that atrial fibrillation induced in FKBP12.6 KO mice can be achieved by inhibiting CaMKII phosphorylation RyR2 instead of PKA, thereby inhibiting SR calcium leakage and delayed depolarization . Decreas2+ is returned to SR through SERCA; excessive Ca2+ can also be expelled from cells through the NCX, resulting in myocardial cell relaxation , SLC8A2 (NCX2), SLC8A3 (NCX3) . In mammNa+:Ca2+ . The dird f-loop .NCX participates intracellular calcium homeostasis regulation and is implicated in the occurrence and development of various diseases, such as heart failure, arrhythmia, diabetes, hypertension, and cerebral ischemia. The role of NCX in cardiovascular diseases is debatable. Some studies have shown that heart-specific NCX knockout mice can resist MI/R injury, which may be associated with calcium overload inhibition . Other s2+ back to the SR, causing myocardial relaxation , ATP2A2 , and ATP2A3 (SERCA3a-f), each of containing multiple subunits. SERCA2a is the most important, mainly expressed in the heart and slow-twitch skeletal muscle, and mainly involved in cardiomyocyte ECC. SERCA1 is mainly expressed in fast-twitch skeletal muscle, SERCA2b mainly in smooth muscle and non-muscle tissue, SERCA2c mainly in epithelial and hematopoietic cells and SERCA3 primarily in hematopoietic cells . SERCA hlaxation . SERCA cal loops .2+ uptake by increasing SERCA activity, and reduce reperfusion-induced calcium overload. SERCA is an essential calcium homeostasis and is associated with the pathological mechanisms of various diseases; SERCA is expected to become an important target for cardiovascular disease.A variety of molecules regulate SERCA; SERCA also affects intracellular calcium homeostasis. Phospholamban (PLB) phosphorylation can release the inhibitory effect on SERCA; PKA affects SERCA sensitivity to calcium . SERCA oPLB located in SR contains 52 amino acids (6.1 kD), easily forms a polymer (22 kD), which interacts with SERCA and inhibits its activity . PLB con2+-releasing proteins such as RyR2, LTCC, IP3R, and regulate calcium recalculation by PLB and SERCA phosphorylation  stores and releases Caunctions .2+ release other than that in the normal calcium cycle is called SR calcium leakage, which is mainly mediated by RyR2. Calcium leakage can cause cardiac systolic dysfunction by reducing effective SR Ca2+ release, cause diastolic dysfunction by increasing diastolic cytosolic Ca2+, and induce arrhythmias  formation. Additionally, mitochondria are also involved in processes such as apoptosis, signal transduction, cell proliferation, and metabolism . The carchondria . CalciumMitochondria are not involved in myocardial ECC regulation under physiological conditions. However, due to the large calcium capacity of mitochondria, which accounts for about 30% of the calcium capacity in plasma and tissue cells, they compensate by alleviating intracellular calcium accumulation during calcium overload in myocardial cells . Studies2+ is driven by a large electrochemical gradient across the plasma membrane into the cells. Cells use this external source of signal Ca2+ by activating various entry channels, such as voltage-operated channels . Excessive intracellular Na+ will promote Na+ excretion and Ca2+ intake by NCX, which significantly increases intracellular calcium levels, hence, leading to calcium overload. When blood flow and oxygen supply to the cardiac tissue returns to normal, extracellular pH level is further increased, HNX and NCX activities are enhanced, and intracellular calcium overload is further aggravated , in the myocardial cells. OFR can decompose cell membrane phospholipid components and damage membrane structure, which leads to increased membrane permeability and excessive extracellular Ca+ influx . OFR als+ influx .2+ plays a vital role in cardiac ECC, so calcium overload will further exacerbate the degree of MI/R injury in cardiac muscle cells. Excessive intracellular calcium will enter the mitochondria, resulting in mitochondrial calcium overload; this inhibits ATP production, exacerbates energy metabolism disorders, and eventually leads to myocardial cell apoptosis  pathway is a database that integrates genomic, chemical, and system function information, linking gene catalogs from genomes that have been fully sequenced to higher-level system functions at cell, species, and ecosystem levels , such as nifedipine, nisoldipine, diltiazem, verapamil, nimodipine, and amlodipine . Therefoecretion . In a wo2+ plays an essential role in maintaining cell proliferation, division, and energy metabolism. The calcium signal pathway-related protein expression and ion channel regulation mechanism connect arrhythmia, myocardial hypertrophy, and heart failure in cardiovascular diseases, making the development of these diseases a cause and effect  is complex, and the targets are not single. For cardiovascular diseases, which involve multiple signaling pathways, TCM has its unique advantages. For example, the past . BesidesIn fact, nearly half of the drugs listed in 2+ movement into and out of the plasma membrane and sarcoplasmic reticulum; calcium circulation disorders can cause various heart diseases. Changes in the Ca2+ steady-state, including decreased SR Ca2+ reuptake, abnormal calcium channels, SR Ca2+ leakage, or significantly decreased SERCA2a expression can cause various heart diseases, including ischemic heart disease, reperfusion injury, hypertrophic cardiomyopathy, diastolic cardiomyopathy, and heart failure , National Key Research and Development Project (grant number 2017YFC1702504 and 2018YFC1707408), and the National Natural Sciences Foundation of China (grant number 81891012).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.


Epigenetic mechanisms including methylation play an essential role in regulating gene expression not only in cancer cells but also in immune cells. Although role of DNA methylation has been extensively studied in tumor cells in tumor microenvironment (TME), the understanding of transcriptional regulation of pro- and anti-cancer immune cells in TME is beginning to unfold. This review focuses on the role of DNA and RNA methylation in regulating immune responses in innate and adaptive immune cells during their activation, differentiation, and function phase in cancer and in non-cancer pathologies. Uncovering these crucial regulatory mechanisms can trigger discovery of novel therapeutic targets which could enhance immunity against cancer to decrease cancer associated morbidity and mortality.DNA and RNA methylation play a vital role in the transcriptional regulation of various cell types including the differentiation and function of immune cells involved in pro- and anti-cancer immunity. Interactions of tumor and immune cells in the tumor microenvironment (TME) are complex. TME shapes the fate of tumors by modulating the dynamic DNA (and RNA) methylation patterns of these immune cells to alter their differentiation into pro-cancer  or anti-cancer  cell types. This review considers the role of DNA and RNA methylation in myeloid and lymphoid cells in the activation, differentiation, and function that control the innate and adaptive immune responses in cancer and non-cancer contexts. Understanding the complex transcriptional regulation modulating differentiation and function of immune cells can help identify and validate therapeutic targets aimed at targeting DNA and RNA methylation to reduce cancer-associated morbidity and mortality. Epigenetic modifications are heritable changes regulating the cellular gene expression patterns required for the normal development and maintenance of various tissue functions ,2,3. WheDNA methylation is the most well-characterized epigenetic mechanism, and was linked to cancer as early as the 1980s . SpecifiIn mammals, the addition of methyl groups to DNA is carried out by “writers”, DNA methyltransferase (DNMT) 1, DNMT3A, and DNMT3B, converting unmodified C into 5-methyl-cytosine (5mC) . DNMT3A DNA methylation can be recognized by readers including methyl-CpG-binding domain (MBD) proteins, certain transcription factors, and zinc finger (ZNF) proteins . GeneralMethyl groups from DNA can be removed either passively or actively. Active DNA demethylation is performed by “erasers”, called ten-eleven translocation (TET), which remove methyl groups from DNA by oxidizing 5mC into 5hmC (5-hydroxymethyl-cytosine), 5fC (5-formylcytosine), and 5caC (5-carboxylcytosine) . The 5fC6A) RNA methylation, plays an essential role in gene expression regulation and development, and human diseases . T. T+ T ce T cells .+ T cells for a long time, CD8+ T cells can become non-functional or exhausted, resulting in reduced effector functions, such as decreased cytokine production (IFNγ and TNF-α) and/or loss of cytotoxicity . Exhausted T cells generally have high surface expression of multiple inhibitory molecules, such as PD-1, TIM3, LAG3, TIGIT, and 2B4, and transcription factors associated with high PD-1 expression are T-bet, Eomes, and YY1 [If an Ag is exposed to CD8 and YY1 ,115,116. and YY1 ,115,116. and YY1 ,115,116.+ TILs become exhausted and lose their effector functions in the TME due to numerous factors, such as immunosuppressive mechanisms by tumor cells. Analyzing the transcriptome and methylome of CD8+ TILs in the TME of colorectal cancer simultaneously, Yang et al. confirmed tumor-reactive TILs have an exhausted tissue-resident memory signature [+ TILs were demethylated and CD8+ TILs had an exhausted phenotype, including high expression of CTLA4, HAVCR2, LAYN, and TOX [+ T cell phenotype and cytotoxic to exhausted CD8+ T cell phenotype, promoter methylation of these cells was compared. Naïve CD8+ T cells showed the most promoter demethylation compared to cytotoxic and exhausted T cells; however, essential cytotoxic CD8+ T cell effector genes, including PRF1, GZMB, IFNG, CCL4, CCL3, CST7, and NKG7, went through hypermethylation to hypomethylation from naïve to cytotoxic CD8+ T cell differentiation, respectively [PDCD1 (encoding PD-1) and CTLA4, were demethylated within cytotoxic CD8+ T cells. Moreover, LAG3 and LAYN were also differentially methylated from naïve to cytotoxic CD8+ T cell transition [CD8ignature . They sh and TOX ,118. To ectively . For exhansition . Therefoansition ,117,118.+ T cells remained unmethylated, whereas the exhausted T cells showed complete demethylation [+ T cells and nonrenewal of CD8+ effector T cells [Interestingly, DNA methylation could determine if T cell exhaustion can be reversed. In chronic LCMV infection, the PD-1 gene promoter of the effector CD8hylation ,119. Furhylation . Low TCF T cells . DNA met6A has various functions, including mRNA stability, translation, splicing, and phase separation, and also takes part in cell differentiation and development [6A RNA methylation can potentially regulate immunity. Although the role of m6A RNA methylation in immunity has not been fully elucidated, few studies have reported its role in both innate and adaptive immune response [melopment ,28,29,30response ,130,131.6A-modified RNA [6A modification exposed to DCs, did not activate their TLR3, TLR7, and TLR8, and led to lower cytokines and activation markers, compared to DC stimulated with unmodified RNA that activated TLRs [6A RNA modifications and lack of METTL3 can result in lack of DC maturation [6A RNA, METTL14, and ALKBH5 were reported to regulate type I IFN production triggered by dsDNA or HCMV [6A modification at the coding sequence and the 3’ UTR of the IFNβ1 gene. Another study reported increased interferon-stimulated genes upon METTL3 (m6A writer) or YTHDF2 (m6A reader) deletion. Specifically, following deletion of METTL3 or YTHDF2, mRNA of IFNβ was modified at m6A, increasing its stability [6A can play a role in the negative regulation of anti-viral response by dictating increased turnover of IFN mRNAs. One study established a key link of m6A to cellular antiviral response by showing that m6A induces antiviral immunity as it regulates crucial proteins of innate immunity [6A demethylase ALKBH5 is recruited by RNA helicase DDX46 to remove m6A from 3’ UTRs of genes encoding TRAF3, TRAF6, and MAVS, thereby reducing export of their transcript out of the nucleus and subsequently preventing production of type I IFNs.Certain DNA and RNA molecules can be detected by the innate immune system as non-self entities via PPRs, such as TLRs. For instance, a study investigated the mammalian innate immune response of DCs through stimulation with DNA, RNA, and modified RNAs, including mfied RNA . Althougfied RNA ,129,130.ted TLRs . Unmodifturation ,128,129. or HCMV ,129,130.tability ,129,130.immunity . Mechani6A RNA methylation has also been shown to regulate adaptive immune responses. Similar to DNA methylation regulating differentiation of CD4+ T cells into various subtypes, m6A RNA methylation was shown to regulate differentiation of CD4+ T cells [+-CRE conditional Mettl3 flox/flox) to delete Mettl3 in CD4+ T cells [lo CD62Lhi) was higher in spleens and lymph nodes compared to WT. When the function of Mettl3-/- CD4+ T cells was compared to WT, they observed normal sensitivity to TCR signaling; however, T helper polarization had abnormalities. For instance, the KO CD4+ T cells had a significant reduction in differentiation into Th1 and Th17 cells, but increased differentiation into Th2 cells. In-depth analysis showed that m6A targets the mRNA of the IL-7 protein, which regulates T cell homeostatic proliferation and differentiation to various subtypes upon numerous external stimuli. SOCS proteins are adaptors which bind to cytokine receptors, such as the IL-7 receptor, thereby preventing STAT5 and downstream signaling [6A modification was shown to regulate the degradation of the Socs genes, via the IL-7-JAK1/STAT5 signaling pathway, and without m6A, Socs mRNA persists, leading to high levels of SOCS proteins and reduced sensitivity to IL-7. This study indicates that m6A not only regulates CD4+ T cells differentiation but also T cell homeostasis [+ T cells and found that Mettl3 -/- Tregs mice developed severe autoimmune disorders compared to WT, suggesting loss of m6A modification can lead to loss of Treg immune suppressive functions [6A, readers have shown potential in regulating immune response. As such, compared to WT, a direct reader of m6A, Ythdf1 KO mice showed better cross-presentation of tumor antigens in DC and better cross-priming with CD8+ T cells, leading to high Ag-specific CD8+ T cells in response to tumors [6A of transcripts encoding lysosomal proteases lead to increased translation of these lysosomal proteases’ (cathepsins) transcripts in DCs, whereas inhibition of Ythdf1 led to inhibition of these cathepsins, resulting in enhanced cross-presentation by DCs and cross-priming of CD8+ T cells by DCs. Indeed, mature DCs were reported to have higher expression of writer complex, including METTL3, than naïve DCs [+ T cells [m T cells . The aut T cells . After vignaling ,129. SOCignaling ,129. Theeostasis . Using aunctions . In addio tumors . Specifiaïve DCs . In addi T cells . Interes T cells .6A RNA methylation in regulating innate and adaptive immune responses. The role of RNA methylation in immunity is still at its infancy and requires further research for discovery of novel therapeutic targets for its translational potential.Collectively, the above studies show the essential role of mAlterations in methylation have been strongly associated with the initiation and progression of cancer . Compare®) and 5-aza-2′deoxycytidine  have been approved by the Food and Drug Administration (FDA) [Several DNA hypomethylating agents have been developed that target DNA hypermethylation. However, among these DNA methyltransferase inhibitors (DNMTi), 5-azacytidine (Vidazaon (FDA) . Becauseon (FDA) . The inhon (FDA) , and 5-aon (FDA) . Both 5-on (FDA) .Following the clinical success of DNMTi with hematologic malignancies, DNMTi were also tested in solid tumors ,137,138.In cancer, promoter hypermethylation of TSGs and silencing of TSGs resulting in tumorigenesis have been the focus of the last few decades, resulting in the discovery of DNMTi ,148,149.Although studies investigating the effect of SAM on the immune system are still lacking, SAM has been shown to modulate the immune system ,166,167.MAT2A, which encodes the catalytic subunit of MATII and is vital for SAM biosynthesis [FOXP3 gene, thereby reducing its protein and mRNA expression in a dose-dependent manner. SAM was also found to decrease expression of an immunosuppressive cytokine, IL-10, and increase expression of IFNγ [Interestingly, studies have shown that SAM is essential for T cell activation and proliferation ,166,167.ynthesis ,164,165.ynthesis . Furtherynthesis . Further of IFNγ .Aberrant methylome is a common consequence of a disrupted SAM cycle associated with transformation of cells towards tumorigenesis ,169,170.6A RNA methylation is still at its infancy. However, novel studies have shown the potential of targeting RNA methylation in cancer. For instance, FTO inhibition through selective inhibitors, such as Meclofenamic acid (MA), MA2, and R-2-hydroxyglutarate (R-2HG), have shown potent anti-cancer activity in several cancers including AML, glioblastoma multiforme (GBM), and colorectal cancer (CRC) [6A RNA methylation levels [The role of DNA methylation in regulating the immune system and cancer has been the focus of research for more than three decades. Regulation of immunity and cancer by mer (CRC) ,30,178. n levels . Rhein in levels . Rhein hn levels . Citraten levels .6A demethylases as a potential anti-cancer target but also their potential in anti-cancer immunity within the TME.Although the inhibitors for RNA methylation regulators have been identified, none of them have been tested in a clinical setting. Furthermore, the effect of these pharmacological inhibitors of RNA methylation on the immune system is yet to be determined. Along this line, recently, RNA demethylase FTO was reported to promote tumorigenesis in melanoma and knockdown of FTO-reduced resistance to ICPi (anti-PD-1) therapy . FTO regIL4 and FOXP3 genes occur in Th2 and Tregs, respectively. In contrast, DNA demethylation and high expression of IFNγ and IL2 genes occur in both Th1 and CD8 T cells, which results in a better anti-cancer immune response. Studies should further investigate the effect of DNA and RNA methylation on transcriptional regulation of immune cells along with tumor cells in a time-dependent manner in order to uncover the complexity of the TME at various stages of cancer growth and progression. As explained earlier, the balance between pro- and anti-cancer immune cells within the TME is key to tumor progression or suppression. However, most studies investigating the role of methylation have focused only on one immune cell subtype. Future studies should investigate various immune subtypes simultaneously. These comprehensive studies will provide deeper insights into the interplay between the immune system and cancer, and allow discovery of novel epi-therapies that can enhance the immune system against cancer and other pathologies. Targeting methylation is a particularly attractive anti-cancer strategy because it is dynamic and reversible. For instance, DNMTi that target DNA hypermethylation can also enhance the efficacy of immunotherapies. Similarly, SAM, targeting DNA hypomethylation, has shown profound effects in combination with ICPi. Along the same line, inhibitors of m6A RNA demethylases have shown potential in enhancing anti-cancer immunity. However, further comprehensive studies are required to delineate the mechanism of action before these inhibitors can be tested in a clinical setting. In addition, SAM, which donates methyl groups to RNA, has shown significant anti-cancer activity in numerous cancer models by regulating DNA methylation. It is yet to be determined if SAM causes inhibition of tumor growth and metastasis through modulating m6A RNA methylation levels. Although the efficacy of epigenetic-based therapeutic strategies targeting tumor and immune cells needs further elucidation, the current state of knowledge provides compelling evidence to suggest that it will be effective in blocking cancer progression and reducing cancer associated morbidity and mortality.The role of DNA and RNA methylation in regulating the differentiation and activity of immune cells within the TME is key to determining the fate of tumor growth or suppression . A pro-c


Background: The superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase  was reported as a major source of oxidative stress in various human diseases. Genetic deletion is widely used to study the impact of NOX2-derived reactive oxygen species (ROS) on disease development and progression in various animal models. Here, we investigate why NOX2 knockout mice show no NOX2 activity but express NOX2 mRNA and protein. Methods and Results: Oxidative burst (NOX2-dependent formation of ROS) was measured by L-012-based chemiluminescence and was largely absent in whole blood of NOX2 knockout mice. Protein expression was still detectable in different tissues of the NOX2 knockout mice, at the expected and a slightly lower molecular weight (determined by Western blot). The NOX2 gene was even largely enhanced at its expressional level in NOX2 knockout mice. RNA sequencing revealed a modified NOX2 mRNA in the knockout mice that is obviously translated to a truncated inactive mutant enzyme. Conclusion: Although the commercial NOX2 knockout mice display no considerable enzymatic NOX2 activity, expression of the NOX2 gene (when using standard primers) and protein  can still be detected, which may lead to confusion among investigators. Using oxygen for enhanced energy production is inevitably linked to the production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, which may have harmful effects on normal cellular function . A tightphox) [In innate immune reactions, neutrophils are cells essential for killing bacteria via activation of a wide variety of effector responses and generation of large amounts of ROS, also named an “oxidative burst” . These hphox) , which aphox) . phox is the main catalytic subunit of the leukocyte NADPH oxidase complex. It is highly expressed in phagocytes  and at much lower levels in lymphocytes, endothelial cells, and colonic epithelial cells [The NOX enzyme family members, NOX1, NOX2, NOX3, NOX4, and NOX5 and the dual oxidase (DUOX) enzymes, DUOX1 and DUOX2, are membrane-associated hetero-oligomeric complexes that generate ROS by using oxygen and NADH/NADPH as the substrate and cofactor ,13. NOX al cells . The sigal cells . In our previous studies, we used the mouse model of inactivation of the mouse cybb gene (NOX2ko) to analyze the role of NOX2-generated ROS in cardiovascular diseases ,17,18,19phox antibody was obtained from BD Biosciences, USA. The secondary antibody peroxidase-labeled goat-anti-mouse (GAM-POX), avidin-biotin complex (ABC) reagent, and DAB reagent (used for IHC) were from Vector Laboratories, CA, USA. The polyclonal NOX-2 antibody used for immunohistochemistry was obtained from LSBio, Seattle, WA, USA. The biotinylated secondary antibody was from Thermo Fisher Scientific, Waltham, MA, USA. The Bradford reagent mix for determination of protein concentration was obtained from BioRad, Munich, Germany.PMSF, leupeptin, aprotinin, agarose, monoclonal anti-β-tubulin antibodies, horseradish-peroxidase-coupled, and anti-mouse IgG were purchased from Sigma, Deisenhofen, Germany. Restriction enzymes, Taq polymerase, Klenow DNA polymerase, and deoxynucleoside triphosphates (dNTPs) were purchased from New England Biolabs, Frankfurt, Germany. All oligonucleotides were from Sigma, Deisenhofen, Germany. The High-Capacity cDNA Reverse Transcription Kit was purchased from Applied Biosystems, Darmstadt, Germany. The PrecisionPLUS 2x qPCR MasterMix with SYBR green was obtained from Primer Design, Chandler’s Ford, United Kingdom. Primer-Probe-Mixes of murine NADPH oxidase (NOX-2 Mm00432775_m1) and murine TATA box binding protein  were purchased from Applied Biosystems, Foster City, CA, USA. The mouse monoclonal gp91tm1Din/J, stock number 002,365 for NOX2ko; B6N.129S2-Ncf1tm1Shl/J, stock number 027,331 for p47phox-ko) and maintained in the Translational Animal Research Center of the JGU Mainz. The NOX2ko(NOX2ko) mice were generated by insertion of a fragment containing a neomycin resistance gene (neoR) under the control of the mouse phosphoglycerate kinase 1 gene (PGK1) promoter and polyadenylation signal (polyA) into exon 3 of the gene  C57BL/6J mice, NOX2ko, and p47phox-ko mice were obtained from Jackson Lab (B6.129S-Cybbor a map ). These All animal studies were permitted by the Ethical Committee and Landesuntersuchungsamt Rheinland-Pfalz . 2+/Mg2+ (1 mM) by L-012 (100 µM) enhanced chemiluminescence (ECL) using a Mithras2 chemiluminescence plate reader . L-012 dione sodium salt) was purchased from Wako Pure Chemical Industries .A leukocyte-dependent oxidative burst was read-out for leukocyte-dependent hydrogen peroxide formation , as desTo proof the sequence data and analyze the NOX2ko mRNA expression, qualitative RT-PCR experiments were performed as described before , using tThe products of the RT-PCR reactions were analyzed by electrophoresis on 2% agarose gels. For comparison, a DNA marker ladder was used.To proof the sequence data and analyze the NOX2ko mRNA expression in leukemia-initiating stem cells, next generation sequencing (NGS) sequence reads published by Adane et al.  analysis using QuantiTect Probe RT-PCR kit (Qiagen), as described previously ,25. Prim(‚∆∆C(T)) method [To calculate the relative expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-, NOX2wt-, or NOX2ko-mRNA, the 2) method  was usedphox, 1:1000, BD Biosciences, USA, directed against a peptide containing the amino acids from position 450 to 556 of the murine NOX2 protein) and visualized using a secondary antibody with a covalently bound peroxidase moiety . All incubation and washing steps were performed according to the manufacturer’s instructions. Visualization of antibody-specific signals was performed using the Super Signal ECL kit from Thermo Scientific and a ChemiLux Imager .The procedures were similar to those described recently ,25. All The first lung segments were fixed in paraformaldehyde (4%) and paraffin-embedded. Paraffin-embedded lung samples were cut to thin sections and stained with the antibody . NOX-2 wData represent means ± SEM. Statistical differences were determined by factorial analysis of variance, followed by “Tukey’s” or “Dunnett’s” multiple comparison test. In the case of two means, classical two-sided t-test analyses were used. All statistical analyses were performed using Graphpad Prism 8. tm1Din/J mice  offered by the Jackson Laboratory as the animal model.In our attempts to analyze the importance of ROS generated by NOX2, we used the B6.129S-CybbAlongside performing genotyping, as recommended by the Jackson Laboratory . We also performed analyses to measure the oxidative burst in whole blood from wt and NOX2ko animals. As shown in To ensure that this block in the oxidative burst in cells of NOX2ko animals resulted from inhibition of NOX2 expression, we performed Western blot experiments with different tissues of wt and NOX2ko animals using a NOX2-specific antibody . Different tissues were used to ensure that our observation was not limited to a cell-specific effect. As shown in To gain further insight into this surprising result of the elevated expression of a NOX2ko protein in NOX2ko animals, we also analyzed the NOX2 mRNA expression in the brains and liver of NOX2ko animals by qRT-PCR, using primer systems described in the Materials and Methods section , as well as NOX2ko_f and NOX2_ko_r  expressed an inactive form of the NOX2 protein. The knockout mice showed NOX2 protein signals in different tissues, as assessed by Western blot analysis and immunohistochemical staining. NOX2 gene expression was even upregulated 6- to 90-fold (NGS or qRT-PCR data) in brain, liver, or leukemia-initiating stem cells obtained from NOX2ko animals. In sharp contrast, whole blood from NOX2ko animals showed no oxidative burst in response to potent activators of NOX2, such as phorbol ester or zymosan A , clearlytg mice [Genetic Nox2 deletion has been used for almost 2 decades to study the impact of NOX2-derived ROS on disease development and progression. A critical role of the NADPH oxidase isoform NOX2 (gp91phox) was demonstrated for angiotensin II (AT-II) induced hypertension by using NOX2ko or overexpressing NOX2tg mice ,30,31,32tg mice . Likewistg mice ,35 or fotg mice . Despitetg mice ,38, and,tg mice ,40,41,42tg mice , as welltg mice ,45,46. phox subunit of the NOX2 complex; however, mutations in other NOX2 subunits can also cause CGD [Staphylococcus aureus, Pseudomonas, and Burkholderia cepacia, as well as fungal species, such as Aspergillus, are common. Thus, the oxidants generated by NADPH oxidase are critical mediators of host anti-microbial defense. Alongside these considerations, the lack of specificity for the different NOX isoforms may represent a major reason for why, so far, NADPH oxidase inhibitors are not used in clinics [The essential role of NADPH oxidase in host antimicrobial responses is shown by the chronic granulomatous disease (CGD) . The mosause CGD ,48,49. C clinics ,50.In addition, data from CGD patients, as well as mouse models , demonstThe enhancement of the expression of the NOX2ko mRNA in comparison to the NOX2wt mRNA, as seen in brain, liver, and leukemia-initiating stem cells , may be 2O2 or ONOO−) after TNF-α or IL-1β exposure induced a robust and long-lasting hyperactivation of NF-κB (by inhibiting the re-synthesis of I-κB). However, preincubation of the cells with the same oxidants before TNF-α or IL-1β exposure leads to a nearly complete inhibition of NF-κB activation [The second explanation of the enhanced NOX2ko mRNA expression may relate to the reduced ROS production in the NOX2ko animals. Our data shown in mice with the inactivated p47phox gene, and therefore the inactivated NOX2 complex, hint this explanation. The reduced ROS production in the p47phox animals lead to an enhanced expression of the endogenous NOX2 mRNA . As showtivation . In additivation , which htivation .Based on our present data, it seems quite likely that inactivation of the NOX2 gene in NOX2ko animals resulted in a reduced inhibition of the NOX2 gene transcription, resulting in enhanced expression of NOX2ko mRNA in these animals.


Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity. Metastatic progression is the leading cause of mortality among breast cancer patients. In order to leave the primary tumor and reach distant organs, cancer cells need to migrate and degrade the surrounding extracellular matrix (ECM). Invadopodia, which are dynamic actin-rich membrane protrusions developed by cancer cells, play a major role in ECM proteolysis ,2,3. InvEfficient invasion of cancer cells not only relies on ECM degradative potential but also on efficient cell migration. Focal adhesions (FAs), which are integrin-linked protein complexes acting as mechanical linkers between the ECM and the actin cytoskeleton, play a major role in cell spreading and migration ,7. InterWhile the actin cytoskeleton is a major player in the regulation of the assembly and dynamics of invadopodia and FAs, microtubules (MTs) and intermediate filaments play more specific roles ,13,14. MWhile the contribution of MTs and +TIPs in FA dynamics are well documented, much less is known concerning their potential function in invadopodia regulation and ECM degradation. We, therefore, investigated the impact of EB1, the central hub in the +TIP network, on invadopodia dependent ECM degradation in breast cancer cell models. We observed that EB1 silencing enhanced the degradation of a reconstituted matrix, by increasing the number of invadopodia. Depletion of the APC and ACF7 +TIPs led to a similar phenotype, whereas depletion of CLASP1/2 and CLIP170 had no impact on invadopodia. Analysis of Src and FAK activities suggest that EB1, APC, and ACF7 control the balance between FAs and invadopodia by promoting FAK activation, favoring Src localization at FAs, and thus negatively impacting invadopodia formation.2 and checked regularly for mycoplasma contamination. Stable MDA-MB-231 cell lines were generated by transfection of the mCherry plasmid constructs by Lipofectamine 2000 , selection with 1 mg/mL geneticin, and sorting by flow cytometry to select cell populations expressing moderate levels of the transgenes. For TGF-β-induced EMT, MCF10A cells were treated for 6 days with 10 ng/mL human recombinant TGF-β derived from HEK293 cells (Peprotech). GM6001 was purchased from Calbiochem (Sigma-Aldrich). Paclitaxel and Nocodazole were purchased from Sigma-Aldrich.Human MDA-MB-231 breast cancer cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) , supplemented with 10% fetal bovine serum . Normal human MCF10A breast epithelial cells (ATCC/LGC Standards) were cultured in DMEM/F12 (ThermoFisher) supplemented with 5% fetal bovine serum, 20 ng/mL EGF , 0.5 µg/mL hydrocortisone , 100 ng/mL cholera toxin (Sigma-Aldrich), and 10 µL/mL insulin (Sigma-Aldrich). Cells were cultured at 37 °C in a humidified atmosphere with 5% COSiRNAs were home-designed and purchased from ThermoFisher. The siRNAs targeting human EB1 (siEB1_1: NM_012325-1262), APC (NM_000038-4610 or -9773), ACF7 (NM_012090-3015), CLASP1 (NM_015282-709), CLASP2 (NM_015097-1294), and LacZ (M55068-427) used as a negative control, have been previously described in Zaoui et al., as well as their efficiency and specificity  [29]. ThWild type FAK and FAK Y397F were amplified by PCR from pAcGFP1-Hyg-C1-FAK wild type and Y397F  with appropriate primers . PCR proRabbit antibodies against Tks5 (M-300), CLASP2 (H-40) and CLASP1 (H-70), mouse antibody against CLIP170 (F3) and rat antibody against EB1 (KT51) were purchased from Santa-Cruz Biotechnology, Dallas, TX, US. Mouse antibody against Cortactin (clone 4F11) was purchased from Millipore. Rabbit antibody against mCherry was from Abcam, Cambridge, United Kingdom. Rabbit antibody against Phospho-FAK (Tyr397) was from ThermoFisher. Mouse antibodies against EB1 and Src (L4A1) and rabbit antibody against p-Y416-Src were from Cell Signaling Technology, Danvers, MA, USA. Anti-α-Tubulin  was from Sigma-Aldrich. Rabbit antibody against FAK (AHO0502) was from BioSource, ThermoFisher.Protein extracts were prepared, quantified, and denatured in SDS loading buffer before running the samples on Novex NuPAGE Bis-Tris 4–12% gels using a MOPS based running buffer . Proteins were then transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies followed by incubation with secondary antibodies coupled to HRP. Antibodies were detected by chemoluminescence. Signal quantification was performed using the Gel analyzing tool from Image J 1.53c.Coverslips coated with Oregon Green 488-conjugated gelatin  were prepared as described in Thuault et al. . After st-test, the unpaired one-tailed t-test, with Welch correction, and the Mann–Whitney test was used. Graphs were plotted using Prism to show the mean and SEM. The mean of each individual experiment was also reported. p-values were indicated on the graph as * p < 0.05, ** p < 0.01, *** p < 0.001.GraphPad Prism software was used to perform all statistical analyses. To determine significant differences between data groups, the paired We investigated the contribution of EB1 to breast cancer cells ability to form invadopodia and degrade the ECM. For that purpose, invasive MDA-MB-231 breast cancer cells transfected with either a control siRNA (siLacZ) or siRNAs directed against EB1 were seeded on an artificial ECM composed of fluorescently-labeled gelatin for 4 h A. InvadoTo strengthen our observations, we investigated the impact of EB1 depletion on the degradative potential of another cellular model, MCF10A normal breast epithelial cells that had undergone epithelial to mesenchymal transition (EMT) following TGF-β treatment, a process inducing migratory and invasive properties. As previously described , normal Overall, these experiments demonstrate that EB1 inhibits ECM proteolysis by restraining invadopodia formation in breast cancer cells. This is in contrast to osteoclasts, in which EB1 is required for podosome belt formation, podosomes being degradative machinery formed by some normal cells, sharing similarities, but also major differences, with invadopodia ,32. HoweEB1 interacts with SxIP-containing proteins, such as CLASP1/2, APC, and ACF7, and CAP-Gly-containing proteins, such as CLIP170 and p150glued in order to regulate the dynamics and stability of MTs ,22. We, Our results uncovered three novel negative regulators of invadopodia belonging to the family of +TIPs factors: EB1, APC, and ACF7. The fact that APC and ACF7 silencing leads to strikingly similar effects to EB1 knockdown suggests that APC and ACF7 might function in collaboration with EB1 to restrain invadopodia formation. EB1, APC, ACF7, CLASP1/2, and CLIP170 have all been implicated in migration-promoting pathways ,35,36,37Currently, few proteins have been reported as negative regulators of invadopodia function. Among them, the Rho GTPase RhoG and its guanine exchange factor SGEF, through the regulation of paxillin phosphorylation, and Ezrin, which promotes Calpain protease activity, favor invadopodia disassembly and thereby restrain ECM degradation ,39,40. FSrc is a critical element of invadopodia formation ,42. We, The decreased activation of FAK in EB1-, APC-, and ACF7-depleted cells could, therefore, be the cause of enhanced invadopodia formation in our cellular model. In order to explore this hypothesis, we evaluated the impact of EB1 silencing on invadopodia formation upon restoration of normal FAK expression levels and/or activity. We generated and selected MDA-MB-231 cells stably expressing wild type FAK fused to mCherry, cells expressing a mutant form of FAK that cannot be phosphorylated on Tyr397 (FAK Y397F), and cells expressing a control vector (mCherry) C. We anaConsidering the inter-connection between +TIPs and the MT cytoskeleton, our observation that depletion of EB1, APC, or ACF7 increases ECM degradation appears in contradiction with previous reports indicating that invadopodia, in similar experimental settings, were insensitive to MT-stabilizing or -destabilizing agents in breast cancer cells ,45. HoweFurthermore, we identified that the +TIPs EB1, APC, and ACF7 are negative regulators of invadopodia formation and function, whereas CLASP1/2 and CLIP170 are not. Globally these factors are required for persistent directional cell migration ,36,37,51In conclusion, we demonstrate that EB1 restricts ECM degradation via the regulation of FAK activity. Previous studies show that EB1 is required for persistent directional migration ,52. Alth


Hepatocellular carcinoma (HCC) is one of the most lethal cancers globally. Hepatitis B virus (HBV) infection might cause chronic hepatitis and cirrhosis, leading to HCC. To screen prognostic genes and therapeutic targets for HCC by bioinformatics analysis and determine the mechanisms underlying HBV-related HCC, three high-throughput RNA-seq based raw datasets, namely GSE25599, GSE77509, and GSE94660, were obtained from the Gene Expression Omnibus database, and one RNA-seq raw dataset was acquired from The Cancer Genome Atlas (TCGA). Overall, 103 genes were up-regulated and 127 were down-regulated. A protein–protein interaction (PPI) network was established using Cytoscape software, and 12 pivotal genes were selected as hub genes. The 230 differentially expressed genes and 12 hub genes were subjected to functional and pathway enrichment analyses, and the results suggested that cell cycle, nuclear division, mitotic nuclear division, oocyte meiosis, retinol metabolism, and p53 signaling-related pathways play important roles in HBV-related HCC progression. Further, among the 12 hub genes, kinesin family member 11 (KIF11), TPX2 microtubule nucleation factor (TPX2), kinesin family member 20A (KIF20A), and cyclin B2 (CCNB2) were identified as independent prognostic genes by survival analysis and univariate and multivariate Cox regression analysis. These four genes showed higher expression levels in HCC than in normal tissue samples, as identified upon analyses with Oncomine. In addition, in comparison with normal tissues, the expression levels of KIF11, TPX2, KIF20A, and CCNB2 were higher in HBV-related HCC than in HCV-related HCC tissues. In conclusion, our results suggest that KIF11, TPX2, KIF20A, and CCNB2 might be involved in the carcinogenesis and development of HBV-related HCC. They can thus be used as independent prognostic genes and novel biomarkers for the diagnosis of HBV-related HCC and development of pertinent therapeutic strategies. Liver cancer is the most common type of cancer across the world, accounting for 8.2% of cancer deaths . HepatocIn recent years, the exploration of genes related to carcinogenesis and development of HCC by bioinformatics methods have been increasing. TP53 , UBE3C , SHP-1 , COL1A1 1. GSE94660  and GSE77509  were established using GPL16791 Illumina HiSeq 2500 (Homo sapiens), while GSE25599  was established using the GPL9052 Illumina Genome Analyzer. RNA-seq raw data and clinical data of 50 normal samples and 374 HCC samples2 were downloaded from TCGA3. For the validation of independent prognostic genes, a dataset named LIRI-JP4, including 202 normal and 243 HCC tissue samples, was downloaded from the International Cancer Genome Consortium (ICGC)5. For comparing the expression levels of independent prognostic genes between HCV- and HBV-related HCC, the GSE69715 dataset [66 normal and 37 HCV-related HCC tissue samples established using GLP570 (HG-U133_Plus_2)] was downloaded from the GEO database.For screening DEGs, three high-throughput RNA-seq-based raw datasets, namely GSE25599 , GSE94662(FC)| ≥ 1.0, p-value ≤ 0.05, and FDR ≤ 0.05 were set as the cutoff criteria for DEGs screening after background correction and data normalization. Overlapped DEGs among GSE25599, GSE77509, GSE94660, and TCGA were identified using the VennDiagram R package  analysis, which involved three categories, namely molecular functions (MF), cellular components (CC), and biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed with the threshold of FDR-value < 0.05 using the clusterProfiler R package , which f6  network of DEGs was constructed using STRING6  and visu6 . DEGs th6  and Cyto6 . Subnets7 and differences in survival rate were evaluated with a log-rank test threshold of p-value < 0.05. To evaluate the accuracy of the survival curves, receiver operating characteristic (ROC) curves were then constructed using the “survival ROC” R package  for survival and ROC curve analyses; for this purpose, we used the survival R package, survival ROC R package, and Perl. Ultimately, a meta-analysis of the independent prognostic genes in Oncomine10 , a cance10 .11 R software. The correlation coefficient (Cor), ranging from −1 (perfect negative correlation) to +1 (perfect positive correlation), indicated how closely data in a scatterplot were arranged along a straight line. p-value < 0.05 for the coefficients indicates a statistically significant relationship.To analyze the potential relationships among the four independent prognostic genes in HCC occurrence and development, TCGA dataset was subjected to correlation analyses using the corrplot12, cowplot13, and ggpubr14 package. The relative expression levels  of these four genes in the tumor tissues comparing with normal tissues were calculated. Wilcoxon test was carried out between the HCC and normal tissues. p-value < 0.05 indicate statistical significance.Since viral Hepatitis B and Hepatitis C are the most commonly implicated risk factors for HCC, to compare the expression levels of the independent prognostic genes between HCV-related HCC and HBV-related HCC, the GSE69715 dataset for HCV and the GSE94660 dataset for HBV were analyzed using gglpot22(FC)| ≥ 1.0, p-value ≤ 0.05 and FDR ≤ 0.05. In total, 230 DEGs were overlapping among the four datasets, of which 103 were up-regulated  were extracted to construct a subnet ; however, multivariate Cox regression analysis suggested that only KIF11, TPX2, KIF20A, and CCNB2 were independent prognostic genes in case of patients with HCC .Further, univariate and multivariate Cox regression analyses were performed to analyze their independent prognostic importance in patients with HCC. As indicated in The Cancer Genome Atlas dataset of normal and HCC tissue samples were subjected to Wilcoxon test; KIF11, TPX2, KIF20A, and CCNB2 were found to have higher mRNA expression levels in HCC than in normal tissue samples . Furtherp-value < 0.05 and AUC ≥ 0.6). Notably, as indicated in p-value < 0.05). Correlation analyses  of KIF11, TPX2, KIF20A, and CCNB2 in the HBV-related HCC dataset ranges from 1.38 to 2.66, while log2(FC) of these genes in the HCV-related HCC dataset ranges from 0.09 to 0.35. Although all of the p-values in the p-value < 0.05), which are statistically significant, we do not believe that they are biologically significant, because the expression level of these four genes in HCV-related HCC showed only a minor increase as compared with that in HBV-related HCC.To deternmine whether KIF11, TPX2, KIF20A, and CCNB2 are specific to HBV-induced HCC comparing with HCV-induced HCC, HCV-related HCC (GSE69715) and HBV-related HCC (GSE94660) datasets were used to analyze the relative expression levels  of these 4 genes in HCC and normal tissue samples. As indicated in Hepatocellular carcinoma is the most common malignant tumor of the liver. The etiological factors of HCC include hepatitis B or C, aflatoxin, alcohol, and metabolic disorders. In HBV endemic areas, chronic hepatitis B infection has been verified to be closely associated with HCC carcinogenesis . IdentifGene Expression Omnibus is a public functional genomics database and includes a large repository of high-throughput, next-generation sequencing results and related information for over 200 organisms . In the A total of 230 DEGs were identified, of which 127 were down-regulated and 103 were up-regulated, and a PPI network was then constructed. Twelve hub genes – BUBIB, CCNA2, CCNB1, CDC20, CDK1, KIF11, KIF20A, PLK1, TOP2A, MAD2L1, TOP2A, and TPX2 – were identified according to a degree score of >45. GO and KEGG pathway enrichment analyses of the 230 DEGs and 12 hub genes suggested that HBV-related HCC occurrence and development are associated with cell cycle, nuclear division, mitosis, p53 pathway, oocyte meiosis, retinol metabolism, and organic acid catabolism. Cell cycle abnormality evidently has a key role during the process of liver cancer , and cycTo analyze the prognosis and clinical significances of the 12 hub genes in HBV-related HCC, clinical data from TCGA were used for survival and ROC curve analyses. It was found that patients in whom the expression levels of the 12 hub genes were up-regulated showed worse survival rate, indicating their prognostic value for HCC. To further analyze the prognostic value of these genes, univariate and multivariate Cox regression analyses were performed using the 12 hub genes and found that KIF11, TPX2, KIF20A, and CCNB2 might be independent prognostic genes and potential targets for the diagnosis of HBV-related HCC. In addition, it was demonstrated that the expression levels of these four genes were higher in HCC than in normal tissue samples, and their expression levels were also higher at different stages of HCC than those in normal tissue samples. Data pertaining to patients with HCC from the ICGC database further validated that KIF11, TPX2, KIF20A, and CCNB2 were associated with worse survival rates in patients with higher gene expression levels. Oncomine analysis demonstrated that the expression levels of these genes were still higher in different patients with HCC.Through continuous data filtering using different procedures and different sources of data, the number of candidate genes reduced, making our results more credible. Besides, correlation analyses of KIF11, TPX2, KIF20A, and CCNB2 indicated the potential relationships among them, and suggested that they together promote the occurrence and development of HCC.The activation of KIF20A–Gli2 axis has been reported to be crucial for hepatoma cell growth, indicating that KIF20A plays a vital role in the development of liver cancer . FurtherDatasets of HCV-related HCC and HBV-related HCC were used to compare the expression levels of these four genes between HCV- and HBV-induced HCC. HCV-related HCC showed only a minor increase in the expression levels as compared with HBV-related HCC, indicating that KIF11, TPX2, KIF20A, and CCNB2 might be specific to HBV-induced HCC. But the underlying mechanisms how these four genes may induce the HBV-related HCC need to be further elucidated.In conclusion, KIF11, TPX2, KIF20A, and CCNB2 seem to play a key role in HBV-related HCC. However, further studies are warranted to explore the mutual influence of these genes and HBV on HBV-related HCC carcinogenesis. Further studies should also identify whether these four genes are induced by factors other than HBV infection in patients with HCC and whether HBV infection itself causes aberrant expression of these genes and promotes HCC progression. Whether HBV-encoded proteins, such as HBV X protein, can interact with intracellular proteins via these four genes and lead to HCC remains to be elucidated.Our findings suggest that KIF11, TPX2, KIF20A, and CCNB2 are involved in the carcinogenesis and development of HBV-related HCC. Thus, they can be used as independent prognostic genes for patients with HBV-related HCC and also as novel biomarkers for the diagnosis of HBV-related HCC and development of pertinent therapeutic strategies.http://www.ncbi.nlm.nih.gov/geo) using the accession numbers GSE25599, GSE94660, GSE77509, and GSE69715, and TCGA (https://cancergenome.nih.gov/), and the ICGC (https://dcc.icgc.org/releases/current/Projects/LIRI-JP) database.The data analyzed in this manuscript can be downloaded from the NCBI Gene Expression Omnibus database (W-NC and XL conceived and designed the investigation. X-CZ and LZ analyzed the data and drafted the manuscript. W-JL, LA, Z-ML, and WK conducted statistical analyses. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oral lichen planus (OLP) is a chronic inflammatory oral mucosal disorder mediated by T cells, with a multifactorial etiology. Hashimoto's thyroiditis (HT) is a common autoimmune disease characterized by hypothyroidism. Although many clinical studies conducted over the past several decades have reported the cooccurrence of OLP and HT, the underlying mechanism remains unclear. This review summarizes potential mechanisms that might be involved in the cooccurrence of OLP and HT. We find that OLP and HT share a common or overlapping pathogenesis in terms of immune, heredity, environmental, and hormonal factors, which might cause cooccurrence. Furthermore, considering the latency of HT, a routine screen for thyroid diseases, particularly HT, is suggested for confirmed OLP patients. Oral lichen planus (OLP) is a relatively common chronic inflammatory disease of the oral mucosa affecting 0.5–2% of the population; middle-aged and elderly female populations are more commonly affected . AlthougThe correlation between OLP and thyroid disease was first reported in 1994 . RobledoThe histopathological features of OLP and HT indicate that the cell-mediated immune response plays an important role in their pathogenesis , 14. TheIn HT, cell- and antibody-mediated humoral immune responses against thyroid gland self-antigens cause thyrocyte destruction, subsequently resulting in hypothyroidism. Human thyroid autoantibodies include thyrotropin receptor antibody (TRAb), thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb), and anti-sodium iodide symporter (NIS) antibodies. TGAb and TPOAb are present in nearly all HT patients. TPOAb are the best serological marker for diagnosing HT, occurring in approximately 95% of HT patients, while TGAb are less sensitive and less specific .In the past decades, many studies have reported a significant link between thyroid diseases, especially autoimmune thyroid diseases (AITD) and autoimmune skin disease . In AITDγ and tumor necrosis factor- (TNF-) α [CD8+ T cells are an important branch of the adaptive immune system; they contribute to the clearance of intracellular pathogens and provide long-term protection . These f(TNF-) α .Both antigen-specific and nonspecific mechanisms may be involved in OLP pathogenesis. A key element of pathogenesis is that T lymphocyte-mediated cytotoxicity leads to keratinocyte apoptosis. Most T cells adjacent to damaged basal keratinocytes are CD8+ T cells. CD8+ cytotoxic T cells can be directly activated by antigens binding to major histocompatibility complex- (MHC-) 1 on keratinocytes and subsequently release chemokines that attract additional lymphocytes and other immune cells into the developing OLP lesion . HT pathγ, TNF-α, and interleukin- (IL-) 2, which mediate cellular immune responses. Th2 cells predominantly produce cytokines, such as IL-4, IL-5, IL-10, and IL-13, which mediate humoral immune responses. The IFN-γ/IL-4 ratio is a simple and direct indicator of the Th1/Th2 balance. Previous studies have shown that both IFN-γ and IL-4 levels are increased in the serum and lesion tissues of OLP patients compared with healthy controls and that the IFN-γ/IL-4 ratio is also significantly increased, indicating that OLP is Th1-biased [γ and lower IL-4 serum levels, further indicating the Th1 bias in HT [CD4+ T helper (Th) cells can differentiate into several distinct subsets, including Th1, Th2, Treg, Th17, Th22, and Tfh, which produce specific cytokines . Th1 cel1-biased . Other s1-biased . HT patias in HT . Collectα and IL-2 and subsequently promote inflammation [Th17 cells are an independent subset of helper T cells distinct from the development of Th1 and Th2 cells. Th17 cells can produce IL-17 and play a critical role in autoimmune diseases. IL-17 can induce keratinocytes, fibroblasts, endothelial cells, and macrophages to secrete TNF-ammation . Furtherammation . Correspammation . IL-17 cTh22 cells are a novel CD4+ T subset that mainly secretes IL-22, which is associated with many autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis . There iβ and IL-10, via direct or indirect cell contact, and subsequently inhibits the immune response [The regulatory T (Treg) cell subset produces cytokines, including transforming growth factor- (TGF-) response . The mairesponse , 42. Oneresponse . Accordiresponse . This suresponse . Howeverresponse  and HT [response  patientsresponse , we hypoFollicular helper T (Tfh) cells are a newly identified T helper cell subset, which can promote the generation of antigen-specific B cells by producing IL-21 ; these cγ-induced chemokine, participates in the pathogenesis of many autoimmune diseases by binding to CXCR3 [Chemokines are proinflammatory cytokines, which are classified into C, CC, CXC, and CX3C subfamilies according to their N-terminal cysteine motifs. Chemokines play a key role in the selective recruitment of T cells via chemokine receptors . CXCL10,to CXCR3 . CXCL10 to CXCR3 . Previouto CXCR3 ; furtherto CXCR3 . Severalto CXCR3 . Anotherto CXCR3 . RANTES to CXCR3 . RANTES to CXCR3 , suggestIn summary, keratinocyte expression of TSHR and TG, which can be recognized and targeted by TRAb and TGAb in HT patients, is the basis for the cooccurrence of OLP and HT . In addiEnvironmental factors play an important role in the pathogenesis of OLP and HT. However, the relationship between smoking and OLP remains unclear. Although some studies have indicated that smoking is unrelated to OLP , 62, NeuHelicobacter pylori [Infectious factors can also result in immune abnormalities . Hepatitr pylori , 75 with2D, derived from 25(OH)D, binds to VDR and regulates the proliferation and differentiation of immune cells, leading to reduced lymphocyte activity , and reduces proinflammatory cytokine expression [2D and 25(OH)D serum levels compared to levels in the controls [Vitamin D has a beneficial effect on the immune system, and inadequate vitamin D intake is involved in many autoimmune diseases . Macrophpression . Previoucontrols . Currentcontrols . Lack ofMany animal and human studies have shown that stress can induce a variety of immunological changes that are associated with several autoimmune diseases –85. ReceThe role of estrogen and progesterone (Pg) in autoimmune disorders, such as multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis, has been established , 91. EpiHuman leukocyte antigen (HLA) is the main histocompatibility complex in humans; HLA polymorphism is a key genetic factor underlying many diseases . HLA-encThis review summarizes and discusses the mechanisms underlying the cooccurrence of these two diseases, which might be related to a range of immune, environmental, endocrine, and genetic factors . These f