Human rotavirus remains a major cause of gastroenteritis worldwide despite the availability of effective vaccines. In this study, we investigated the genetic diversity of rotaviruses circulating in Lebanon. We genetically characterized the VP4 and VP7 genes encoding the outer capsid proteins of 132 rotavirus-associated gastroenteritis specimens, previously identified in hospitalized children (<5 years) from 2011 to 2013 in Lebanon. These included 43 vaccine-breakthrough specimens and the remainder were from non-vaccinated subjects. Phylogenetic analysis of VP4 and VP7 genes revealed distinct clustering compared to the vaccine strains, and several substitutions were identified in the antigenic epitopes of Lebanese specimens. No unique changes were identified in the breakthrough specimens compared to non-breakthroughs that could explain the occurrence of infection in vaccinated children. Further, we report the emergence of a rare P[8] OP354-like strain with a G9 VP7 in Lebanon, possessing high genetic variability in their VP4 compared to vaccine strains. Therefore, human rotavirus strains circulating in Lebanon and globally have accumulated numerous substitutions in their antigenic sites compared to those currently used in the licensed vaccines. The successful spread and continued genetic drift of these strains over time might undermine the effectiveness of the vaccines. The effect of such changes in the antigenic sites on vaccine efficacy remains to be assessed. Reoviridae family, are non-enveloped triple-layered particles that encompass a genome of eleven double-stranded RNA (dsRNA) segments coding for six structural viral proteins  and five or six non-structural proteins (NSPs 1-5/6). RVAs are classified into G (glycoprotein) and P (protease-sensitive) genotypes based on the genes coding for the two outer capsid proteins, VP7 and VP4, respectively . Nonetheless, epidemiological studies show that at least six human RVA G genotypes  and three P genotypes  predominate globally in human infections (Rotavirus-associated gastroenteritis (RVGE) constitutes a major public health burden in both industrialized and developing countries . Rotavirs NSPs 1-/6. RVAs Velasquez et al. showed that natural RVA infections can protect against subsequent infections . These fCurrently, two live-attenuated oral RVA vaccines, RotaTeq® and Rotarix®, are licensed by the Food and Drug Administration (FDA) and approved by the Advisory Committee on Immunization Practices (ACIP) for human use , 10. RotRotavirus is continuously evolving . TherefoThe specimens were detected during a prospective, multicenter hospital-based RVA surveillance study that was conducted from January 2011 to June 2013. The study population included hospitalized children <5 years of age admitted with acute GE recruited at seven centers distributed over North, Central, and South Lebanon . Subject* region) was carried out using previously described primers (* region (945 bp) of the VP4 gene was amplified using primers designed specifically for this study to cover all epitopes present in the VP5* region: VP5-F (5′-GGTGGAAGARTATGGACATTTCA-3′) and VP5-R (5′-CATAATTGGAGTCTGATAATCATC-3′).Viral RNA was extracted from 10% (w/v for solid and v/v for liquid) stool suspensions for all RVA enzyme immunoassay positive samples using a QIAamp Viral RNA Mini Kit  according to manufacturer's instructions. The extracted RNA was denatured at 97°C for 5 min and reverse transcription followed by polymerase chain reaction (RT-PCR) was carried out using the Qiagen OneStep RT-PCR Kit  with the following thermal cycling conditions: initial reverse transcription step at 42°C for 30 min, followed by initial denaturation at 95°C for 15 min, 35 cycles of amplification and a final extension at 72°C for 7 min. Amplification of the complete VP7 gene and partial VP4 gene  for automated genotyping of RVA and confirmed using the Basic Local Alignment Search Tool (BLAST) available on GenBank database (http://blast.ncbi.nlm.nih.gov/). To determine the extent of intra- and inter-genotypic diversity among VP7 and VP4 genes, phylogenetic trees were constructed using the neighbor-joining (NJ) algorithm, and evolutionary distances were computed using the Maximum Composite Likelihood method. To further investigate the genetic relatedness of the isolates, VP7 and VP4 gene sequences were compared with sequences of Rotarix® and RotaTeq®, sequences of potential candidate RV vaccine strains, namely the human neonatal strains RV3 . Nucleotide sequences were assembled using ClustalW tool included in BioEdit Sequence Alignment Editor v7.2.5. The corresponding G and P genotypes were assigned by RotaC v2.0 software .The N-linked glycosylation sites  were predicted using the NetNGlyc 1.0 server  of each participating center before initiating the study. Specimens were collected upon obtaining a signed consent from the legal guardian/parent approving participation in the study.JN849114 and JN849113), RotaTeq® , RV3 (FJ998278) and 116E (L14072). VP4 and VP7 sequences were submitted to GenBank under accession numbers: VP4  and VP7 sequences .Reference and potential candidate vaccine strains were retrieved from GenBank: Rotarix® (Forty-three RVA specimens from breakthrough infections and 89 specimens from non-breakthrough infections were included in the study. Thirty-five (83%) of the breakthrough specimens were from subjects vaccinated with Rotarix®, seven (15%) with RotaTeq®, and one from an undocumented vaccine type. These strains included 8 G1P[8], 15 G2P[4], 1 G2P[8], 7 G4P[8], 11 G9P[8], and 1 G9P[4]. The average Vesikari score was reported to be 13.8±2.7 for the breakthrough cases  comparedn = 4) from 2011 to 2012, which clustered with an Indian strain (mani-375) with 98.3–98.7% nucleotide similarity. Lebanese G1 RVA specimens exhibited higher nucleotide (93.9–97.9%) and amino acid similarities (94.3–97.8%) to G1 strain of Rotarix® than that included in RotaTeq® (91.1–93.0% nucleotide and 93.1–95.3% amino acid identities)  within the G1-genotype. The Lebanese strains belonged to lineages 1, 4, and 5; whereas, Rotarix® and RotaTeq® G1 sequences belonged to lineages 1 and 2, respectively. Two G1 non-breakthrough specimens from 2011 clustered in lineage 1 along with the G1 of Rotarix® and other global strains, while the remaining specimens belonged to non-vaccine lineages . Lineagentities) .n = 12) clustered with strains from Australia, Canada, USA, Russia, Uruguay, Malawi, and Far East countries with 95.8–99.9% nucleotide identity. Strains in G2-lineage 3 (n = 22) clustered with strains from Europe, Thailand, and Australia with 96.0–99.4% nucleotide similarity. Collectively, the Lebanese G2 RVA specimens showed 93.5–94.3% nucleotide and 94.6–95.6% amino acid identities with the G2 strain of RotaTeq®.Lebanese G2 RVA specimens clustered in two lineages (2 and 3) that were distinct from G2 of RotaTeq® (lineage 1). Strains in G2-lineage 2 .n = 20) and G9-lineage 4 (n = 11) clustering with RVA strains from Europe, Ethiopia, Iran, and Japan with 98.4–99.5% nucleotide similarity. The G9 RVA specimens had 87.8–88.9% nucleotide and 91.5–92.4% amino acid similarities with the vaccine candidate strain G9-116E  belonged to lineage 3 clustering with strains from Europe with 88.6–99.8% nucleotide similarity. Lebanese strains belonging to P[8]-lineage 4 (n = 20) clustered with strains from the Middle East, Europe, Japan, Pakistan, Bangladesh, and South Africa with 96.9–99.2% nucleotide similarity. P[8]-lineage 5 was exclusively formed of non-breakthrough specimens (n = 14) while lineage 6 included only one breakthrough isolate. In contrast to the VP7 gene, pairwise comparison revealed that the VP4 gene of P[8] breakthrough and non-breakthrough specimens shared slightly higher nucleotide and amino acid identities  to the P[8] of RotaTeq® than that included in Rotarix®   .n = 15) clustering in P[4]-lineage 3. This lineage did not include any strains from other countries. P[4]-lineage 1 included six of the Lebanese RVAs along with strains from the USA, Australia, Africa, and South Asia with 98.5–99.0% nucleotide similarity. The remainder Lebanese P[4] RVAs (n = 12) co-clustered in lineage 2 with strains from Europe, Australia, Japan, and South Africa with 98.5–99.0% nucleotide similarity.Lebanese P[4] RVA specimens were accommodated in three lineages with the majority  and one G1 (N049) specimens clustered among G9 specimens in P[8]-lineage 4 comprised mainly of G9 specimens. This cluster also included a G1P[8] specimen from Belgium (BE2003). This tight clustering pattern suggests an intra-genotypic reassortment between G1P[8], G2P[8] and G9P[8] strains involving at least the VP4 gene. Similarly, G9 H017 and A095 specimens clustered tightly with G2 specimens in P[4] lineages 1 and 2, respectively, indicative of another potential reassortment event . P[4] liThe VP7 protein plays a crucial role in stabilizing the viral outer capsid and is an important target for protective antibodies. Comparative studies of the VP7 protein indicated that substitutions occurring in two structurally defined antigenic epitopes [7-1 (7-1a and 7-1b) and 7-2], encompassing 29 amino acid residues, permit escape from neutralization by monoclonal antibodies (mAbs) , 25. mAbAlignment of the deduced amino acid sequences of the VP7 protein of the Lebanese breakthrough and non-breakthrough specimens with the vaccine strains showed that only four amino acids were fully conserved amongst all the detected genotypes . The G1 All G2 breakthrough and non-breakthrough specimens possessed three substitutions in their VP7 antigenic epitopes when compared to G2 of RotaTeq®: A87T and D96N located in the 7-1a epitope and S213D in the 7-1b epitope. In addition, 23 of the 34 G2 specimens displayed an additional antigenic site substitution S242N in the 7-1b region; and two G2 breakthrough specimens possessed a unique change, S148F, in the 7-2 region . The G2 n = 12) featured an additional S221G substitution in the 7-2 epitope region . These three N-glycosylation sites are conserved in the G2 of RotaTeq® vaccine. The VP7 of the G4 specimens possessed only the potential N-linked glycosylation site at position 69, which is also present in the G4 component of RotaTeq® vaccine. Noteworthy, all the Lebanese G4 strains exhibited an insertion of an asparagine (N) residue at position 76 adjacent to the mentioned N-glycosylation site in the hydrophilic region which was in congruency with global strains clustered in the same lineage. This substitution was also observed in all the G4 strains from other countries. Of note, eight Lebanese G9 RVA specimens, of which two are breakthroughs, possessed a unique alanine to valine substitution at position 68 (A68V) adjacent to the N-linked glycosylation site spanning residues 69-71, previously speculated to potentially alter the glycosylation and antigenicity of VP7 . Interes* and VP5*, respectively  and five (5-1 to 5-5) epitopes, respectively, which are targets for neutralizing mAbs. Neutralizing antibodies directed against the 5-1 epitope region seem to block virion membrane penetration  of the VP7 protein and very few were mapped to the spike's body or VP5*  shared a similar VP4 antigenic residues profile compared to the P[8] strains of the vaccines. The breakthrough G2P[4] specimen (A081) possessed an aspartic acid (D) at position 196, similar to RotaTeq®, while the remainder Lebanese specimens had an asparagine (N) like Rotarix® .Currently, two RV vaccines Rotarix® and RotaTeq® are available worldwide for the prevention of RV infections with proven safety profiles , 12, 37.Vaccine use can impose a selective pressure on circulating strains, which could result in a shift toward otherwise less common genotypes or selection of mutant viruses that are not effectively neutralized. For instance, the introduction of Rotarix® in Belgium was associated with a decrease in the vaccine homotypic strains . SimilarIn Lebanon, where the majority of infants receive Rotarix®, G1P[8] was the dominant genotype post-vaccine introduction compared to G4P[8] in the years before vaccine introduction , 42. In *; all of which correlated with neutralization-resistance against a panel of mAbs and polyclonal sera  of the G2 VP7 gene, G2 strains have been rapidly evolving and spreading during the last decade (Considering RVA frequency by VP7 and VP4 combination, G2P[4] was the most frequently detected RVA breakthrough genotype in Lebanon . A highet decade , 50. Glot decade , with lit decade –52. MoreThe VP4 gene is more likely to be influenced by negative selection due to the important structural and functional roles it plays, including attachment, penetration, and maturation. Therefore, it is less diverse, compared to the VP7 , 53. In Glycosylation of the RV glycoproteins modifies their antigenicity and promotes their resistance to neutralizing mAbs . Gain orA new strain of RVA, known as OP354-like, possessing a genetically divergent P[8] VP4 has recently emerged worldwide . Zeller This is the first report on RVA genetic diversity in Lebanon. RVAs circulating in Lebanon including breakthrough strains are diverse and possess many substitutions in the antigenic epitopes that were previously associated with escape from neutralizing mAbs. However, a limitation of this study was the lack of serum specimens from vaccinated and non-vaccinated patients to assess the immunologic status of these patients and explain the potential cause of breakthrough infections. The Lebanese strains were similar to RVAs circulating in other countries. We also report for the first time the spread of the recently emerging and rare OP354-like P[8] RV genotype to the Middle East, including Lebanon. Continuous monitoring of circulating RVAs in parallel with the assessment of their vaccine matching using genotype-specific antibodies and neutralization assays are warranted to maintain the effectiveness of current vaccines.The datasets generated for this study can be found in the GenBank.The studies involving human participants were reviewed and approved by the Institutional Review Board, American University of Beirut. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.HZ, GD, and GM conceived and designed the study. HH, LR, and AS conducted the experiments. ZA and MH collected the samples. HH and HZ analyzed the data and wrote the manuscript. All authors revised and approved the final draft.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.


XRCC5 (3R/2R/1R/0R); two single nucleotide polymorphisms (SNPs); XRCC6 (C/G) and XRCC7 (G/T)] with DNA damage, repair and mRNA expression in human PBMCs in dose and adaptive response studies. Genomic DNA extracted from venous blood samples of 20 random healthy donors (16 adaptive and 4 non-adaptive) and genotyping of NHEJ repair genes was carried out using PCR amplified length polymorphism.Radio-adaptive response (RAR) is transient phenomena, where cells conditioned with a small dose (priming) of ionizing radiation shows significantly reduced DNA damage with a subsequent high challenging dose. The role of DNA double strand break repair gene polymorphism in RAR is not known. In the present study attempt was made to find out the influence of NHEJ repair gene polymorphisms [a VNTR; XRRC5 (3R/2R/1R/0R), XRCC6(C/G) and XRCC7 (G/T) with DNA damage. Donors having genotypes with 2R allele at XRCC5 showed significant positive correlation with mRNA expression level  for dose response. Genotypes C/C and C/G of XRCC6 showed a significant positive correlation (P = 0.0001), whereas, genotype T/T of XRCC7 showed significant negative correlation  with mRNA expression.The dose response study revealed significant positive correlation of genotypes at XRCC6 showed significantly higher (P < 0.05) mRNA expression level in primed cells suggesting their role in RAR. In addition, NHEJ repair gene polymorphisms play crucial role with radio-sensitivity and RAR in human PBMCs.Interestingly, adaptive donors having C/G genotype of  Environmental exposures such as chemical and physical mutagens including ionizing radiation may (IR) pose concern to human health. However, it is important to understand the underlying biological mechanisms regarding the individual susceptibility to radiation exposure. IR induces a variety of DNA damages/lesions such as single strand breaks (SSBs), double strand breaks (DSB) and base damages in human cells in addition to the basal endogenous DNA damages produced by oxidative stress. Radiation induced SSBs and oxidative DNA damages produced in a cell are large in number per 1.0 Gy of low Linear energy transfer (LET) IR, as compared to DNA DSBs, which are very few in number , 3. Thes0/G1 phase of the cell cycle  with respect DNA damage, DNA repair capacity (DRC) and mRNA expression and their influence in RAR.The objective of the present study was to find out the correlation of genotypes observed at three NHEJ repair gene polymorphisms  was carried out using SYBR green based real time q-PCR for dose response, post irradiation time point kinetics and RAR studies. Details of the primer sequences and PCR cycling conditions were as described elsewhere [. The results are expressed in normalized ratio as described by Pfaffle et al. (2001) [Total RNA was extracted and cDNA was prepared from irradiated as well as sham irradiated control PBMCs. Analysis of mRNA expression pattern of the NHEJ repair genes [lsewhere . The res. (2001)  and the Approximately 2 ml of venous blood samples were collected in EDTA containing vaccutainers from each donor. Genomic DNA was extracted from whole blood using non-enzymatic salt precipitation method . DNA wasXRCC6 (C/G), XRCC7 (G/T) and a VNTR at XRCC5 (3R/2R/1R/0R). The SNPs of XRCC6 (C/G), and XRCC7 (G/T) polymorphism were analyzed by PCR amplified restriction fragment length polymorphism (PCR-RFLP). The VNTR at XRCC5 (3R/2R/1R/0R) polymorphism was studied by PCR amplified fragment length polymorphism (Amp-FLP). All the PCR reactions were performed on Master cycler gradient thermocycler  in a final volume of 25 μl containing locus specific primers (5 picomoles of each primer), 50 ng genomic DNA, 1.5 mM MgCl2, 200 μmoles each dNTPs and 0.5 unit of Taq DNA Polymerase. Primer sequences and PCR conditions used for polymorphism study are given in Table Genetic polymorphisms were studied at selected SNPs for C5 3R/2R/R/0R. TheXRCC6(C/G) and XRCC7(G/T) polymorphisms, respectively. Undigested and restriction enzyme (RE) digested PCR product sizes for XRCC5 (0R/1R/2R/3R), XRCC6 (C/G) and XRCC7 (G/T) polymorphisms are given in Table XRCC5, XRCC6 and XRCC7) are given in Fig. Approximately 8 μl of amplicons were digested overnight with BanI and PvuII restriction endonucleases  at 37 °C for XRCC5 0R/R/2R/3R, P ≤ 0.05 was set for significance level.Linear regression analysis was performed to study dose response at DNA damage. For mRNA expression analysis, the normalized ratio was calculated as described by Pfaffle (2001) . PearsonXRCC5 (KU80), XRCC6 (KU70) and XRCC7 (PRKDC) genes were carried out among 20 donors and the genotypes were correlated with the DNA damage, repair and transcriptional profile. Linear regression was carried out for dose response study at DNA damage and gene expression level at each of the genotypes to find out the association of polymorphisms at these above genes with DNA damage, mRNA expression and inter-individual variation if any. RAR study was also carried out in primed and non-primed cells of 16 adaptive and 4 non-adaptive donors, where genotypes observed at NHEJ polymorphisms were correlated with biological end points such as DNA damage, repair and mRNA expression.DNA polymorphism study of In the present study, the distribution of genotype frequencies at each of the polymorphisms was determined among 20 donors studied and correlated with DNA damage, repair and mRNA expression level.XRCC5 gene with the DNA damage among the 20 donors was studied. For XRCC5 (0R/1R/2R/3R) polymorphism, five genotypes  were observed among these 20 donors.The genotypic frequencies of XRCC5 at 0R/1R, 0R/2R, 0R/3R, 1R/1R, 1R/2R, 1R/3R, 2R/2R and genotypes were 5, 5, 0, 40, 30, 0, 20 and 0%, respectively, among the 20 donors studied  and repair pattern among these groups  showed increasing trend in mRNA expression level at all the doses as compared to other genotypes though not statistically significant (P = 0.285). Nevertheless, correlation analysis revealed that genotype (0R/0R) showed decreased XRCC5 mRNA expression level with increasing doses , whereas donors with genotype (1R/1R) showed positive correlation  between mRNA expression at XRCC5 andradiation doses studied. However, donors with genotype 2R allele showed positive correlation between mRNA expression level at XRCC5 and at different radiation doses studied  . Correlation analysis revealed variation in the level of mRNA expression of XRCC5 among the genotypes observed  with mRNA expression and post-irradiation time after 2.0 Gy. Donors with genotype (1R/1R) showed positive correlation  between mRNA expression of XRCC5 and post-irradiation time points. As observed from the dose response data of XRCC5 at transcript level, donors having genotype with 2R allele  showed strong correlation between XRCC5 mRNA expression and post-irradiation time points as compared to other genotypes.At C/C, C/G, and G/G genotypes were 50, 40, and 10%, respectively, among 20 donors  polymorphism, the frequencies of the XRCC6 gene with respect to DNA damage was assessed among the 20 donors studied. For XRCC6 (C/G) polymorphism, C/C, C/G and G/G polymorphisms were observed among 20 donors. Figure XRCC6 polymorphism showed significant positive correlation between DNA damage and various doses, thus indicating XRCC6 (C/G) polymorphism is not associated with the induction of DNA damage.Association of polymorphism at XRCC6 with respect to DNA repair was also studied among these donors. As shown in Fig. Similarly, association of genotypes of XRCC6 was studied at 0 h and 4 h post irradiation. Figure XRCC6 at various doses studied at 4 h post-irradiation. As shown in Fig. C/C and C/G showed positive correlation  between mRNA expression level and radiation doses, whereas G/G genotype showed decreasing mRNA expression level with increasing radiation dose   between mRNA expression and post-irradiation time points. Donors with G/G genotype showed negative correlation  between mRNA expression and post-irradiation time after 2.0 Gy, which was not statistically significant.Figure XRCC7 G/T polymorphism, the frequencies of the G/G, G/T and T/T genotypes were 25, 50, and 25%, respectively, among the 20 donors studied  between XRCC7 mRNA expression and radiation doses, whereas genotype T/T showed decreasing mRNA expression at XRCC7 with increasing doses   and (G/T) genotypes showed no significant correlation  between mRNA expression of XRCC7 and post-irradiation time points. Donors with (T/T) genotype showed negative correlation  between mRNA expression of XRCC7 and post-irradiation time points after 2.0 Gy of irradiation, which was not statistically significant.Figure XRCC5, XRCC6 and XRCC7 with the adaptive and non-adaptive donors with respect to DNA damage, repair and mRNA expression profile in primed and non-primed cells. Figure RAR study was performed in primed (cells exposed with a priming dose of 0.1 Gy followed by 2.0 Gy after 4 h incubation) and non-primed cells (cells exposed with a 2.0 Gy) among 20 healthy donors. Sixteen of them showed significant reduction of DNA damage in primed cells (cells exposed with a priming dose of 0.1 Gy followed by 2.0 Gy after 4 h incubation) and are called as adaptive donors, whereas four donors did not show any significant reduction of DNA damage and are called as non-adaptive donors. In the present study, emphasis was given to study association of the genotypes of XRCC5, XRCC6 and XRCC7. As shown in Table XRCC7 polymorphisms among each group (adaptive or non-adaptive donors). But the repair percentage was comparatively higher among non-adaptive donors.Table XRCC5 showed similar mRNA expression among the adaptive vs non-adaptive donors (1.04 ± 0.13 vs 1.04 ± 0.0). There was also significant difference (p < 0.05) in the percentage of DNA damage (%T) among adaptive vs non-adaptive donors. At XRCC6 polymorphism, donors with C/C and C/G genotypes showed significantly higher (P < 0.05) mRNA expression level among adaptive donors as compared to non-adaptive donors . At XRCC7, all the three genotypes  did not show significant mRNA expression level among adaptive vs non-adaptive donors as shown in Table But when compared between adaptive and non-adaptive donors in non-primed cells at 2.0 Gy, the genotypes 1R/2R of XRCC5, XRCC6 and XRCC7, average DNA damage and relative expression in primed cells (adaptive donors) and non-primed cells (non-adaptive donors) are given in Table XRCC5, the genotypes 1R/1R and 1R/2R showed no significant difference in mRNA expression level in primed cells of adaptive vs non adaptive donors . However, at XRCC6, the donors with C/G genotype showed significantly increased (P < 0.05) mRNA expression in primed cells among adaptive vs non-adaptive donors (C/G: 1.22 ± 0.26 vs 0.95 ± 0.00). It showed significantly higher % of DNA damage in primed cells of non-adaptive donors as compared to adaptive donors. At XRCC7, mRNA expression is marginally higher in primed cells of adaptive donors as compared to non-adaptive donors for G/G genotype. But higher % of DNA damage was observed among non-adaptive donors as compared to adaptive donors.Distribution of genotypes at 0/G1 and early S-phase of the cell cycle [DSBs are one of the most lethal DNA lesions induced by IR, as well as endogenous reactive oxygen species (ROS) , which all cycle –39.DDR induced by genotoxic stress including IR lead to alteration of gene expression profile in human cells , 18. SevSeveral studies have been carried out to find out the role of cellular activities involving the action of DNA repair and cell cycle checkpoints –45. IndiXPC  polymorphism where the haplotypes “T-A”  was associated with the lowest DNA damages, thus suggesting that the DNA repair capacity of host cells might be modulated by specific XPC polymorphisms [, reported that polymorphisms in XRCC1 DNA repair genes could influence individual DNA repair capacity [Some of the DNA repair gene polymorphism such as XPC, XRCC1 has shown significant correlations between genotypes and induced DNA damages. For instance, at orphisms . Similarcapacity . HoweverXRCC5 contains seven copies of cis elements, which are essential for basal expression and are involved in CpG methylation [XRCC5, which lead to a phenotypic variation that could affect susceptibility to cancer [XRCC5 was associated with enhanced levels of the XRCC5 protein in bladder cancer patients [XRCC5 (3R/2R/1R/0R) polymorphism and its mRNA expression showed significant positive correlation for (2R) allele at various doses and post-irradiation time intervals.The promoter of hylation . Studieso cancer . Furtherpatients . HoweverXRCC6-61C/G polymorphism is associated with an increased risk of cancers, including breast cancer and gliomas [XRCC6 (C/G) polymorphism studies indicate significant positive correlation of the XRCC6 transcript level with various irradiation doses as well as post-irradiation time for wild type allele (C), however, XRCC6 transcript level was negatively correlated for variant (G) allele. Accordingly, radiation induced DNA damage was observed to be lower in donors with polymorphic group containing (C) allele as compared to the polymorphic group containing (G) allele. Consequently, DNA repair was faster in donors with polymorphic group containing (C) allele as compared to the polymorphic group containing (G) allele. All these findings indicate that wild type (G) allele may be associated with the radio-sensitivity and DNA repair capacity of an individual.There are reports which show that  gliomas . Our resXRCC7 (rs7003908) is located in the intron 8 of the gene and may play a role in regulating splicing and therefore cause mRNA instability [XRCC7 with several types of cancers [XRCC7 (G/T) polymorphism indicate no significant correlation of the XRCC7 transcript level with different dose groups as well as post-irradiation time for wild type allele (G). However, XRCC7 transcript level was negatively correlated for variant (T) allele. Despite these variations in the transcript levels, the radiation induced DNA damage and its repair was not significantly different among these genotypes.DNA polymorphism at G6721T of tability , 53. NHEtability . Few stu cancers , 55–60. XRCC5 (3R/2R/1R/0R), XRCC6 (C/G) and XRCC7 (G/T) polymorphism with DNA damage, repair capacity and its mRNA expression showed that wild type (G) allele may be associated with the radio-sensitivity and DNA repair capacity of an individual. At the transcript level, (2R) allele of XRCC5 polymorphism and (T) allele of XRCC7 polymorphism also showed significant association at various doses of IR and post-irradiation time intervals.In summary, our studies on association of It is quite intriguing that individual variation due to intrinsic DNA repair capacity decides the fate of a person’s disease status. While DNA repair assays are required to be considered as potential clinical tools for prevention or treatment of disease of an individual , geneticThe present study demonstrated that radio-adaptive experiments showed changes at transcript level among adaptive donors in many of the genotypes as compared to non-adaptive donors. However, further studies at post-translational modification and proteomics level are required to understand the cause of genetic variability. Radio-sensitivity is an important aspect for patients undergoing radio-therapeutic treatments as efficacy of chemotherapy as well as radiation therapy depends upon individual response, which can decide treatment dosages and time span of treatment required. The present study also assessed the role of NHEJ repair gene polymorphism in RAR.DNA DSBs are highly deleterious and involvement of NHEJ repair gene polymorphism are of high importance for genome integrity. Further, high throughput studies on larger number of gene polymorphisms may be helpful for studying radio-sensitivity studies. NHEJ repair gene polymorphisms play an important role in RAR and thus can be used as potential biomarkers to identify radiosensitive and radio-resistant individuals in a population as well as among cancer patients undergoing radio-therapeutic treatments.


GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.Mutations in the  The inward current carried by Na+ and Ca2+ influx through cGMP-gated channels partially depolarizes rods and cones in the dark , a rapidly progressing loss of vision caused by the degeneration of functional cones and rods    , 31, whe1 (LCA1) . The LCAin vitro . Recessis (CSNB) . Unlike  . The “extracellular” domain, located in the intradiskal space of the photoreceptor disks, connects with the cytoplasmic portion of the cyclase via a short transmembrane region. The cytoplasmic part of the enzyme includes a protein kinase homology domain, a catalytic domain, and a short dimerization domain located between the kinase homology and the catalytic domains .The primary structure of RetGC1 includes several domains homologous to other membrane guanylyl cyclases A. The “e domains , 4, 32. c domain . The CSNc domain , 40, 41.c domain , 34, 35.c domain B.A), the LCA1 R768W RetGC1  failed to bind GCAP1 in cyto  (A), but not the LCA1 RetGC1 variants . However, they also completely failed to activate the two LCA1 RetGC1 variants (B and C). It is worth pointing out here that the inability of a human Mg2+ GCAP2 to activate WT human RetGC1 in vitro reported in Ref. Mg0.04 μm) A, but novariants , 41, 46 patients , R768W apatients . GCAP2, patients , 47, 48,patients , 50, botvariants , B and CA–C). A low level of activity was detectable in GCAP1-stimulated R666W RetGC1, but the apparent affinity for GCAP1 was strongly reduced . The R761W and L911F RetGC1 were completely inactive in the presence of all three GCAP isoforms. The lack of activity in the L911F RetGC1 (A) appeared to be at odds with the clearly defined GCAP1 co-localization pattern in cyto (C). To ensure that the GCAP1-GFP binds with the L911F RetGC1 in cyto specifically via GCAP1 rather than GFP moiety, we used V77E GCAP1 lacking the ability to bind WT RetGC1   .Figure 7GUCY2D LCA1 allele products  and CORD6 (Ser838) subunits also contribute to the change. Their co-expression produces the Arg838:Ser838 heterodimers mixed with Arg838:Arg838 and Ser838:Ser838 homodimers. Those dimers cannot be biochemically separated to test their individual Ca2+ sensitivities, largely because the detergents required for extraction of RetGC1 from the membrane destroy cyclase regulation . Each subunit (subunits a and b) coordinates the guanine base (the g site) of one GTP molecule and the ribose-5-triphosphate (the r site) of the other GTP molecule (A and B). Substitutions E925K and C997D disable coordination of the guanine moiety (g—), and D929A disables coordination of the ribose-5-triposphate moiety (r—) (— r+) nor (g+ r—) RetGC1 homodimer can bind GTP and produce cGMP (C). However, cGMP can be produced when (g— r+) and (g+ r—) subunits are combined as a heterodimer. In this case, the remaining functional r site of the subunit a pairs with the remaining functional g site on the opposite subunit b (D). We reasoned that the functional complementation between the Arg838 and Ser838 subunits (E and F) should allow for selectively testing the Ca2+ sensitivity of GCAP1-activated heterodimers in the presence of the inactive homodimers.In the RetGC1 catalytic domain structure , the act pockets A. Each smolecule , A and Bety (r—) , 51. Conuce cGMP , 51  and (g+ r—) RetGC1 subunits. The Ser838 did not prevent binding of GCAP1  nor Ser838(g+ r—) RetGC1 produced cGMP in the presence of Mg2+ GCAP1 when expressed separately. Only their co-expression enabled cGMP synthesis through the formation of the heterodimer , making it possible to compare the regulatory properties of different dimeric RetGC1 combinations . It is worth mentioning that the E925K/C997D substitutions enable binding of ATP instead of GTP via the g— site :Ser838(g+ r—) heterodimer harboring a single GTP-binding site as compared with the Arg838:Arg838 dimers binding one or two GTP molecules (C). The apparent affinity of RetGC1 for Mg2+ GCAP1 was increased even further in the Ser838(g— r+):Arg838(g+ r—) heterodimer (C), which recognized GTP via the Arg838 subunit (E). R838S substitution also did not prevent RD3 binding to RetGC1 in cyto :Arg838(g+ r—) heterodimer than the Arg838:Arg838 and even the Ser838(g— r+):Ser838(g+ r—) dimers .R838S substitution increases the affinity of RetGC1 for Mg2+GCAP1 , 36, 37.olecules C. The aperodimer C, which  subunit E. R838S 838(g— r+):Arg838(g+ r—) RetGC1 that binds only one GTP molecule per active site (C), the Ca2+ sensitivity of its regulation by GCAP remained identical to the WT (A). In contrast, it was strongly reduced in the dimers harboring Ser838. The [Ca2+]1/2 in Ser838(g+ r+):Ser838(g+ r+) and Ser838(g— r+):Ser838(g+ r—) dimers was increased ∼2.5-fold compared with the WT, also regardless of whether they bound two or just one GTP per active site (A and B). The [Ca2+]1/2 increased even further, to ∼6.5-fold higher than the WT, in the Arg838:Ser838 RetGC1 heterodimers (A and B). In contrast to the single-residue substitution, R838S, a deletion of ∼40-kDa fragment from the cyclase extracellular domain did not alter the Ca2+ sensitivity of the Ser838(g+ r—):Arg838(g— r+) heterodimer (A), because the extracellular domain does not contribute to the regulation of RetGC1 by GCAP  isozyme  was similar to that of previously characterized LCA1 mutant R1091X . Activation by GCAP3, expressed exclusively in a subset of human cones .The role of d vision  is much  alleles  merely c alleles . The low alleles A was simt R1091X , and itst R1091X . The twot R1091X . The L91an cones , would hA) after its co-expression with G982VfsX39 , is consistent with the additional increase of the heterodimer's affinity for Mg2+ GCAP1 (C).The gain-of-function substitutions of Argeceptors , 36, 37.mutation , 35, 36.omodimer . The veromodimer A, is con2+ GCAP1 C.838:Ser838 coiled-coil dimerization domain is more optimal than WT for the regulatory binding of GCAP1. We find in our study that pairing WT and CORD6-specific dimerization domains is even more conducive to GCAP1 binding.Mutational analyses of RetGC1 indicate that its dimerization domain and the kinase homology domain are most critical for GCAP binding , 45. PreGUCY2D allele would likely stimulate Ca2+ influx in the dark even more than Ser838:Ser838 homodimers. It would therefore seem unreasonable to expect that decreasing the fraction of the Ser838:Ser838 homodimers by increasing the level of WT GUCY2D allele expression instead of reducing the expression of the CORD6 allele could alleviate photoreceptor degeneration.To summarize, the heterodimers formed in photoreceptors that harbor heterozygous CORD6 in cyto . In vivo studies strongly indicate that one of the essential RD3 functions in photoreceptors prevents photoreceptor degeneration by suppressing aberrant guanylyl cyclase activation by GCAPs  were from Millipore/Sigma or Fisher Scientific, restriction endonucleases were from New England Biolabs, Phusion Flash polymerase was from Thermo Scientific, and oligonucleotide primers were from Integrated DNA Technologies.UCY2D) cDNA inserted in pRCCMV vector (Invitrogen) was modified to introduce new restriction sites for subsequent cloning without changing the encoded protein sequence. LCA1-, CSNB-, and CORD6-specific substitutions were then introduced using “splicing by overlap extension” method , 100 μm zaprinast and dipyridamole was incubated for 30 min at 30 °C, and the reaction was stopped by heat-inactivation for 2 min at 95°. The [32P]cGMP product was separated by TLC using fluorescently backed polyethyleneimine cellulose plates (Merck) developed in 0.2 m LiCl. The cGMP spots visualized under UV light were cut out from the plate, and the [32P]cGMP was eluted with 0.5 ml of 2 m LiCl in 20-ml scintillation vials. The radioactivity was counted by liquid scintillation in 10 ml of UniverSol . Data fitting was performed using Synergy Kaleidagraph 4 software.Human RetGC1 , and the chemiluminescence image developed using a Pierce SuperSignal Femto kit was acquired using a Fotodyne Luminous FX instrument.HEK293 cell fractions containing the recombinant RetGC1 were subjected to electrophoresis in 7% SDS-PAGE and transferred to Immobilon-P membrane (Millipore Sigma) at 60 V for 20 h. The membranes were blocked using SuperBlock blend (Fisher Scientific), and probed by rabbit polyclonal antibody raised against the recombinant C-terminal fragment, MetEscherichia coli strain (Agilent Technologies), extracted from the inclusion bodies, and purified as previously described  vector in BL21(DE3) CodonPlus escribed , 59 withN-myristoylated GCAPs for the in vitro assays were expressed in BLR(DE3) E. coli strain harboring pBB131 plasmid coding for a yeast N-myristoyl transferase and urea-extracted from the inclusion bodies. GCAP1 was purified by calcium precipitation, butyl–Sepharose chromatography, and Sephacryl S–100 chromatography using previously published procedure  centrifugal filter and then centrifuged for 10 min at 200,000 × g at 4 °C in a Beckman Optima ultracentrifuge and loaded on a Sephacryl S-100 column (2.6 × 60 cm) equilibrated with 20 mm Tris-HCl, pH 7.5, containing 100 mm NaCl and 0.3 mm CaCl2. After the main peak containing GCAP2 was collected, dithiothreitol was added to 4 mm and EDTA was added to 2 mm to remove Ca2+ bound to GCAP2. The excess of EDTA was then removed by 3–4 cycles of 20-fold concentration/dilution in 10 mm Tris-HCl, pH 7.5, containing 30 μm EDTA using an Amicon Ultra-15  centrifugal filter at 4 °C. Human GCAP3 was purified using similar procedure except that 1.0 × 30-cm Superdex-200 column equilibrated with 20 mm Tris-HCl, pH 7.5, and 100 mm NaCl was used for size-exclusion chromatography instead of Sephacryl S-100.Human GCAP1 (E6S), GCAP2, and GCAP3 cDNAs were inserted into the NcoI/BamHI sites of the pET11d vector . The rocedure  modifiedrocedure  with the2/MgCl2 mixtures maintaining required free Ca2+ concentrations at 1 mm free Mg2+ were prepared using Tsien and Pozzan method  utilizing coordinates of the model structure reported by Liu et al.   upon reasonable request.The data referred to in this article are contained within the article. Unprocessed data can be available from the corresponding author  (EY011522) to Alexander M. Dizhoor10.13039/100004897Pennsylvania Department of Health (CURE Formula Grant) to Alexander M. Dizhoor


TCF7L2 rs290487 C allele. Analysis of 195 cirrhotic patients revealed a higher insulin resistance index and incidence of hepatogenous diabetes in patients with the rs290487 C/C genotype compared to those with the C/T or T/T genotype. The in vitro experiments using targeted mutant PLC-PRF-5 cell line showed that cells with the rs290487 C/C genotype (C/C cells) had higher glucose production, lower glucose uptake, and lower TCF7L2 mRNA and protein levels than those with the C/T genotype (C/T cells). Integrated multi-omics analysis of ChIP-seq, ATAC-seq, RNA-seq, and metabolomics data showed genome-wide alterations in the DNA binding affinity of TCF7L2 in the C/C cells, including gain  and loss  of binding sites in several glucose metabolism-related genes. These allele-specific changes in transcriptional regulation lead to increased expression of gluconeogenesis-related genes  and their downstream metabolites . These findings demonstrate that the TCF7L2 rs290487 C allele enhances gluconeogenesis through allele-specific changes in transcription and chromatin binding.In this study, we investigated the mechanisms underlying the altered hepatic glucose metabolism and enhanced diabetes risk in individuals with the  The rc groups , 5. The c groups . In recec groups , 4, 6. Ic groups . While mc groups , a couplc groups , 10.Functional studies suggest that the rs290487 C allele may cause insulin resistance –13. Our TCF7L2 rs290487 SNPs and clinical parameters in 195 cirrhotic patients who were susceptible to HD. As shown in P < 0.05, We analyzed the correlation between the TCF7L2 SNPs such as rs7903146 and rs12255372 are associated with changes in alternate-spliced TCF7L2 transcripts and TCF7L2 mRNA expression [TCF7L2 transcripts in 54 patient liver tissue samples. We did not detect transcripts containing exons 11-14 (transcript No. 12) and exons 13b-14 (pancreas-specific transcript No. 9 and 10) that have been described in a previous study [TCF7L2 mRNA and those of liver-specific alternate-spliced TCF7L2 transcripts such as transcript No. 2, 4, and 5 [TCF7L2 mRNA and some liver transcripts were significantly higher in patients with HD than in patients without HD  using CRISPR/Cas9 knock-in approach . TargeteTCF7L2 mRNA and transcript No. 5 and 7 in C/C cells compared to the C/T cells , including 1934 C/C- and 1669 C/T-specific binding sites  that were enriched for binding site motifs for a variety of transcription factors including TCF7L2 and its partners or competitors such as FOXA1/2, FOXO1, and HNF4A.To further assess allele-specific binding, we performed genome-wide ATAC-seq. The C/C cells showed 30% fewer open chromatin regions (peaks) than the C/T cells . The comP < 0.05, G6PC, PCK1, PPARGC1A, and HNF4A were significantly up-regulated and the genes related to insulin signaling transduction pathway such as PPP1R3B and MLXIPL were significantly down-regulated in the C/C cells compared to the C/T cells  was used for quality control . We usedic acids . Mummichic acids , especiaic acids . Then, wic acids  and founic acids .P < 0.05, PCK1 showed the highest correlation degree within the biological network. Based on this network and pathway analyses, we propose that the rs290487 C allele enhances gluconeogenesis  and long C-terminal  as well as the corresponding small and large protein products. The smaller nuclear TCF7L2 isoform (58kDa) functions as a potent suppressor of hepatic gluconeogenesis by activating downstream target genes of the β-catenin/TCF pathway [In this study, we demonstrate that  pathway . Our datPPARGC1A and CREB, as well as loss of TCF7L2 binding sites in genes such as PGK1 and PGM1, all of which regulate glucose metabolism. Motif analysis indicates altered binding affinity in TCF7L2-related TFs and its competitors , which regulate hepatic glucose metabolism. Consequently, we demonstrate allele-specific changes in the transcriptome and metabolome. Multi-omics analysis demonstrates that this rs290487 SNP specifically alters hepatic glucose metabolism, especially, glycolysis, gluconeogenesis and insulin signaling transduction. The increased expression of genes such as PCK1, G6PC, and ENO2/3, and their related metabolites such as oxaloacetate and 3-Phospho-D-glyceroyl phosphate shows that rs290487 C allele enhances gluconeogenesis, which consistent with the phenotype.We also demonstrate genome-wide TCF7L2 rs290487-specific DNA binding. The T to C transition changes the binding affinity of TCF7L2 to known target genes , 22. MorG6PC, PCK1, and HNF4A. However, TCF7L2 silencing does not directly or indirectly affect PPARGC1A expression, as analyzed by ChIP-seq and RNA-seq [PPARGC1A, probably by opening the chromatin at its promoter region (ATAC-seq) and by the presence of an additional TCF7L2 binding site (ChIP-seq). Moreover, transcriptome analysis of TCF7L2 silencing highlights a panel of transcription factors that act as transcriptional partners of TCF7L2 based on the analysis of co-enriched motifs of TCF7L2 binding sites [In comparison with the results from two previous TCF7L2 silencing studies , 22, our RNA-seq , 22. In ng sites . Our stuTCF7L2 mRNA levels are elevated in patients with the C/C genotype compared to those with the T/T wild type. Moreover, positive correlation between liver TCF7L2 mRNA levels and HOMA-IR index further suggests metabolic regulation of TCF7L2 expression. Therefore, we speculate that the reason for people with risk SNPs being more susceptible to diabetes is aberrant regulation of hepatic gluconeogenesis. On the contrary, diabetes-related hyperglycemia and/or hyperinsulinemia increases TCF7L2 expression and activates β-catenin/TCF7L2 signaling [Our study also shows that environmental factors such as hormones modulate TCF7L2 expression. We demonstrate that the TCF7L2 protein levels are significantly lower in the C/C cells compared to the C/T cells. However, high glucose and insulin treatment dramatically increases the TCF7L2 protein expression in both C/C and C/T cells. These results are consistent with the findings from previous studies , 23. Morignaling .CYP3A5, UGT1A1, UGT2A3, GSTM2 and GSTM4 were differentially expressed between C/C and C/T cells. The analysis of differential peaks in both ChIP-seq and ATAC-seq revealed highly enriched motifs for NFAT and CREB, which are the known targets of calcineurin inhibitors that are used as immunosuppressants after solid organ transplantation [Our previous study demonstrated the link between the TCF7L2 SNPs and new-onset diabetes after transplantation . In thisantation . We alsoThe study has several advantages and limitations. First, only cirrhotic patients who are known to have a high incidence of HD were included in the study to better elucidate the correlation between SNP and hepatic glucose metabolic disorders. However, the role of rs290487 in hepatic metabolism still needs to be investigated in patients with type 2 diabetes or healthy subjects. Second, we successfully established the point mutation model in PLC-PRF-5 cells but failed to establish it in other liver cell lines such as HepG2, SK-HEP-1, and Hep3B2.1-7 (data not shown). PLC-PRF-5 is a human hepatocarcinoma cell line whose genome contains integrated hepatitis B virus DNA and secretes virus envelope proteins , and is TCF7L2 SNPs on hepatic glucose metabolism by performing multi-omics analysis of an point mutation in vitro model. The proposed mechanism is shown in TCF7L2 rs290487 SNPs may lead to changes in alternative splicing and alter protein content and structure, which subsequently changes the transcription factor-DNA binding affinity and the number of DNA-binding sites, thereby promoting gluconeogenesis. Some connections need further exploration and verification.In summary, this is the first study to directly evaluate the effect of risk Between 2015 and 2017, we enrolled adult hepatitis B virus-related cirrhotic patients who were admitted as liver transplant candidates to the First Affiliated Hospital, Zhejiang University School of Medicine. We excluded patients with pre-existing type diabetes mellitus 2, those in need of intensive care unit (ICU) support, or those who were not followed-up. Finally, we included 195 cirrhotic patients for this study and obtained their blood samples. We also collected 54 liver samples from abdominal surgery out of the 195 patients. The samples were immediately frozen in liquid nitrogen and stored at -80°C. We analyzed genetic polymorphisms using Applied Biosystems SNaP-Shot and TaqMan technology . We obtaThe PLC-PRF-5 cell line was purchased from the American Type Culture Collection  and maintained in DMEM medium supplemented with 10% fetal bovine serum. For experiments, cells were seeded in 24-well plates for 24 h. Then, we changed the medium to serum- and glucose-free DMEM medium for another 8 h. The glucose uptake and production tests have been described in our previous study .TCF7L2 mKI targeting vector was constructed by ligating the homologous arm into the LscKO-3G vector. Then we electroporated the sgRNA2 and TCF7L2 mKI targeting vector into the PLC-PRF-5 cell line using the Celetrix CTX-1500A electroporator . Then, the transformed cells were selected by growing in DMEM medium containing 1μg/mL Puromycin. The positive colonies were screened by limiting dilution and genotyping using the Junction PCR (The genome editing of PLC-PRF-5 cells was performed by the Beijing Biocytogen Co. Ltd. using the CRISPR/Cas9 system. We designed and synthesized four guide RNAs  and sgRNtion PCR . EventuaTCF7L2 mini promoter (1324bp) is shown in Dual-luciferase expression assay was performed as described previously . The relQuantitative real-time PCR was performed as described previously . The qPCWestern blotting was carried out as described previously . The nucChIP-seq was performed by Omigen Inc.  using the ChIP-IT Express Enzymatic shearing kit  according to manufacturer’s protocols. Briefly, the cells were harvested and fixed. Then, the cells were lysed and homogenized using the Dounce homogenizer. The cell debris was removed by centrifugation and the soluble chromatin was quantified in the Nanodrop 2000 (ThermoFisher Scientific). ChIP was performed using Dynabeads Protein G (ThermoFisher Scientific), 4 μL anti-TCF4/TCF7L2 antibody  or rabbit IgG , and 20-50 μg soluble chromatin. The beads with the protein G -antibody-chromatin complexes were washed in wash buffer according to manufacturer’s protocols. The chromatin was then reverse cross-linked by treatment with RNase A and proteinase K at 65°C overnight. The DNA was then cleaned up using the QIAGEN MinElute PCR Purification Kit . ChIP-seq libraries were prepared using the KAPA Hyper Prep Kit  and sequenced using the Illumina HiSeq X-Ten instrument (Illumina).ATAC-seq was performed by Omigen Inc. Briefly, 50,000 cells from each sample were pelleted by centrifugation and lysed in buffer containing 0.1% NP40, 0.1% Tween 20, and 0.01% Digitonin to obtain the nuclei. The nuclei were then processed using the TruePrep DNA Library Prep Kit V2 for Illumina kit  according to the manufacturer’s protocols. Immediately after transposition, the DNA fragments were purified using the MinElute PCR Purification Kit  and PCR-amplified with the barcode and primers for 10-12 cycles. The resulting libraries were purified and assessed by the Qubit 3 Fluorometer (Invitrogen) and Agilent 2100 Bioanalyzer (Agilent), and sequenced using the Illumina HiSeq X-Ten instrument (Illumina).The clean sequenced reads from the ChIP- or ATAC-seq were aligned to human reference genome using Bowtie2 (v2.3.4.1) and the enriched chromatin regions were identified using MACS2 (v2.1.1). The peak visualization was performed using Integrative Genomics Viewer (v2.3.5). The differential peak analysis was performed using the MAnorm software (v1.2.0). The motifs were analyzed using the findMotifsGenome.pl from HOMER software (v4.10).Transcriptome was sequenced by Omigen Inc. using the Illumina paired-end RNA-seq approach according to the standard protocol. Briefly, the mRNAs were purified from the total cellular RNA using the magnetic beads attached to the poly-dT oligo. Then, the double-stranded complementary DNAs were synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (Invitrogen). Then, the cDNA library was constructed and sequenced using the Illumina HiSeq X-Ten instrument (Illumina).. The sequencing data was analyzed by first indexing and aligning the paired-end clean reads to the reference genome using the TopHat2 (v2.0.9) and Bowtie2 (v2.3.4.1) software. Then, the HTSeq (v0.6.1) software was used to count the number of reads that map to each gene. The DESeq2 R (1.10.1) software package was used to analyze differentially expressed genes. Enrichment analysis of the differentially expressed genes was performed using KOBAS 3.0 software.2O  and frozen in liquid nitrogen, and stored at -80°C. The ultra-performance liquid chromatography-mass spectrometry (UPLC/MS)-based metabolomics analysis was performed as described previously [https://www.mzcloud.org/) and the ChemSpider (http://www.chemspider.com/) database was used for qualitative and quantitative analysis. Functional and pathway analysis was performed using MetaboAnalystR 2.0 [http://www.omicsnet.ca) and Metscape 3 [http://metscape.med.umich.edu/) web tools.The cells were rinsed with PBS, pelleted by centrifugation the cell pellets were quickly extracted using 1mL chilled MeOH/Heviously . The rawystR 2.0 , SIMCA 1ystR 2.0  and mummystR 2.0   and compared using the Pearson’s χ2 test. Correlation analysis was performed using Pearson linear regression. The sequence data generated and/or analyzed in this study have been submitted to the GEO repository (GSE138781).SPSS version 13.0 software  was used for statistical analysis. Supplementary FiguresSupplementary Tables


The CD4+ T cells at Days 8 and 21 expanded after prime vaccination and this expansion correlated strongly with early post-vaccination HAI and Neut titers (p ≤ 0.002). In an adult population, the rapid serological response observed after initial H3N2v vaccination correlates with post-vaccination plasmablasts and CD4+ T cell responses.The cellular immune responses elicited by an investigational vaccine against an emergent variant of influenza (H3N2v) are not fully understood. Twenty-five subjects, enrolled in an investigational influenza A/H3N2v vaccine study, who received two doses of vaccine 21 days apart, were included in a sub-study of cellular immune responses. H3N2v-specific plasmablasts were determined by ELISpot 8 days after each vaccine dose and H3N2v specific CD4+ T cells were quantified by intracellular cytokine and CD154 (CD40 ligand) staining before vaccination, 8 and 21 days after each vaccine dose. Results: 95% (19/20) and 96% (24/25) subjects had pre-existing H3N2v specific memory B, and T cell responses, respectively. Plasmablast responses at Day 8 after the first vaccine administration were detected against contemporary H3N2 strains and correlated with hemagglutination inhibition HAI (IgG:  Since 2011, a swine variant of influenza A/H3N2 (H3N2v) has caused 430 infections associated with exposure to pigs at agricultural fairs in 18 different US states with 26 hospitalizations and one death between 2011 and 2018 . One-thiClinicaltrials.gov Registration: NCT01746082) ) were obtained from the Centers for Diseases Control and Prevention International Reagent Resource (IRR ). The rePeripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood using a Ficoll-Hypaque gradient and stored in a liquid nitrogen tank. Cryopreserved PBMCs were thawed in a 37 °C water bath and washed. Before use, cells were counted and checked for viability by Trypan blue dye exclusion.HAI and Neut assays were performed at Cincinnati Children’s Hospital Medical Center using previously described methods . 2 incubator. ASCs were detected with biotinylated anti-human IgG, IgM, or IgA antibody  followed by incubation with Avidin-D-HRP conjugate , and then developed using AEC substrate . Developed plates were scanned and analyzed using an automated ELISpot counter . A direct enzyme-linked immunospot (ELISpot) assay was used to enumerate the numbers of total IgG, IgA and IgM ASCs and the H3N2-specific IgG, IgA, and IgM ASCs present in fresh PBMCs, performed as described previously . 96-wellICS and CD154 staining assay were performed, as previously described . Cryopre6 cells per mL in R-10 supplemented with 1/100,000 pokeweed mitogen extract (PWM), 6 μg/mL phosphothiolated CpG ODN-200626 , and 1/10,000 fixed Staphylococcus aureus Cowan (SAC)  for 6 days. Eight replicate wells were cultured per subject. Stimulated cells were harvested, washed and applied to ELISpot filter plates which had been coated overnight with A/Minnesota/11/2010 H3N2v subunit vaccine or rHAs of contemporary H3N2 strains in PBS, or goat anti-human Ig . Total and virus-specific IgG-secreting MBCs were quantified by ELISpot assay as described above in plasmablasts ELISpot assay. Insufficient PBMCs did not allow quantification of MBCs in 5 out of 25 subjects.MBCs were also tested by ELISpot after polyclonal stimulation of PBMC in vitro as previously described  by incubAs an exploratory sub-study, the sample size was limited to the number of subjects enrolled in the parent clinical trial at the Emory site and not designed to test a statistical hypothesis. Analyses includes descriptive and graphical summaries and Pearson correlations. Confidence intervals for binomial proportions were calculated using the exact Clopper-Pearson method.Post-vaccination responses were compared with baseline using Wilcoxon signed-rank test. HAI, Neut, IgA, IgM and IgG responses were log-transformed for analysis. Statistical significance was considered at a level alpha = 0.05, without adjustment for multiple comparisons. Analyses were performed using SAS 9.3 or higher . Twenty-five subjects were enrolled in this immunogenicity sub-study. Baseline characteristics of subjects included in this subset are summarized in HAI and Neut antibody responses against H3N2v for the 25 subjects included in the sub-study  were conAt baseline no subjects had detectable variant or seasonal H3N2-specific IgG or IgA ASCs (plasmablasts) in blood as expected. At Day 8, the number of influenza-specific IgG-secreting plasmablasts against the H3N2v/Minnesota/2010 strain had a geometric mean (GM) of 205 per million PBMCs . At Day 8, the number of influenza-specific IgA-secreting plasmablasts against the H3N2v/Minnesota/2010 strain had a GM of 56 per million PBMCs  . PlasmabTotal IgM secreting ASCs were detected, as well as the total IgA and IgG (data not shown), however, H3N2 specific IgM ASCs were not detected, as their peak likely occurred prior to the detection of H3N2 specific IgG and IgA ASCs. The magnitudes of H3N2v-specific IgG- and IgA-secreting ASCs at Day 8 correlated with H3N2v HAI and Neut antibody titers at Day 8 . p < 0.001; IgG: r = 0.74, p = 0.0001)  A,B, whic6 CD4+ T cells) (6 CD4+ T cells). Both Day 8 and 21 H3N2v-specific responses were significantly higher compared to baseline values (p < 0.001 at both Days 8 and 21), demonstrating that vaccine-specific CD4+ T-cell responses occurred following H3N2v vaccination. Interestingly, the magnitude of H3N2v-specific CD4+ T cells declined to levels similar to pre-vaccination following the second dose   were used to identify H3N2v specific CD4+ T cells. At baseline, immune marker positive CD4+ T cells were detected in 24 out of 25 subjects  A. After = 0.34)] A, suggesIt appeared that a lower baseline memory H3N2-specific CD4+ T cell response likely resulted in a higher HAI or Neut post-vaccination titer at all time points checked. This inverse correlation did not reach statistical significance, possibly due to the relatively small number of subjects in the sub-study . HoweverIn addition, the CD4+ T cell response is polyfunctional. as determined by the frequency of CD4+ T cells producing 1, 2, 3 or 4 immune markers among CD154, IFN-gamma, IL-2 and IL-21 B.Influenza A viruses have been isolated from swine since the 1930s. H1N1 virus was the predominant subtype, isolated from U.S. swine until the late 1990s, when human H3N2 viruses spread widely among North American pigs. Since then, triple re-assortant swine influenza A/H3N2 viruses emerged and began circulating in pigs containing genes from human, swine and avian influenza viruses . TransmiSimilar to the primary study , our stuIt is known that pre-vaccination serological status has a large impact on the immune response to seasonal influenza vaccination and seroconversion rates are negatively associated with past influenza vaccinations, baseline HAI Ab titers ≥ 40 and baseline HAI Ab titers ,17. HereWe previously reported that pre-existing H3N2v MBCs correlated with the Day 8 fold-change of HAI antibody , and furThough some evidence suggests that preexisting influenza-specific CD4+ T cells correlate with disease protection in an influenza human challenge model , other sIncreased in ASCs and MBCs detection to contemporary H3N2 strains such as A/Perth/16/2009 H3N2 and A/Victoria/361/2011 H3N2 after H3N2v vaccination . In pediThe study had several limitations. A major limitation of our study is that we did not conduct HAI and Neut antibody assays against the contemporary strains. We had a limited number of subjects resulting in limited power to detect correlations. We included only subjects between the ages of 18–64 years .Our data demonstrated pre-existing memory B and T cells could modulate antibody response to vaccination of newly emerged variant influenza strain, and may have implications for the design of vaccination strategies against potential future influenza pandemics and the identification of early markers of vaccine efficacy. Since there is a great interest in defining an early biomarker that will predict a successful response to a vaccine, the presence of antigen-specific MBCs at baseline , the mag


Although m6A modification plays important roles in tumor development, its role in therapeutic resistance remains unknown. In this study, we aimed to examine the expression level of m6A-modification related proteins and elucidate the effect of m6A-related proteins on radiation response in nasopharyngeal carcinoma (NPC). Among the genes that participated in m6A modification, YTHDC2, a m6A reader, was found to be consistently highly expressed in radioresistant NPC cells. Knocking down of YTHDC2 expression in radioresistant NPC cells improved the therapeutic effect of radiotherapy in vitro and in vivo, whereas overexpression of YTHDC2 in radiosensitive NPC cells exerted an opposite effect. Bioinformatics and mechanistic studies revealed that YTHDC2 could physically bound to insulin-like growth factor 1 receptor (IGF1R) messenger RNA and promoted translation initiation of IGF1R mRNA, which in turn activated the IGF1R-AKT/S6 signaling pathway. Thus, the present study suggests that YTHDC2 promotes radiotherapy resistance of NPC cells by activating the IGF1R/ATK/S6 signaling axis and may serve as a potential therapeutic target in radiosensitization of NPC cells.N6-methyladenosine (m Nasopharyngeal carcinoma (NPC) is a malignant tumor arising from the epithelium of the nasopharynx, and most cases of NPC usually occurred in Southeast Asia and Southern China –3. NPC i6-methyladenosine (m6A) refers to methylation of the adenosine base at the nitrogen-6 position. It is the most abundant modification in messenger RNA (6A RNA modification is a dynamic and reversible process coordinated by methyltransferase complex (m6A “writer”), demethylases (m6A “eraser”), and m6A-binding proteins (m6A “reader”)  , 11. Then in RNA , 13. To em cells . In contem cells . A similarcinoma , 19. Forferation . Althoug6A modification. Previous studies demonstrated YTHDC2 as an essential regulator involved in the switch from mitosis to meiosis during spermatogenesis  messenger RNA and promote translation initiation of IGF1R mRNA, which in turn activated the IGF1R-AKT/S6 signaling pathway. Thus, the present study suggests that YTHDC2 promotes radiotherapy resistance of NPC cells by activating the IGF1R/ATK/S6 signaling axis.YTH domain containing 2 (YTHDC2) is a member of DExD/H box RNA helicase family proteins, containing ATP binding motifs and RNA binding motifs. YTHDC2 regulates mRNA translation and stability by recognizing mogenesis –23. Moreogenesis . A recenr YTHDC2 . In the 6A reader YTHDC2 promotes radioresistance of nasopharyngeal carcinoma via a translation-dependent pathway.In summary, our study demonstrates that the mA total of 105 paraffin-embedded primary nasopharyngeal carcinoma (NPC) samples were obtained from Xiangya Hospital. A total of 105 NPC cases were recruited between January 2017 and July 2019. NPC samples were collected with complete pathological and clinical data. The follow-up period for assessing radiotherapy efficacy was defined as the first time NPC patients were treated with radiation to half year after radiation course. All samples were deidentified and all patients signed informed consents. The study was approved by the Institute Research Medical Ethics Committee of Central South University.The inclusion criteria of 105 cases included in our study were as follows: (1) pathologically proven squamous cell carcinoma (SCC) of locally advanced nasopharyngeal carcinoma (AJCC 7th staging system III–IVa); (2) the existence of tissue blocks available for our research; (3) first line IC and cisplatin-based concurrent chemotherapy regimen; (4) the MRI imagine evaluation data after radiochemotherapy are available, and the response is partial response or complete response; (5) without anticancer treatment prior to primary RT, or surgery after RT; (6) adequate organ function. This retrospective study was approved by the Clinical Research Ethics Committee of the Xiangya Hospital of Central South University.2] and cisplatin . The target volumes were defined with reference to International Commission on Radiation Units and Measurements (ICRU) reports No. 50 and No. 62. A total dose of 70.4 Gy in 32 fractions to the PGTVnx (GTVnx +3 mm margin), 66 Gy to 70.4 Gy in 32 fractions to the PGTVnd, 60.8 Gy in 32 fractions to the PTV1, and 54Gy in 30 fractions to the PTV2 were prescribed. All patients were treated with one fraction daily, 5 days per week. Dose limits for the critical tissue structures and plan evaluation were as defined by the Radiation Therapy Oncology Group (RTOG) 0225 (All patients received three cycles or two cycles of the IC regimen: TP (consisting of docetaxel [1 day of 75 mg/mOG) 0225 . The patOG) 0225 , which c2. All cell lines accepted cell line authentication service.CNE2 was established from a 68-year-old male patient with nasopharyngeal carcinoma  and CNE2Human wildtype YTHDC2 was generated using the following primers: forward: 5′-ATGTCCAGGCCGAGCAGCGTCTCCCCGCGG-3′ and reverse: 5′-TCAATCAGTTGTGTTTTTTTCTCCCAAGGG-3′, and then subsequently cloned into lentivector-based pLVX-IRES-Puro (Addgene). ShRNA targeting YTHDC2 was designed using software provided by Qiagen  and cloned into the pLKO.1-TRC vector. The sequences of shRNA are as follows:ShYTHDC2_1#(pLKO.1): 5′-CCGGCGGAAGCTAAATCGAGCCTTTCTCGAGAAAGGCTCGATTTAGCTTCCGTTTTTTG-3′ShYTHDC2_3#(pLKO.1): 5′-CCGGGCCTTGGATGTAAATCTCTTTCTCGAGAAAGAGATTTACATCCAAGGCTTTTTTG-3'.Lentivirus for shRNA-scramble, shRNA-YTHDC2_1#, shRNA-YTHDC2_3#, pLVX-Vec, pLVX-YTHDC2 were packaged with pMD2.G and psPAX2 (purchased by addgene). Briefly, 2.24 μg psPAX2, 0.76 μg pMD2.G and 1.5 μg pLKO-shYTHDC2 were co-transfected into HEK-293T cells in 60 mm dish with ViaFect™ Transfection Reagent (Promega). The Lentivirus particles were harvested at 48 and 72 h after transfection and concentrated with PEG8000 Virus Precipitation Solution (the concentrations of PEG-8000 and NaCl in the stock solution are 40% (W/V) and 1.2M, respectively). Finally, the concentrated lentivirus particles were directly added into the NPC cells and incubated at 37°C for 48 hr before they were washed by PBS. One μg/ml puromycin  was added into culture medium for the selection of stable NPC cell lines.NPC cell lines were lysed in a standard RIPA buffer containing proteinase and phosphatase inhibitors , and the protein concentrations were quantified using Bradford reagent (Bio-Rad). Then prepared protein was separated via SDS-PAGE and transferred to NC membrane . The primary antibodies used in the study include anti-METTL3 , METTL14 , FTO, ALKBH5 , WTAP , YTHDF1 , YTHDF2 , YTHDF3 , YTHDC1 , YTHDC2 , IGF1R , AKT , Phospho-AKT , S6 Ribosomal protein , and Phospho-S6 Ribosomal protein (ser240/244). The bound antibodies were detected with HRP-conjugated secondary antibodies (Abcam). Densitometric analysis of WB results was performed with ImageJ software.t-test was used to calculate statistical significance. Cell apoptosis assay was carried out according to the apoptosis kit (BD biosciences).Cell viability was detected by CCK-8 (Biomake) following the manufacturer's guideline. All viability experiments were repeated in three independent times, and Student's −ΔΔCT method was used to calculate the relative expression of YTHDC2. The ΔΔCt (ddCt) method was as described below:Total RNA was isolated using TRIzol (Invitrogen). A total of 1,000 ng RNA was reverse-transcribed into cDNA in a total reaction volume of 20 μl with PrimeScriptTM RT Reagent Kit according to manufacturer's instructions (Takara). The qPCR primer sequences were listed in ΔCt = Ct (target gene)-Ct (ref gene) andΔΔCt = ΔCt (target sample)-ΔCt (ref sample).We got the relative mRNA expression between two samples  by calculation of the following formula:Relative mRNA expression = B/A.−ΔΔCT.A : 2−ΔΔCT.B (radioresistant cells):22PO4, 8 mmol/L Na2HPO4, and 150 mmol/L NaCl). Then, 0.01 mol/L PBS supplemented with 5% normal goat serum and 0.1% Triton X-100 was used to block the sections, followed by an incubation with anti-YTHDC2  overnight at 4°C. After three times washing in PBS and treatment with adjuvant for 20 min at 37°C, the sections were exposed to secondary antibody for 2 h at 37°C. The immunohistochemical reaction was visualized with 3, 3, 0-diaminobenzidine (DAB) for 3 min. All sections were counterstained with haematoxylin. Both the staining intensity and extent of protein expression were automatically scored by Vectra 2 system .Paraffin-embedded NPC samples were collected from Xiangya Hospital of Central South University. Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval, and endogenous peroxidases were blocked. The sections were washed three times with 0.01 mol/L PBS . Colonies consisting of more than 50 cells were defined as surviving colonies. All viability measurements are normalized with the 0 Gy group. Cell survival curves were fitted by Graph Pad software.NPC cells were seeded in 6-well plates at densities of 1 × 102)/2.Two thousand cells/well were plated in 24-well, flat-bottomed plates using a two-layer soft agar system in a volume of 400 μl/well. After the incubation for 15 days, colonies were counted and measured. Four fields were randomly selected for colony counts (>50 μm diameter and >100 μm diameter) under an inverted microscope  at 40× magnification. The freeware ImageJ  and Adobe Photoshop  were used in colony diameter measurement. Thirteen colonies were randomly selected for diameter measurement. Colony volume was calculated using the formula: volume =  and analyzed by RT-PCR. The turnover rate was assessed according to a previously published paper .2, 100 mg/ml CHX, 1% Triton X-100, with freshly added 1:100 protease inhibitor (Roche) and 40 U/ml SUPERasin (Ambion). The sample was then fractionated into 24 fractions (0.5 mL per fraction) and analyzed by Gradient Station (BioCamp) equipped with ECONOUV monitor  and Gilson FC203B fraction collector . RNA was purified from whole cell, 40S, 60S, 80S, and Polysome fractions and subjected to qPCR analysis. Expression of IGF1R in each fraction was normalized to TUBULIN as well as Input.We followed the reported protocols with the following modifications , 32. CNE7) were UV–cross-linked at 254 nm . After three washing with cold PBS, the cells were scraped with 1 ml ice-cold PBS and collected in 1.5 ml EP tube. Centrifuge cells at 300 g for 5 min at 4°C, then remove the supernatant and resuspend cells in 200 μl of lysis solution  for 30 min vigorous shaking. After centrifuging the 1.5 ml tube, we transferred supernatant to a new 1.5 ml tube and added DNA I for 30 min. Next, whole cell lysate was incubated with anti-flag antibody for 4 h at 4°C with continuous mixing (rotating or roller mixer). Forty microliters of protein A/G agarose beads were added to recruit RNA–protein complexes. RNAs associated with PTBP1 were recovered with Trizol-chloroform and analyzed by RT-PCR or qPCR.We following the previous protocols with the following modifications , 34. CNE6 cells resuspended in 50 μl of Matrigel (Corning) were subcutaneously injected into 4–6 weeks old male nude mice. When tumor volumes reached 150–200 mm3, animals were divided into control group and radiotherapy group. In the radiotherapy group, tumors were treated with a single irradiation (4 Gy) when tumor volumes reached approximately 150–200 mm3. The tumor stopped growing in the next few days and then restarted growth. Three weeks later, the tumors were harvested, and tumor weight and volume were measured. Tumor volumes were calculated by l × w2/2 .All animal procedures were approved by the Animal Ethics Committee of Central South University; 2 × 10GSE102349 . The used primers are listed in P-values are presented as star marks in figures: *P < 0.05, **P < 0.01, ***P < 0.001. No statistical analysis was used to predetermine sample size and the suitability of statistical approaches.Statistical analyses were performed using SPSS 20.0 (SPSS) and GraphPad Prism, version 7. Flow cytometry data was analyzed using FlowJo 10.01. The details of statistical methods for each experiment were indicated in the respective figure legends. Results are expressed as means ± SD. For comparisons between groups, statistical analyses were performed using the Mann–Whitney test. 6A modification, an analysis of an NPC transcriptome-sequencing dataset (GSE48501 including radiosensitive cell line CNE2 and radioresistant cell line CNE2-IRR) from the GEO database was performed. Among all the genes, METTL3 and YTHDC2 were found to be expressed at remarkably higher levels in the CNE2-IRR cell line than in the parental cell line CNE2  assay. We found that YTHDC2 expression was elevated in patient specimens of partial response (PR) compared to those of complete response (CR) . FurtherAn association between YTHDC2 expression and the radiotherapy effect in clinical samples and cell lines prompted us to explore the impact of YTHDC2 on radioresistance. We first knocked down the endogenous YTHDC2 by using lentiviral infection of two different short hairpin (sh)-RNAs in CNE2-IRR and HK1-IRR cells . The YTHNext, we examined whether the enforced expression of YTHDC2 impacted the response of cancer cells to irradiation. YTHDC2 was overexpressed in the radiosensitive NPC cell line CNE2 and HK1 . As show6A reader by binding to specific sites of RNAs, we searched the m6AVar and RMbase databases  and found that G/C rich regions were distributed in the transcriptional starting site of YTHDC2 gene, and a methylated CpG island (CGI) was located at the promoter of YTHDC2  subfamily of proteins, part of the DEAD (Asp-Glu-Ala-Asp) box family of RNA helicases. It was reported that several DEAH/D box RNA helicases promote translation initiation of mRNA , 44. In r YTHDC2 . Thus, wr YTHDC2 . The RNAr YTHDC2 . Furtherr YTHDC2 . In addir YTHDC2 . Taken tin vivo.To confirm our observations of the effect of YTHDC2 on radiosensitivity, an NPC xenograft mouse model was established by using a subcutaneous injection of CNE2-IRR cells infected with YTHDC2 shCON, shYTHDC2-1, and shYTHDC2-3 lentivirus, respectively. Firstly, we found that knockdown of YTHDC2 had little effect on tumor growth without radiotherapy . However6A methylation of RNAs plays important roles in various cancers. However, it is unclear whether functional proteins participating in m6A modification are involved in regulating radiation resistance. Herein, we found the aberrantly high expression of YTHDC2, an m6A reader protein, in radioresistant NPC cells, which might be due to hypomethylation of the YTHDC2 promoter. YTHDC2 could activate the PI3K-AKT/S6 pathway by regulating the translation of IGF1R mRNA. In consistence of our conclusion, several studies revealed that the PI3K-AKT pathway played an important role in a tumor's radiation response . In fact, all CpG dinucleotides are methylated with the general exception of CGIs, and the high methylation of CGIs is generally associated with the inhibition of promoter activity . Moreovein vivo and in vitro.In our study, we found that YTHDC2 regulated the protein expression of IGF1R by affecting translation IGF1R mRNA, which is consistent with the previous studies. IGF1R is a transmembrane receptor activated by a hormone called insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. Ligand binding activates the receptor kinase (such as IGF1R), subsequently leading to increasing activation of downstream pathways (such as the PI3K-AKT/PKB pathway and Ras-MAPK pathway). The resultant activation of the MAPK pathway will lead to cell proliferation, whereas activation of the PI3K pathway inhibits apoptosis and stimulates protein synthesis. Hyperactive PI3K/AKT signaling may account for the increased radiation resistance of cancer cells. It has been suggested that AKT is the key molecule in PI3K-mediated radioresistance by promotion of the repair of IR-induced DNA double-strand breaks, maintenance of the stemness of cancer cells, and inhibition of IR-induced autophagy , 54. In 6a reader YTHDC2 in NPC cells promotes the translation efficiency of IGF1R mRNA, resulting in resistance to irradiation (In summary, our findings demonstrated for the first time that the high expression of the madiation .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The human study was approved by the Institute Research Medical Ethics Committee of Central South University. All samples were deidentified and all patients signed informed consents.J-JH, Y-MD, QZ, and L-QS designed the study. J-JH, Y-MD, and ZL performed the experiments. J-JH, QF, and Y-MD performed the animal experiments. Z-XR, JG, YM, Y-DG, X-XR, Y-YZ, and L-YL performed the immunohistochemistry. J-JH, Y-MD, and MZ completed the statistics and interpreted the data. Y-ZD provided the critical suggestions. All authors contributed to the article and approved the submitted version.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.


Lamin A/C has been implicated in the epigenetic regulation of muscle gene expression through dynamic interaction with chromatin domains and epigenetic enzymes. We previously showed that lamin A/C interacts with histone deacetylase 2 (HDAC2). In this study, we deepened the relevance and regulation of lamin A/C-HDAC2 interaction in human muscle cells. We present evidence that HDAC2 binding to lamin A/C is related to HDAC2 acetylation on lysine 75 and expression of p300-CBP associated factor (PCAF), an acetyltransferase known to acetylate HDAC2. Our findings show that lamin A and farnesylated prelamin A promote PCAF recruitment to the nuclear lamina and lamin A/C binding in human myoblasts committed to myogenic differentiation, while protein interaction is decreased in differentiating myotubes. Interestingly, PCAF translocation to the nuclear envelope, as well as lamin A/C-PCAF interaction, are reduced by transient expression of lamin A mutated forms causing Emery Dreifuss muscular dystrophy. Consistent with this observation, lamin A/C interaction with both PCAF and HDAC2 is significantly reduced in Emery–Dreifuss muscular dystrophy myoblasts. Overall, these results support the view that, by recruiting PCAF and HDAC2 in a molecular platform, lamin A/C might contribute to regulate their epigenetic activity required in the early phase of muscle differentiation. A fine regulation of acetyltransferases (HAT) and histone deacetylases (HDACs) activity is at the basis of all physiological cellular processes including skeletal myogenesis ,2. It haLMNA gene mutations are responsible for the onset of autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD2), which is characterized by skeletal muscle weakness and wasting, joint contractures and cardiomyopathy [LMNA mutations causing EDMD2, has been reported [Lamin A/C is the major constituent of the nuclear lamina. Mature lamin A is produced from its precursor prelamin A by complex post-translational processing including farnesylation, double endoprotease cleavage and carboxymethylation . First, myopathy ,19. A coreported ,21. Morereported ,23.We have previously demonstrated that lamin A/C binds HDAC2 in human fibroblasts . While lLMNA mutations causing EDMD2 and lamin A/C-HDAC2 interaction was impaired in EDMD2 myoblasts. These results add to the evidence that a functional lamina is required for epigenetic enzyme regulation during myogenesis.A main role of lamin A/C interplay with epigenetic enzymes in myogenesis has been demonstrated by several studies ,27. LamiLMNA pathogenetic mutants used in this study were: LA-R527P and LA-R401C, linked to EDMD2 [mHDAC2-K75R-F: 5’-AGCGATGAGTATATCAGGTTTCTACGATCAATA-3’mHDAC2 K75R-R: 5’-TATTGATCGTAGAAACCTGATATACTCATCGCT-3’mHDCA2-K75Q-F: 5’-AGCGATGAGTATATCCAGTTTCTACGATCAATA-3’mHDCA2-K75Q-R: 5’-TATTGATCGTAGAAACTGGATATACTCATCGCT-3’HEK293 cells (human embryonic kidney cells) were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (FBS), and were transfected with HA-PCAF or FLAG-tagged plasmids containing: wild-type prelamin A, LA-WT, which undergoes normal maturation; LA-C661M, which cannot be farnesylated; LA-L647R, which is farnesylated but cannot undergo endoproteolysis. to EDMD2 . HA taggto EDMD2 ,30. FlagAfter mutagenesis, ORF of each plasmid was verified by a DNA sequencing service .LMNA (Patient1) and L140P-LMNA (Patient2), were obtained from the BioLaM biobank (approved by the “Rizzoli Orthopedic Institute Ethics Committee” on 05/09/2016. Prot. gen “0018250-01-13”). All EU and local ethical rules were respected. Cell cultures established as reported [Transfection of HEK293 cells was performed using Fugene 6 solution  according to the manufacturer’s instructions and cells were incubated for 24 h after transfection. C2C12 cells (mouse myoblast cell line) were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% FBS and differentiated with 2% of horse serum (HS) for 12 h to obtain committed myoblasts and 24 h to obtain early stage myotubes. Human myoblast cultures from control donor and EDMD2 patients Y259D-reported  were culAntibodies employed were: anti-PCAF (sc-13124) from Santa Cruz  used 1:50 for immunofluorescence (IF) or 1:100 for western blotting (WB); anti-lamin A/C, goat polyclonal (SC-6215) from Santa Cruz  used at 1:100 dilution for and in situ proximity ligation assay (PLA); anti-HDAC2, rabbit polyclonal (AB16032) from Abcam  used at 1:2000 for WB and 1:200 for PLA analysis; anti-H3K9 acetylated, rabbit polyclonal (07-352) from Merck Millipore  used at 1:200 for IF; anti-FLAG tag (F3165) from Sigma  and anti-HA tag (sc-7392) from Santa Cruz  were used at 1:1000 in WB and 1:300 in IF; anti-GFP (sc-9996) from Santa Cruz  was used at 1:500 in WB; anti acetyl-lysine (9824S) from Cell Signaling  was used at 1:2000 in WB; anti myogenin from Santa Cruz  was used at 1:100 in IF.A total of 2 μM of Mevinolin , a drug able to induce non-farnesylated prelamin A accumulation through inhibition of farnesyl production, was added to HEK293 cells for 18 h. A total of 100 μM of Sirtinol, a SIRT1 inhibitor, able to induce PCAF acetylation , was add® In Situ Detection Reagents Orange (DUO92007) according to manufacturer instructions. Briefly, methanol-fixed samples were treated with 4% BSA in PBS to saturate non-specific binding and were incubated with primary antibodies overnight at 4 °C. Thereafter, slides were incubated for 1 h at 37 °C with secondary probes diluted to final concentrations of 1:5. Ligation solution was added to each sample and slides were incubated in a humidity chamber for 30 min at 37 °C. Later, ligation solution was removed with wash buffer A and amplification solution was added to each sample. Slides were incubated in a humidity chamber for 100 min at 37 °C and then washed with wash buffers. Duolink in situ mounting medium with DAPI was added to the slides and samples were observed by a Nikon Eclipse Ni fluorescence microscope equipped with a digital CCD camera and NIS Elements AR 4.3 software. Quantitative analysis of PLA results was performed using Duolink Image Tool software  by counting 200 nuclei per sample.In situ proximity ligation assay (PLA) was performed using kits from Sigma : DuolinkTransfected HEK293 cells were subjected to IP lysis buffer containing: 50mM Tris-HCl (ph = 8), 150 mM NaCl; 1% NP-40; 1.5 mM MgCl; 1 mM DTT; 1 mM PMSF; 20 mM NaF; 0.05% SDS; phosphatases and proteases inhibitors. After sonication, clarification and protein quantification, 700 μg of lysate for samples were immunoprecipitated with 1 μL/mL of anti-FLAG antibody  over-night at 4 °C. After the addition of 30 µL/mL of protein A/G  for 60 min at 4 °C, the immunoprecipitated proteins were washed with lysis buffer 3 times. Subsequently, immunoprecipitated samples were added to Laemmli’s buffer and subjected to immunoblotting analysis. Incubation with primary and secondary antibodies was performed and immunoblotted bands were revealed by Invitrogen ECL detection system. Densitometric analysis was performed with ImageJ2 program.Cells grown on coverslips were fixed with absolute methanol at room temperature for 10 min or with 4% paraformaldehyde for 15 min at 4 °C (plus 5 min with 0.02% triton x-100 for permeabilization). After saturation of non-specific binding with PBS, containing 4% BSA for 20 min, coverslips were incubated with primary antibodies overnight at 4 °C or 1 h, and revealed with FITC, Cy5 or TRIC-conjugated secondary antibodies diluted at 1:100 (incubated for 1 h at RT). Samples were mounted with an anti-fade reagent  and observed with different microscopes.Immunofluorescence and PLA analysis were performed using a Nikon Eclipse Ni epifluorescence microscope with 40× and 100× objectives. The images captured with NIS-Elements 4.3 AR software, were elaborated using Photoshop CS.Confocal analysis was performed using a Nikon A1 confocal laser scanning microscope, equipped with a 60×, 1.4 NA objective and with 405, 488, and 561 nm laser lines. Z-stacks were collected at optical resolution of 80 nm/pixel, stored at 12-bit with 4096 different gray levels, pinhole diameter set to 1 Airy unit and z-step size to 200 nm.Nikon-Structured Illumination Microscopy (3D N-SIM) which permits observing fluorescent samples at resolutions below the limit the diffraction of light imposed by optical microscopy (85–100 nm) was performed using a Plan-Apochromat 100×/1.49 Oil TIRF objective and 488 and 561 nm laser lines. For each axial plane of a 3D stack 1024 × 1024 pixel images and 4096 gray levels were acquired in 3 rotations and 5 different phases. Final images (recorded at z-step size of 125 nm) were reconstructed using NIS Elements Advanced Research software . Protein colocalization was evaluated by comparing the equivalent pixel positions of green and red signals of fluorophores in each of the acquired images . A two-dimensional scatter plot diagram of the individual pixels from the paired images was generated and a threshold level of signal to be included in the analysis was selected. Pixels with intensity values greater than 50% grey levels  were selected for both signals, and the colocalization binary maps that indicate regions containing highly colocalized signals were imaged and merged (in white) to the green and red signals ,33. ImagOur recent findings supported the hypothesis that HDAC2 acetylation could influence lamin A/C-HDAC2 interplay . To testSince it has been demonstrated that the acetyltransferase PCAF induces HDAC2 acetylation on lysine 75 , we deciTo further support the evidence that PCAF activity might influence HDAC2 affinity for lamin A/C, human myoblasts were treated with sirtinol, a SIRT1 inhibitor able to induce PCAF autoacetylation and activation ,34. HighDuring muscle differentiation, PCAF and HDAC2 have opposite regulatory effects and the balancing between these enzymes regulates MyoD acetylation and its transactivation activity ,6. For tInterestingly, enrichment at the nuclear periphery of lamin A/C-PCAF and lamin A/C-HDAC2 complexes was observed in committed cells c. MoreovTo deepen the relationship between PCAF and lamin A/C, HEK293 cells were co-transfected with HA-tagged PCAF and FLAG-Lamin A and immunofluorescence analysis was performed with antibodies against the respective tags. In cells overexpressing PCAF alone, PCAF had a typical nucleoplasmic localization , while cIt has been reported that lamin A processing intermediates are accumulated in myoblasts during myogenic differentiation ,17. In pLMNA mutations could affect PCAF recruitment to the nuclear lamina and enzymatic activity. To test this possibility, we performed immunofluorescence analysis of HEK293 cells co-expressing HA-PCAF and EDMD2-linked LA-R527P and LA-R401C. In cells expressing lamin A pathogenetic mutants, we observed a reduced percentage of nuclei with PCAF recruitment to the nuclear envelope (relative to cells expressing wild-type lamin A)  a. Interelamin A) b. To suple cells .Since we observed that HDAC2 acetylation by PCAF influences its binding to lamin A/C, we decided to investigate HDAC2-lamin A/C interaction in EDMD2 cells. Interestingly, PLA demonstrated a reduction of lamin A/C-HDAC2 binding in EDMD2 cells with respect to controls, both in committed myoblasts and myotubes . These rRecent literature demonstrates an emerging role of lamin A/C in epigenetic regulation of myogenesis ,26. We pOur findings allow us to speculate that lamin A/C could contribute to HDAC2-PCAF dynamic interaction aimed at the regulation of muscle-specific transcription factors. For instance, during myogenesis, an antagonistic function of PCAF and HDAC2 on MyoD activation has been demonstrated ,6,9. In Interestingly, we observe that PCAF recruitment to the nuclear lamina is significantly increased by farnesylated prelamin A, the lamin A precursor, which is accumulated during differentiation of human muscle cells ,17. WhilIn this context, it is possible that lamin A/C-HDAC2 complexes, in differentiating muscle cells could also serve to move HDAC2 away from transcriptionally inactive loci of muscle genes, thus promoting gene expression. In support of this hypothesis, it has been demonstrated that lamin A/C binding to muscle gene promoters was reduced when myoblasts were induced to differentiate, while histone deacetylase inhibitors, reduced lamin A/C localization to the myogenic promoters .The altered lamin A/C interaction with HDAC2 and PCAF in EDMD2 myoblasts suggests that this function is also relevant to disease pathogenesis. Despite our previous results that showed that lamin A/C-HDAC2 interaction is not affected in human fibroblasts carrying pathogenetic EDMD2 mutations , here weLMNA mutations affect lamin A/C-PCAF binding and PCAF or HDAC2 recruitment might differently impair the expression of different muscle-specific genes. In this perspective, aberrant epigenetic enzyme regulation by mutated lamin A/C could contribute to the phenotypic variability observed in laminopathic patients. Finally, given the role of lamin A/C in epigenetic enzyme regulation, epigenetic drugs could be a new challenge for a therapeutic approach to laminopathies. For instance, modulating lamin A/C-HDAC2 binding by TSA in EDMD2 could be tested as an approach that might improve the transcriptional activity of muscle cells through improved PCAF-dependent HDAC2 acetylation.The extent to which