Nirmatrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro), is clinically useful against infection with SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to many monoclonal antibody therapies, potential SARS-CoV-2 resistance to nirmatrelvir is a major public health concern. Several amino acid substitutions have been identified as being responsible for reduced susceptibility to nirmatrelvir. Among them, we selected L50F/E166V and L50F/E166A/L167F in the 3CLpro because these combinations of substitutions are unlikely to affect virus fitness. We prepared and characterized delta variants possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F. Both mutant viruses showed decreased susceptibility to nirmatrelvir and their growth in VeroE6/TMPRSS2 cells was delayed. Both mutant viruses showed attenuated phenotypes in a male hamster infection model, maintained airborne transmissibility, and were outcompeted by wild-type virus in co-infection experiments in the absence of nirmatrelvir, but less so in the presence of the drug. These results suggest that viruses possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F do not become dominant in nature. However, it is important to closely monitor the emergence of nirmatrelvir-resistant SARS-CoV-2 variants because resistant viruses with additional compensatory mutations could emerge, outcompete the wild-type virus, and become dominant. Resistance to nirmatrelvir, an oral antiviral agent that targets SARS-CoV-2 and is clinically useful against infection with Omicron variants, is currently not well understood. In this study, the authors characterize mutant viruses with reduced sensitivity to nirmatrelvir in vitro and in vivo. Omicron variants possess more than 30 amino acid substitutions in the spike protein, and its subvariants BA.4/5, BA.4.6, and BA.2.75 are less susceptible to therapeutic monoclonal antibodies including REGEN-COV [casirivimab (REGN10933) plus imdevimab (REGN10987)], bamlanivimab (LY-CoV555) plus etesevimab (LY-CoV016), evusheld [tixagevimab (COV2-2196) plus cilgavimab (COV2-2130)], and xevudy [sotrovimab (S309)]6. BA.4/5, BA.4.6, and BA.2.75 are susceptible to bebtelovimab (LY-CoV1404) but the latest subvariants BQ.1.1 and XBB escape from it7. The antiviral compounds nirmatrelvir, remdesivir, molnupiravir, and ensitrelvir, which target virus proteins other than the spike protein, are effective against such variants and subvariants.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, is still prevalent three years since its emergence and continues to spread around the world. It accumulates amino acid substitutions frequently and many variants of concern (VOCs) have appeared and caused several waves of infection. Among these variants, omicron (lineage B.1.1.529) was identified at the end of 2021 and became globally dominant9 and nirmatrelvir11.Several antivirals have been widely used for to treat COVID-19 including GS-441524 (remdesivir), EIDD-1931 (molnupiravir), PF-07321332 (nirmatrelvir), and S-217622 (ensitrelvir). Remdesivir and molnupiravir inhibit virus RNA-dependent RNA polymerase, whereas nirmatrelvir and ensitrelvir interfere with 3CL protease . As these drugs are increasingly used in clinical settings, there is concern about the emergence of viruses with reduced sensitivity or resistance. Amino acid substitutions associated with resistance were reported for remdesivir12. When treatment is initiated within three days of onset, approximately 90% protection against severe COVID-19 and hospitalization has been reported13. Amino acid substitutions that are responsible for resistance to nirmatrelvir have been reported in 3CLpro (nsp5). In silico analysis showed that the N142L, E166M, Q189E, Q189I, and Q192T substitutions reduced the potency of nirmatrelvir in vitro14. A screen using a VSV-based system revealed that the Y54C, G138S, L167F, and Q192R substitutions confer resistance to nirmatrelvir15. Naturally occurring amino acid substitutions such as S144A, A173V, and E166D/G also contribute to resistance to nirmatrelvir16. Deep mutational analysis has shown that the E166V, P252L, and T304I substitutions reduce the IC50 value to nirmatrelvir18. In another study, resistant viruses possessing the E166V and T304I substitutions in combination with other amino acid substitutions in nsp5 were selected through virus passaging in the presence of nirmatrelvir in cell culture19. The E166V and T304I substitutions conferred strong resistance but resulted in a loss of viral fitness; viral fitness was restored by compensatory substitutions such as L50F and T21I18. The L50F and T21I substitutions had little effect on the sensitivity to nirmatrelvir18. Another resistant virus selection experiment in vitro identified the combination of the L50F, E166A, and L167F substitutions as being associated with resistance to nirmatrelvir20. Each E166A and L167F substitution conferred reduced sensitivity to nirmatrelvir20. Recent studies have reported that the virus fitness of viruses possessing the L50F and E166V substitutions and the L50F, E166A, and L167F substitutions is comparable to that of wild-type virus in cell culture20. Another preprint reported that a recombinant virus possessing the L50F, E166A, and L167F substitutions showed similar pathogenicity in hamsters and transmitted to co-housed hamsters21. Therefore, among the many resistant mutations reported, we chose to study Nsp5-L50F/E166V19 and Nsp5-L50F/E166A/L167F20 because they appeared unlikely to affect virus fitness in vitro. By using reverse-genetics based on the delta variant, we prepared mutant viruses possessing these substitution combinations and characterized their replication in vitro, their pathogenicity in hamsters, and their airborne transmissibility in hamsters. We also assessed their replication in co-infection experiments with wild-type virus in the presence or absence of nirmatrelvir in the hamster model.Nirmatrelvir (PF-07321332) is co-formulated with the pharmacokinetic enhancer ritonavir 50 value of PF-07321332 (nirmatrelvir) against wild-type virus was 8.08 μM , whereas those against Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F were greater than 100 μM. Although EIDD-1931 (molnupiravir) and GS-441524 (remdesivir) showed similar IC50 values against all three viruses tested, the sensitivity of Nsp5-L50F/E166V to S-217622 (ensitrelvir) was moderately reduced and that of Nsp5-L50F/E166A/L167F was appreciably reduced; the IC50 values were 1.41 μM  and 19.34 μM . These results indicate that both mutant viruses have reduced sensitivity to PF-07321332 (nirmatrelvir) and show reduced sensitivity to S-217622 (ensitrelvir) as well.To characterize mutant viruses possessing amino acids mutations that are responsible for reduced sensitivity to nirmatrelvir, we prepared a wild-type delta B.1.617.2) variant and its mutant viruses possessing the L50F and E166V substitutions in Nsp5 (Nsp5-L50F/E166V) or the L50F, E166A, and L167F substitutions (Nsp5-L50F/E166A/L167F) by using a BAC-based reverse genetics system. No nucleotide mixtures of more than 10% were identified in the three stock viruses. These three viruses were tested for sensitivity to the antivirals PF-07321332 (nirmatrelvir), EIDD-1931 (molnupiravir), GS-441524 (remdesivir), and S-217622 (ensitrelvir) in a focus reduction assay  and lungs ((p = 0.00794 or p = 0.0476) of the treated hamsters compared to the untreated animals, the wild-type virus was predominant in most of the hamster nasal turbinates. However, in the lungs of three hamsters, Nsp5-L50F/E166V became dominant, and in the lungs of one hamster, Nsp5-L50F/E166A/L167F became dominant. These results demonstrate that although Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F are less fit than the wild-type virus, Nsp5-L50F/E166V could be problematic by dominating the wild-type virus in lungs in the presence of nirmatrelvir.We next examined the competitive growth capability of each virus by co-infecting the wild-type virus with a mutant virus at a ratio of 3:7 based on virus infectious titers. The ratios of each mutant virus to wild-type virus in the inoculum based on deep sequence analysis were 48:52 (wild-type: Nsp5-L50F/E166V) and 42:58 (wild-type: Nsp5-L50F/E166A/L167F) , since it is the antiviral drug used most often to treat COVID-1924. Some of these drug-resistant viruses are less fit than the wild-type virus25, but others are not28. Regardless of whether viral fitness is reduced, such resistant viruses rarely spread worldwide. If a resistant virus does spread globally, either the selective pressure is extremely high due to the widespread use of the antiviral (which is unlikely), or the resistant virus acquires greater fitness through the acquisition of additional amino acid substitutions29. Combination therapy is thought to be a better strategy to treat viral infections and reduce the likelihood of emergence of resistant viruses30, but it is possible for viruses resistant to both antivirals to emerge32.Viruses, especially RNA viruses, generate variants. Although SARS-CoV-2 has a proofreading mechanism, many people have been infected and each individual producing a large amount of virus could lead to the emergence of viruses resistant to monoclonal antibodies or antivirals when used as monotherapy. In influenza patients, viruses resistant to neuraminidase (NA) or PA inhibitors have been detected after treatment with the antiviralsIn conclusion, Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F are unlikely to dominate in nature. However, secondary adaptive mutations may make these mutant viruses dominant to wild-type virus. Moreover, mutant viruses possessing other resistance-conferring substitutions could emerge and dominate in the human population. Therefore, we need to closely monitor the amino acid mutations that confer resistance to nirmatrelvir and secondary adaptive mutations by undertaking active virus surveillance to detect such viruses as soon as possible after emergence.All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan.2. The cells were regularly tested for mycoplasma contamination by using PCR and confirmed to be mycoplasma free.VeroE6/TMPRSS2 (JCRB 1819) cells were propagated in the presence of 1 mg/ml geneticin  and 5 μg/ml plasmocin prophylactic (Invivogen) in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. VeroE6-TMPRSS2-T2A-ACE2 cells were cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin–streptomycin, and 10 µg/mL puromycin. HEK293T cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS. All cells were maintained at 37 °C with 5% COActive components of remdesivir and molnupiravir , and nirmatrelvir (PF-07321332) were purchased from MedChemExpress. S-217622 (ensitrelvir) was kindly provided by Shionogi Co., Ltd. Compounds were dissolved in dimethyl sulfoxide (in vitro) or 0.5% methylcellulose (in vivo) prior to use.34. The mutations responsible for the L50F/E166V and L50F/E166A/L167F substitutions in 3CLpro were introduced during the PCR step. To rescue these viruses, pBeloBAC11 encoding the wild-type virus , Nsp5-L50F/E166V (Accession No. OR116092), or Nsp5-L50F/E166A/L167F (Accession No. OR116093) was transfected into HEK293T cells. At 3 days post-transfection, the supernatant containing the viruses was collected and inoculated onto VeroE6/TMPRSS2 at 37 °C to prepare virus stocks. The stock viruses were deep sequenced to confirm the absence of unwanted mutations, and no position contained unwanted nucleotides that exceeded 10% of the population in all stock viruses.The full-genome nucleotide sequence of SARS-CoV-2  was assembled into the pBeloBAC11 vector to generate infectious cDNA clones under the control of a cytomegalovirus (CMV) promoter as described previously35. In brief, viral RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN). cDNA was synthesized by using a Lunar-Script RT SuperMix Kit (New England BioLabs) and subjected to a multiplexed PCR in two pools using ARTIC-N1 primers v5 and Q5 Hot Start DNA polymerase (New England BioLabs). The DNA libraries for Illumina NGS were prepared from pooled amplicons by using a QIAseq FX DNA Library Kit (QIAGEN) and then analyzed by using the iSeq100 System in 150-bp paired-end mode using an iSeq 100 i1 Reagent v2 (300-cycle) kit (Illumina). The reads were assembled by CLC Genomics Workbench . The average coverage depths of the wild-type virus, L50F/E166V, and L50F/E166A/L167F were 8,078×, 10,742×, and 8,731×, respectively.The whole genome of SARS-CoV-2 was amplified by using a modified ARTIC network protocol in which some primers were replaced or added36. Briefly, VeroE6-TMPRSS2-T2A-ACE2 cells in 96-well plates were infected with SARS-CoV-2 at 100–400 focus forming unit/well. After a 1-h incubation at 37 °C, the inoculum was replaced with 1% Methyl Cellulose 400  in culture medium containing serial dilutions of the antiviral compounds. The cells were incubated for 18 h at 37 °C and then fixed with formalin. The cells were stained with a mouse monoclonal antibody against SARS-CoV-2 nucleoprotein, clone N45 , followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories Inc.). Foci were visualized by using TrueBlue Substrate (SeraCare Life Sciences). The focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The 50% inhibitory concentration (IC50) values and 95% confidence intervals were calculated by using GraphPad Prism 9.3.0 (GraphPad Software).Antiviral susceptibilities were determined by using a focus reduction assay as previously reportedVeroE6/TMPRSS2 cells were infected with the indicated virus at a multiplicity of infection (MOI) of 0.001. After a 1-h incubation at 37 °C, the inoculum was replaced with medium ± 20 μM nirmatrelvir. Cell culture supernatants were collected at 8, 16, 24, 32, 40, 48, 56, and 72 h post-infection. Virus titers were determined by the use of a plaque assay in VeroE6/TMPRSS2 cells.5 plaque forming unit (PFU) of the indicated virus. Body weights were measured daily before inoculation and 10 days after inoculation. Respiratory parameters [Penh (a nonspecific assessment of breathing patterns) and Rpef (a measure of airway obstruction)] were also measured by using a whole-body plethysmography system (PrimeBioscience) as previously described38.Five- to six-week-old male Syrian hamsters (Japan SLC) were used in this study. Since SARS-CoV-2 occasionally fails to replicate in female hamsters, we used only male hamsters in this study. Under isoflurane anesthesia, five hamsters per group were intranasally inoculated with 105 PFU of the indicated virus. At 3 and 6 days post-infection, the animals were euthanized and nasal turbinates and lungs were collected. The virus titers in these organs were determined by use of plaque assays on VeroE6/TMPRSS2 cells.For the virus titration, five hamsters per group were intranasally infected with 1040. Briefly, five hamsters per group were intranasally infected with 105 PFU of each indicated virus and placed in cages. One day after infection, naïve hamsters were placed in adjacent cages, 5-cm apart, separated by a double-layer partition with unidirectional air flow. Infected and exposed hamsters were euthanized at 4 days post-infection or 3 days after exposure. Nasal turbinates and lungs were collected for virus titration on VeroE6/TMPRSS2 cells.The transmission study was performed as previously described5 PFU) was inoculated into ten hamsters per group. One day after inoculation, five hamsters per group were treated orally with nirmatrelvir at 250 mg/kg twice daily as previously described42. The remaining five hamsters per group were left untreated. The animals were euthanized at 4 dpi and their nasal turbinates and lungs were collected for deep sequence analysis. The ratio of wild-type to Nsp5-L50F/E166V or Nsp5-L50F/E166A/L167F was calculated from the differences at positions 10202 and 10551 or 10202, 10551, and 10555, respectively. Samples with read depths of more than 1000 were analyzed.For the co-infection study, wild-type virus and Nsp5-L50F/E166V or Nsp5-L50F/E166A/L167F were mixed at a ratio of 3:7 based on their titers, and the virus mixture (total 2 × 10P values. We compared virus titers in hamsters with the control by using a one-way ANOVA followed by Tukey’s multiple comparisons. Body weight and virus growth kinetics were compared by using a two-way ANOVA followed by Tukey’s multiple comparisons. Transmission efficiency was compared by using a two-sided Fisher’s exact test. The frequency of wild-type virus in the nasal turbinate and lungs of the treated hamsters was compared with that of the untreated hamsters by using the Mann–Whitney test followed by the two-stage step-up procedure of the Benjamini, Krieger, and Yekutieli test. Differences between groups were considered significant for P values < 0.05.GraphPad Prism software version 9.3.0 was used to calculate All reagents described in this paper are available through Material Transfer Agreements.Further information on research design is available in the Reporting SummaryPeer Review FileSouce data file


The IL-10/IL-10 receptor (IL-10R) axis plays an important role in attenuating neuroinflammation in animal models of Multiple Sclerosis (MS) and increased IL-10 has been associated with a positive response to MS disease modifying therapy. Because environmental factors play an important role in MS susceptibility and disease course, identification of environmental factors that impact the IL-10/IL-10R axis has therapeutic potential. In this review, we provide historical and updated perspectives of how IL-10R signaling impacts neuroinflammation, discuss environmental factors and intestinal microbes with known impacts on the IL-10/IL-10R axis, and provide a hypothetical model for how B cells, via their production of IL-10, may be important in conveying environmental “information” to the inflamed central nervous system. One such context is multiple sclerosis (MS), a chronic, inflammatory disease of the central nervous system (CNS) that affects over 2 million people worldwide . Early s1.1in vitro (+/CD8+ T cells and B cells) have been identified as producers of IL-10 (+ T cells by inducing their anergy (+ Foxp3+ regulatory T cells (Tregs) (+ T regulatory type 1 (TR1) cells  , 31 and 1) cells .1.2+ B cells leads to disease recovery , a hetero-tetramer consisting of two alpha and two beta subunits . While tin vitro , 29.Upon IL-10 binding, a cascade of intracellular signaling events occurs Figure 11.4IL10RA and IL10RB genes have both been strongly associated with infant colitis associated with defects in downregulation of proinflammatory cytokine secretion by monocytes , where the constant interaction between immune cells and the gut microbiota demands strict regulation within the local intestinal milieu. In humans, mutations to the onocytes –50. Othemulation –53.+ Tregs is critical for suppressing pathogenic Th17 cells and IL-10R signaling in Th17 cells directly suppresses their expansion  and B cells (Bregs) , 35, 38.t manner . Furthert manner .+ T cells derived from the blood of MS patients are relatively hyporesponsive to the immunosuppressive function of IL-10 in vitro compared to healthy controls, and this hyporesponsiveness is associated with impaired STAT3 phosphorylation  to promote their survival and proliferation in the CNS  or potentially independent of the microbiome (section 3.3-3.4)A person’s sex, age, diet, exercise, prior infections, geographic location, antibiotic use, exposure to pollution and early life factors  can all influence the composition of one’s microbiome . As such3.1Akkermansia, Acinetobacter, and Parabacteriodes taxa , was found to be responsible for protection against EAE by triggering the activation of IL-10+ Tregs through the Toll-like receptor 2 pathway  mice with P. distasonis significantly increased the amount of IL-10+ CD4+ T cells in the spleen and mesenteric lymph nodes  including acetate, butyrate, and proprionate are produced by the colon during the bacterial fermentation of dietary fibers. Progressive MS patients have been shown to have lower levels of SCFAs in the blood. Of note, oral SCFA administration to mice increases the number of IL-10ring EAE . Furtherin vitro . In the in vitro . In MS, in vitro .Lachnospiraceae that correlated with a decrease in IL-17+ CD4+ T cells and an increase in IL-10+CD14+ monocytes in the blood as well as a reduction in relapse rate compared to “western diet” MS patients 2D3 was found to dampen the differentiation and maturation of APCs resulting in increased production of IL-10 concomitant with a reduced generation of alloreactive T cells dalities –141. Simenotypes . Further T cells . A causaIn summary, Vitamin D has a direct impact on the IL-10/IL-10R axis in MS and EAE. Vitamin D may also have an indirect impact on the IL-10/IL-10R axis via the microbiome , which i3.4+ B cells to persist and contribute to CNS autoimmunity. Exploring how vIL-10 influences B cell populations during MS may allow us to further understand how EBV influences MS disease.Epstein-Barr Virus (EBV) is a common human gammaherpesvirus that persists in more than 90% of the population worldwide . Recentl4+ B cells, a heterogenous collection of Bregs have been described in various disease contexts, and the specific signals controlling their development- several which can be influenced by the environment, are now of significant interest  respond to IL-10 to alter neuroinflammation, and the environmental factors that impact IL-10R signals, is less comprehensive. In this vein, it is critical to not only understand the specific environmental contexts that influence IL-10 production, but also what cell types receive IL-10. The conflicting evidence between IL-10 knockout studies and cell specific IL-10R knockout studies in EAE indicate that there is more to be understood about IL-10R signaling during CNS autoimmunity. Exploring how these IL-10R-expressing cell types respond to environmental stimuli reframes the focus from the IL-10 producing cell type(s) to the cell-specific downstream effects of IL-10R signaling, and how environmental factors impacts these signals. Furthermore, we propose that B cells are the critical link between environmental stimuli and IL-10R signaling during MS/EAE. Identifying environmental factors that modulate the IL-10/IL-10R axis has the potential to provide new insights into therapeutic intervention for MS patients.EB wrote the article with editorial oversight from AW and JG. All authors contributed to the article and approved the submitted version.


Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Experimental Design: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Results: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. Conclusions: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.Purpose: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of  Senescence is a stress response that limits the replication of damaged or aged cells by implementing permanent cell cycle arrest . SenesceSenescent cells undergo dramatic alterations to their chromatin landscape that affect genome accessibility and transcriptional programs . NucleosPrevious studies have shown that histone variant H2A.J accumulates in human fibroblasts in vitro as well as in murine and human skin tissue in vivo during replicative, oncogene- and radiation-induced senescence, and affects the inflammatory gene expression of senescent cells ,14,15. I2 and 5% CO2 in MEM  with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1% penicillin/streptomycin. A total of 1 µg/mL doxycycline was added to medium for 1 week prior to IR to express shRNA sequences or to overexpress H2A or H2A.J. The cells were homogenously grown on coverslips and used for experiments once 90% confluency was achieved.Primary WI-38 human lung fibroblasts were obtained from ATCC. Immortalized WI-38 hTERT-fibroblasts were genetically modified to prepare H2A.J knock-down (H2A.J-KD) or knock-in (H2A.J-KI), canonical H2A knock-in (H2A-KI) and non-targeted controls (NT) as described previously . Stable Cells were exposed to IR using the linear accelerator Artiste™  . Cells were analyzed at different time-points  following IR (20 Gy) and compared to non-irradiated controls (non-IR).®-20, and incubated overnight with a primary antibody  followed by Alexa-Fluor®488 or Alexa-Fluor®568 secondary antibody . Subsequently, cells were mounted in a VECTAshield™ mounting medium with 4′, 6-diamidino-2-phenylindole . Fluorescence images were captured with a Nikon-Eclipse Ni fluorescence-microscope equipped with a Nikon DS-Qi2 camera . For evaluating H2A.J-, 53BP1-, SAHF-, and p21-positivity, at least 200 cells were captured for each sample (positive cells in %). For quantifying 53BP1-foci per cell, at least 50 foci and/or 50 cells were analyzed per sample. For the identification of DNA-SCARS, 53BP1/PML co-localization was analyzed in 50 nuclei, and numbers of 53BP1/PML co-localizing foci relative to total 53BP1-foci were expressed in percentages.Cells were fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton X-100, washed with 0.1% TweenFollowing 5 min fixation with 2% paraformaldehyde and 0.2% glutaraldehyde, cells were incubated with X-Gal staining solution  at 37 °C overnight. After 30 s of methanol incubation, the dried samples were permeabilised with 0.2% Triton X-100 and washed with 1% BSA. Samples were blocked with 4% BSA for 1 h, followed by an overnight incubation with H2A.J primary antibody. Incubation with Dako immunoglobulin/bioatinylated secondary antibody  was followed by Vectastain ABC Peroxidase standard  and SIGMAFAST™ 3.3′Diaminobenzidine  incubations, respectively. Samples were finally mounted in Dako Faramount Mounting Medium .RNA extraction, library preparation, and paired-end  sequencing using NovaSeq  were performed by GENEWIZ Germany GmBH . Subsequently, raw reads were trimmed with Cutadapt (v3.5) to clip low-quality ends from reads that were below a Phred quality score of 20. Additionally, a minimum read length of 50 was set. Data quality was inspected before and after trimming using FastQC (v0.11.3) . SequencWI-38 hTERT-fibroblasts were washed and incubated with 1xDNase . Tagmentation, library preparation, and paired-end  sequencing using NovaSeq500  were performed by ActiveMotif . The obtained sequences were aligned to the human genome sequence GRC38.p13  using BWp value ≤ 0.1), and motif locations from HOMER analysis.Fragments per million mapped fragments were obtained with the fpm function of DESeq2  and visuConditioned media (CM) were collected from three independent cultures of NT, H2A.J-KD and H2A.J-KI fibroblasts. CM from cultured fibroblasts was collected at 2 weeks after 20 Gy IR compared to non-irradiated fibroblasts. Cell numbers in flasks were determined at the timepoint of the CM collection. CM samples were frozen at −80 °C until analysis. A Multi-Analyte ELISArray™ Kit  was used to screen SASP factors with a standard ELISA plate reader, according to the supplier’s protocol.® Reverse Transcription Kit  was used for cDNA synthesis and Advanced Universal SYBR Green Supermix  was used for subsequent quantitative PCR in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories GmbH), as previously described [RNA extraction was completed using TRIzol™  and phenol/chloroform phase separation. A QuantiTectescribed .C57BL/6-N mice (2-month-old) were purchased from Charles River Laboratories  and housed in pathogen-free rooms to minimize infection risks, and supplied with a standard laboratory diet and water ad libitum. For whole-body irradiation  using an Artiste™ linear accelerator , the animals were placed in an 18 cm-diameter Plexiglass cylinder covered by 1.5 cm thick plastic to improve dose homogeneity. Computed-tomography-based three-dimensional dose calculations were made with the Pinnacle planning system . Animals were irradiated with single-doses (10 Gy or 2 Gy) or with fractionated IR . At defined time-points after the last IR exposure, mice were anesthetized, perfused, and the lungs were removed, fixed overnight in 4% paraformaldehyde , and processed for further analysis. At least three biological  and three technical (whole lung slices) replicates were examined for each irradiation regimen. The experimental protocol was approved by Medical Sciences Animal Care and Use Committee of Saarland University.Formalin-fixed tissues were embedded in paraffin and sectioned at 4-μm thickness. After deparaffinization in xylene and rehydration in decreasing alcohol concentrations, sections were boiled in a citrate buffer  and incubated with Roti-Immunoblock . For IFM analysis, sections were incubated with primary antibodies  followed by AlexaFluor-488 secondary antibody  and mounted in VECTAshield with 4′,6-diamidino-2-phenylindole . For IHC analysis, sections were incubated with anti-H2A.J antibody followed by biotin-labeled antibodies  and staining was completed by incubation with 3,3′-diaminobenzidine and substrate chromogen. Finally, sections were counterstained with haematoxylin and mounted with Aqueous-Mounting-Medium . For qualitative analysis, H2A.J-positive cells were visualized under the Nikon E600 epifluorescent microscope .® image analysis software . In these regions of interest (ROI), the brown DAB staining signal for H2A.J was compared to the hematoxylin counterstain for each nucleus, and cells were categorised into no (blue), weak (yellow), moderate (orange) and strong (red) H2A.J-stained nuclei. The percentage of cells was calculated for each staining category in relation to the total cell numbers in ROIs.Whole digital slide images were obtained from H2A.J-stained lung sections of irradiated  and non-irradiated mice using Axioscan 7 . In lung parenchyma, 10 representative regions were annotated manually (excluding bronchiolar ducts and blood vessels) using the multiplex IHC module of HALOp value of <0.05 was considered statistically significant, <0.01 as statistically highly significant, and <0.001 as extremely statistically significant. Significant statistical differences compared to non-irradiated controls  or between cell lines (asterisks with square brackets) are presented in the figures as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).GraphPad Prism  was used to collect and analyse data. Data are presented as the mean of three experiments ±SEM. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used for comparison among different groups. A Cip1/Waf1. Accordingly, p21 staining was established to verify our finding of reduced senescence induction in H2A.J-KI fibroblasts after IR exposure. While all non-irradiated fibroblast populations revealed very low p21 levels (≤7%), the proportion of p21+ cells increased to 75–90% for NT, H2A.J-KD and H2A-KI after IR, reflecting their strong senescence response. Significantly, for H2A.J-KI fibroblasts, the p21 expression level remained very low even after IR exposure (≈14%), suggesting that H2A.J-KI fibroblasts are largely resistant to radiation-induced senescence.Immortalized fibroblasts with H2A.J knock-down (H2A.J-KD) or knock-in (H2A.J-KI) and canonical H2A knock-in (H2A-KI) were analyzed compared to non-targeted controls (NT). Confluent H2A.J-KD, H2A.J-KI, H2A-KI and NT fibroblasts were irradiated with 20Gy, and H2A.J expression was analyzed at 24 h, 1 w and 2 w post-IR by IFM. For NT fibroblasts, we observed time-dependent increases of pan-nuclear H2A.J staining intensities , which modulate transcription by recognizing and binding to specific DNA sequences. Changes in chromatin accessibility for TF binding sites were studied by ATAC-seq, permitting the genome-wide analysis of transcriptionally open/active and closed/repressed regulatory elements. Using the motif discovery tool HOMER, the sequence-based prediction for TF motif frequency or activity was mapped and compared for different conditions: for each line of fibroblasts in their post-IR versus non-IR status A and betTranscriptionally active genes are characterized by accessible chromatin at their promoters, nearby enhancers, and some internal regions within the gene body itself. To determine whether differentially accessible chromatin configurations were correlated with their corresponding gene expression levels, ATAC-seq and mRNA-seq datasets were integrated for NT, H2A.J-KD and H2A.J-KI fibroblasts in their irradiated versus non-irradiated state. RNA-seq analysis was performed in NT, H2A.J-KD and H2A.J-KI fibroblasts for measuring gene expression changes in response to IR exposure. Volcano plots show unchanged, up- and down-regulated genes in NT, H2A.J-KD, and H2A.J-KI fibroblasts at 2 w post-IR, compared to their non-irradiated controls . During After high-dose IR exposure fibroblasts become senescent, but remain viable for long intervals and develop SASP with the secretion of inflammatory cytokines. For all lines of fibroblasts (most pronounced for NT fibroblasts) transcribed SASP genes  were generally upregulated following IR compared to their non-irradiated counterparts. Subsequently, transcribed SASP genes were correlated with corresponding SASP proteins by investigating the secretion of interleukin-6 (IL6), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP1), and interleukin-8 (IL8) in culture media by ELISA, as readout for the inflammatory secretome. Non-irradiated NT, H2A.J-KD and H2A.J-KI fibroblasts expressed only low levels of these common SASP factors, and hence no senescence-messaging secretome B. NT fibPrevious studies noted organ-specific differences in H2A.J protein expression in normal tissues generally, with strong labeling of luminal epithelial cells, even without IR exposure . Here, wChromatin organization and transcriptional profiles undergo tremendous rearrangements during senescence progression. To explore the potential epigenetic mechanisms of histone variant H2A.J in the regulatory networks of radiation-induced senescence, lung fibroblasts with down- and upregulated H2A.J expression levels were analyzed for their dynamic changes in chromatin organization, epigenetic and transcriptional landscape, and their cellular function following IR exposure. Through integrative ATAC-seq and RNA-seq analysis and functional validation of the senescence state, we identified and analyzed mechanistic interrelations between H2A.J incorporation and altered chromatin architecture with modified genome accessibility and transcription factor recruitment, changed SASP expression, and senescence competence. Our findings demonstrate that the fine-tuned incorporation of histone variant H2A.J defines the epigenetic landscape for driving the senescence programme in response to DNA-damaging stress. H2A.J overexpression, in contrast, was associated with reduced senescence-associated chromatin alterations, significant changes in the SASP secretome, and the potential to overcome senescence-associated growth arrest. Analyzing lung tissues following IR exposure, we observed increased H2A.J expression in specific cell populations (particularly in pneumocytes), most pronounced after high doses, but even after moderate and low doses. These findings suggest that H2A.J-associated epigenetic mechanisms play decisive roles even at therapeutic doses used during fractionated radiotherapy. It is increasingly recognized that DNA-damage triggered events stimulate inflammation and modulate cellular functions in injured normal tissues over months to years . Future Collectively, our discoveries about the H2A.J-associated epigenetic mechanisms that drive radiation-induced damage development complement the traditional radiobiological conception of normal tissue injury. Understanding the precise molecular events responsible for the perpetuation of normal tissue injury is critical for developing therapeutic strategies to prevent, mitigate, and treat radiation toxicities.Histone variants greatly expand the roles and dynamics of nucleosomes by wrapping more or less DNA, by having greater or lesser stability, through unique post-translational modifications, or by interacting with specific chromatin components . PreviouThe topological organization of nucleosomes across the genome regulate chromatin accessibility through a variety of chromatin mechanisms, such as altering TF binding by modulating steric hindrance. ATAC-seq measurements of open and closed chromatin accessibility reflect regulatory capacities and distinct functional states of chromatin organization . Our finA substantial number of anticancer interventions, such as radiotherapy, induce senescence in normal but also in cancer cells by triggering genotoxic stress, leading to stable cell cycle arrest and SASP induction. Therefore, therapy-induced senescence serves as an initial antitumour mechanism to halt proliferation and prevent further genomic instability. However, we here present the first experimental evidence that H2A.J is progressively incorporated into the chromatin following IR exposure, and that H2A.J overexpression can reverse this radiation-induced cell cycle arrest. Therefore, H2A.J overexpression may promote tumorigenesis or even improve resistance to cancer treatments. Within the scope of our current studies on irradiated skin tissue, we observed that mitochondria-associated genes are also strongly modulated by H2A.J. Increasing evidence suggests that an altered mitochondrial metabolism is associated with the onset of resistance to radiotherapy . HoweverGenome-wide chromatin accessibility profiling for TF-binding motifs using ATAC-seq emphasize the functional role of the histone variant H2A.J for regulatory senescence networks. Collectively, integrative analysis of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility in regulatory regions were clearly associated with modified inflammatory gene expression profiles. Our in vitro model with knock-down or knock-in of the H2A.J gene in a defined cell type makes it possible to study very precisely the functional effects of H2A.J on chromatin organization, the recruitment of transcription factors, and the pro-inflammatory secretome after IR exposure. However, fibroblasts are a rather radioresistant cell population that does not reflect the radiation reactions of diverse cell populations found in complex tissues. In future research work, knock-out mice will be examined before and after IR exposure in order to investigate the importance of H2A.J for the radiation response in the context of complex tissue homeostasis.Cellular senescence is a stable cell growth arrest that is characterized by the silencing of proliferation-promoting genes through the compaction of chromosomes into SAHF ,38. Our


Policosanols (PCs) are long-chain linear aliphatic alcohols that are present in the primary leaves of cereal crops, such as barley and wheat, sugar cane wax, and beeswax. PCs have been used as a nutraceutical for improving hyperlipidemia and hypercholesterolemia. However, the PC content in mutant wheat lines has not been investigated. To select highly functional wheat sprouts with a high content of PCs in wheat mutant lines developed via gamma-irradiated mutation breeding, we cultivated the sprouts of wheat mutant lines in a growth chamber with white LED light (6000 K) and analyzed the PC content in these samples using GC-MS. We studied the PC content in 91 wheat sprout samples: the original variety , commercially available cv. Geumgang (WS87) and cv. Cheongwoo (WS91), and mutant lines (WS02–WS86 and WS88–WS90) developed from WS01 and WS87. Compared to WS01, 18 mutant lines exhibited a high total PC content (506.08–873.24 mg/100 g dry weight). Among them, the top 10 mutant lines were evaluated for their PC production after cultivating under blue (440 nm), green (520 nm), and red (660 nm) LED light irradiation; however, these colored LED lights reduced the total PC production by 35.8–49.7%, suggesting that the cultivation with white LED lights was more efficient in promoting PCs’ yield, compared to different LED lights. Therefore, our findings show the potential of radiation-bred wheat varieties as functional foods against hyperlipidemia and obesity and the optimal light conditions for high PC production. Triticum aestivum L.) is a staple crop that ranks third in global grain production and serves as a source of energy and an important nutrient in the human diet + fragmentation, indicating the loss of a -CH3 group, and exhibited characteristic fragment ions at m/z 103 [CH2OSi-(CH3)3+], m/z 75 [HO–Si(CH3)2+], and m/z 15 [C4H9+], indicating TMS alcohols (tR) for the PC-TMS derivatives are listed in R2 = 0.996−1.000).The PCs in the alcohols . The GC-Several studies have analyzed the PC contents of different varieties of wheat and barley sprouts cultivated at different growth times and under different LED light conditions ,7,25,26.Among the 85 sprout samples of the wheat mutant lines (WS02–WS86) derived from the original variety (WS01), 19 mutant lines showed a higher total PC content than that of WS01. WS74 exhibited the highest total PC content, followed by WS37, WS69, WS72, WS76, WS57, WS81, WS78, WS49, WS75, WS31, WS79, WS51, WS46, WS63, WS48, WS70, WS66, and WS40. Octacosanol (C28-OH) was the most abundant PC in all the mutant lines, accounting for approximately 75% of the average total PC content. Triacontanol (C30-OH) and hexacosanol (C26-OH) were the second and third major components, accounting for ~13.5% and ~6.5% of the average total PC content, respectively. The remaining PCs accounted for <2% of the average total PC content, indicating that they are very minor components.The concentration values of the PCs of the 91 wheat sprout samples were exported for hierarchical clustering analysis. HCA with a heatmap was performed to determine the sample classification and to highlight variations in wheat sprout samples’ PC composition . Among tT. aestivum for sprouts containing high contents of functional ingredients, the top 10 mutant lines with a >20% higher total PC content than that of the original cultivar were selected. The GC-MS chromatograms for each sample are shown in To develop new varieties of Among the 10 selected mutant lines, the highest total PC content was observed in WS74 (873.24 mg/100 g). The octacosanol , tricosanol , and docosanol  contents in WS74 were more than twice as high as those in WS01, and the content of the remaining four components slightly increased (<1.3 times) or decreased. WS37 ranked second in the total PC content, and its octacosanol , hexacosanol , and triacontanol  contents increased by ~1.8, 1.7, and 1.3 times, respectively, compared to those in WS01; however, the contents of the other compounds were similar to those in WS01 or decreased. WS76 had the highest contents of docosanol  and tetracosanol , which were more than three times higher than those in WS01. Furthermore, WS53 contained the highest contents of hexacosanol  and heptacosanol , which were twice as high as those in WS01. The highest tricosanol (C23-OH) content (17.07 mg/100 g) was observed in WS49. These results suggest that γ-irradiation breeding influences genetic mutation for the increased accumulation of beneficial metabolites, as previously reported for wheat and perilla .Compared to the commercially available cultivars cv. Geumkang (WS87) and cv. Cheongwoo (WS91), the original cultivar , had a higher total PC content. However, compared to those in WS01 and WS87, WS91 showed 60–70% lower octacosanol (C28-OH) and triacontanol (C30-OH) contents, which are major components, and slightly higher tricosanol (C23-OH), tetracosanol (C24-OH), and heptacosanol (C27-OH) contents. The three mutant lines (WS88–WS90) derived from cv. Geumkang (WS87) had a slightly lower total PC content than that in WS87. WS88 and WS90 showed a similar PC content distribution to that in WS87. However, WS89 had 1.85 times the tricosanol (C23-OH) present in WS87, but the contents of the other components decreased compared to those in WS87.To evaluate the effects of different LED conditions on PC accumulation in wheat sprouts, we quantified the PC content in the top 10 wheat mutant lines and the original variety, which were grown in a well-controlled growth chamber under white (6000 K), blue (440 nm), green (520 nm), and red (660 nm) LED light irradiation for 7 d, using GC-MS . The GC-The total PC content in the original variety WS01 decreased more under green-light irradiation than under blue- or red-light irradiation. WS78 and WS81 exhibited similar patterns to WS01, whereas the total PC content in WS37, WS49, WS57, WS74, and WS76 decreased slightly as the wavelength of the colored LED lights increased in the order of blue (440 nm) < green (520 nm) < red (660 nm). In contrast, the total PC content in WS72 showed a slight decreasing trend as the wavelength of the colored LED lights decreased from red to green to blue. The total PC content in WS69 and WS75 decreased slightly under green LED light compared to that under blue and red LED lights. Hence, the individual and total PC content in wheat sprout samples varied without a specific trend under different LED light conditions, and white LED light was the best condition for PC accumulation. The inconsistent or negative changes in the PC content under LED cultivation observed in this study were similar to those reported in previous studies on barley and wheat sprouts [T. aestivum) is one of the most important energy sources; however, its sprout has received considerable attention due to its nutritional and functional aspects. PCs are a mixture of primary alcohols of a very long-chain fatty acid consisting of eicosanol (C20-OH), heneicosanol (C21-OH), docosanol (C22-OH), tricosanol (C23-OH), tetracosanol (C24-OH), hexacosanol (C26-OH), heptacosanol (C27-OH), octacosanol (C28-OH), and triacontanol (C30-OH). They are found in cuticular waxes in sprouts of cereal crops and waxes of insects. Among them, octacosanol (C28-OH) was the most abundant PC in wheat sprouts [Coix lacryma-jobi), the main components were docosanol (C22-OH), tetracosanol (C24-OH), hexacosanol (C26-OH), and octacosanol (C28-OH) [Cannabis sativa) were tetracosanol (C24-OH), hexacosanol (C26-OH), and dotriacontanol (C32-OH) [Arachis hypogaea) was docosanol (C22-OH) [Spinacia oleracea), the major components were tetracosanol (C24-OH) and hexacosanol (C26-OH) [Wheat  . Corn ke(C28-OH) . The mos(C32-OH) . The mai(C22-OH) . For Kor(C26-OH) . In thisSaccharomyces cerevisiae) led to the production of C22-OH [S. cerevisiae), C24-OH and C26-OH were produced [Solanum lycopersicum) and rice (O. sativa) increased the accumulation of C24-OH–C30-OH [Aegilops tauschii leaf surface, a heterologous expression of five Ae.tFARs in yeast (S. cerevisiae) which resulted in Ae.tFAR1, Ae.tFAR2, Ae.tFAR3, Ae.tFAR4, and Ae.tFAR6, produced C16-OH, C18-OH, C26-OH, C24-OH, and C28-OH, respectively [Brachypodium distachyon, and the transgenic BdFAR2 and BdFAR3 lines of Brachypodium showed significantly increased amounts of C26-OH and C28-OH compared to the wild species [Hordeum vulgare) genome information, HvFAR2, HvFAR3, HvFAR4, HvFAR5, and HvFAR6 were identified, and the HvFAR3 expression increased the accumulation of C26-OH in barley sprouts [Previous studies on the biosynthesis of long-chain primary alcohols found in wheat sprout cuticle wax have identified and revealed new functions of fatty acyl coenzyme A reductase (FAR) . TaFAR5,f C22-OH . Additioproduced . HoweverH–C30-OH . Five waectively . Three B species . From av sprouts . TherefoThe content of octacosanol (C28-OH), which was the major constituent of wheat sprouts in this study, increased in the top 10 mutant lines  along with an increased total PC content, compared to the original variety. Octacosanol (C28-OH) induces a dose-dependent activation of AMPK phosphorylation in human hepatoma HepG2 cells, suggesting it is a lipid/cholesterol-lowering agent . In addiRed/far-red light-sensing phytochromes and blue light-sensing cryptochromes play important roles in regulating light-mediated physiological responses through regulated transcriptional networks . PhytochH. vulgare) and wheat (T. aestivum) subjected to differential LED light conditions showed that the hexacosanol (C26-OH) content in barley and the octacosanol (C28-OH) content in wheat were not consistent with light qualities [Additionally, growing plants under different wavelengths of light exposure results in physiological changes . For exaualities . In thisIn conclusion, the differences in the individual and total PC contents of the wheat sprout samples, including the original variety (WS01), commercially available varieties cv. Geumgang (WS87) and cv. Cheongwoo (WS91), and 85 mutant lines (WS02–WS86 and WS88–WS90) were evaluated for the first time in the literature. Compared to WS01, 18 mutant lines exhibited a higher total PC content with an increased octacosanol (C28-OH) content, suggesting that these mutant lines can serve as a functional source of PCs. Moreover, the top 10 mutant lines with the highest range of a total PC were subjected to differential LED light conditions . However, their individual and total PC contents were reduced compared to those with the white LED light irradiation, indicating that the correlation between colored LED lights and PC biosynthesis in wheat sprouts is negative. Our study reveals the beneficial effect of radiation breeding on increasing the metabolite accumulation in wheat sprouts. However, further studies on the genetic factors controlling radiation-induced PC biosynthesis in wheat sprouts are required.60Co) irradiation. The original cultivar of colored wheat was developed by a cross-breeding of Woori-mil (Korea RDA accession no. IT172221) and D-7 (a wheat line developed by Korea University) [Wheat mutant lines were developed by radiation breeding of the original cultivar of colored wheat using seeds treated with 200 Gy of gamma (versity) . The mutN-methyl-N-(trimethylsilyl) trifluoroacetamide  were added to each hexane extract for the silylation of the PCs [y) and concentrations  of the PC standards were calculated using second-degree polynomial regression equations , and their correlation coefficients are shown below: eicosanol ; heneicosanol ; docosanol ; tricosanol ; tetracosanol ; hexacosanol ; heptacosanol ; octacosanol ; and triacosanol .For the quantitative analysis of the PCs, sprouts (0.1 g) of wheat samples cultivated in a growth chamber under a white LED light (6000 K) were extracted into 10 mL of hexane with shaking at 24 °C for 24 h. Each hexane extract was filtered and evaporated under a vacuum. Chloroform (0.5 mL) and 250 μL of  the PCs . The soly = 75727x − 17152, R² = 0.9997); heneicosanol ; docosanol ; tricosanol ; tetracosanol ; hexacosanol ; heptacosanol ; octacosanol ; and triacosanol .Wheat sprouts cultivated in a growth chamber irradiated with blue (440 nm), green (520 nm), and red (660 nm) LED lights were extracted and silylated using the procedure described above. Calibration curves were prepared using a series of standard solutions at four different concentrations. The regression equations of the calibration curves of the PCs and the coefficients were calculated as follows: eicosanol . The PCs were identified by comparing the retention time and fragmented mass values of the peaks with those of the PC standards.The GC-MS analysis of wheat sprout samples cultivated in a growth chamber under a white LED light (6000 K) was performed on a GCMS-QP2010 Ultra . The GC-MS analysis of wheat sprout samples cultivated in a growth chamber with blue, green, and red LED lights was performed using a GCMS-QP2020 NX (Shimadzu). An HP-5 MS capillary GC column  was used with 99.99% high-purity helium at a flow rate of 1.2 mL/min. The sample (1 μL) was injected in split mode (1:5 ratio). The oven temperature was initially set to 230 °C, was increased to 260 °C at a heating rate of 25 °C/min, and was maintained at this temperature for 10 min. The transfer line temperature was set to 280 °C. The MS data were acquired in the electron ionization (EI) mode with an ionization voltage of 70 eV, an ion source temperature of 230 °C, and a scan range of p < 0.05, ** p < 0.005, *** p < 0.001, and **** p < 0.0001). The data set of the quantification of the PCs obtained for the 91 wheat sprout samples was normalized and subjected to a hierarchical clustering analysis (HCA) with a heatmap using “pheatmap” in R software (version 4.0.2) [All experiments were conducted in triplicate, and the mean values were reported. One-way analysis of variance was performed using GraphPad Prism 9 software  to assess significant differences (* n 4.0.2) .


The phenotype of IL1B+3954(T) was converted from weak to strong in one host, and of the non-T allele from strong to weak in the other . Together with homozygous alternate or reference SNPs at IL10-1082 or CD14-260 in 4 hosts, all hosts were identified as strong or weak GCF responders. The GCF response is therefore a strong or weak genetic trait that indicates strong or weak innate immunity in EG and controllable or uncontrollable periodontal disease, dental implant survival and late-life comorbidities.Chronic periodontitis is a bacterial infection associated with dentally adherent biofilm (plaque) accumulation and age-related comorbidities. The disease begins as an inflammatory exudate from gingival margins, gingival crevicular fluid (GCF) in response to biofilm lysine. After a week of experimental gingivitis , biofilm lysine concentration was linearly related to biofilm accumulation (plaque index) but to GCF as an arch-shaped double curve which separated 9 strong from 6 weak GCF responders (hosts). Host DNA was examined for single nucleotide polymorphisms (SNPs) of alleles reported in 7 periodontitis-associated genes. Across all 15 hosts, an adenine SNP (A) at IL1B-511 (rs16944), was significant for strong GCF (Fisher’s exact test,  Porphorymonas gingivalis which can spread throughout the body [Chronic adult periodontitis is a bacterially induced inflammatory disease that slowly destroys the connective tissue and bone-supporting teeth in the oral cavity. It is preceded by gingival inflammation (gingivitis) which first appears in teenagers and young adults whose oral hygiene is inadequate. the body .Interleukins 1A and 1B (IL1A and IL1B) were found to detect individuals (hosts) with tooth loss due to severe periodontitis after adjusting for smoking, diabetes and professional dental care [A study of the severity of periodontitis in identical and non-identical adult twins indicates almost 50% heritability, even after adjustment for smoking and behavioral variables . Corresptal care . How thahttps://iadr.abstractarchives.com/abstract/18iags-2958594/high-or-low-gingival-crevicular-fluid-response-after-experimental-gingivitis (accessed on 1 May 2023). A better way of looking at genotypes associated with periodontitis could be to determine if they have a role in experimental gingivitis (EG) . This prIL1 gene family [Recent studies of EG in Danish and US cohorts identified fast and slow rates of gingivitis development associated with fast and slow inflammatory mediator development ,13,14,15e family ,17. StroIn health, mastication intermittently damages the dentally attached (DAT) cells of junctional epithelium JE, A, causinAn EG study from China detailed how a strong or weak gingivitis response after 3 weeks was related to differences in microbial colonization . Using rEikenella corrodens appear within a day or two [As bacteria increase at the gingival margin region after EG as described in the y or two . Its outy or two  to non-ny or two ,25. Prioy or two ,27, indiy or two . After ay or two .The plaque index (PI) measure of biofilm accumulation increases collinearly with lysine and GCF exudation but above 0.11 mM (0.11 µmol/g biofilm), the PI and GCF measurements diverge. PI and lysine keep increasing , but GCFy-axis (Although the arched curve in y-axis  clearly IL1B+3954 (numbered downstream from the first nucleotide encoding its IL1β protein) is associated with many inflammatory diseases, including periodontitis where periodontopathogens [IL1B+3954(T) in a study using 117 periodontitis patients and 175 controls. The need for so many IL1B+3954(T) individuals and controls suggests the presence of unknown confounding SNP variants from the same or different genes [Histories of infections produce distinctive frequencies of SNP alleles within genes from different populations or ethnic groups . The altathogens ,31 enhanathogens . The assnt genes .IL1B+3954(T), the IL1A-889(A), encoded protein IL1α, is associated with several chronic inflammatory diseases [IL1A-889(A)+IL1B+3954(T). Of 5117 US patients (hosts), who were 34–55 years of age and mostly US Caucasians, 534 patients exhibited tooth loss, severe periodontitis [Independently of diseases ,34. The Our aim was to determine whether strong and weak GCF exudation responses to similar biofilm lysine concentrations were linked to SNPs of genes associated with periodontitis. If so, they could explain whether the difference in GCF exudation predicts structurally different microbiomes after EG resembling those in natural gingivitis pre-EG ,22, and IL10 (IL10-597) was tested but not included because it replicated IL10-1082. The Reference SNP (rs) Report and the aggregate allele frequency for Europeans (ALFA Project) is provided by the US National Center for Biotechnology Information (NBCI), https://www.ncbi.nlm.nih.gov/snp/rs1800587 (accessed on 14 July 2023) or https://www.ncbi.nlm.nih.gov/snp/rs1143634 (accessed on 14 July 2023), etc. for each rs number in IL1A-889 and IL1B+3954 are each hetero- or homozygous for increased expression of their respectively encoded proteins [IL1B-511 [IL10-1082(TT) [CD14-260(AA) [IL6-1363 [COX2+8473 [MMP8-799 [IL1A-889 and IL1B+3954. Of the 7 genes chosen because of their influence on periodontitis , either proteins ,33,34,35IL1B-511  which po1082(TT) , and CD1-260(AA) . In all OX2+8473  and MMP8MMP8-799  associatIL1B-511 did significantly associate with strong GCF response (IL1B+3954(T) and IL10-1082(T) were each a major fraction of weak GCF responses, but neither was significant  allele of IL1B+3954  in IL1B+3954. Thus, SNPs in IL1B at −511 and +3954 were exclusively associated with strong or weak GCF exudation in 9 of the 15 hosts. Of the six hosts remaining, five numbered 1, 2, 3, 10 and 12, possessed both ILB-511(A), significant for strong GCF response, and IL1B+3954(T), significant for weak GCF response. In hosts 2 and 3, this allelic conflict was resolved as a strong response by the homozygous alternate SNP allele (CC) of IL10-1082. In host 10, the reference SNP allele (TT) of IL10-1082 gave a weak GCF response, similar to the SNP reference allele (AA) of CD14-260 in host 12). Combinations of alleles are a feature of IL1B allelic conflict, and host 11 was an expected strong GCF responder like the six other IL1B-511(A)+IL1B+3954(CC) hosts. It seems likely that latter’s phenotype was converted to weak by IL6-1363(T) in the genome, and that the host 1 phenotype, IL1B+3954(T), was converted to strong despite the presence of IL10-1082(TT), which should have confirmed a weak response as in host 10. These phenotype reversals exemplify a genetic phenomenon called specific epistasis [IL6-1363(T) and the C or T SNP alleles at IL1B+3954 [IL1B+3954(T) significant with respect to GCF response  mediating epistasis of IL1B+3954(CC) to weak with IL1A-889(A) in the genome. IL1B+3954(T) in the genome not only associates with severe periodontitis [Besides the tooth loss SNP genotype in host 14, a second such genotype was detected in host 11 by dontitis  but alsoIL1B-511 alleles (GA or AA) in the genome with the IL1B+3954 reference allele (CC). The weak trait was indicated in hosts 13 through 15 with the reference allele (GG) at IL1B-511 present with alternate alleles (TC or TT) at IL1B+3954. In host 11, a 7th IL1B-511(GA) allele paired with IL1B +3954(CC) was converted to weak by IL6-1363(GT) epistasis of IL1B+3954 (CC) and one of the five hosts possessing the allelic conflict, IL1B-511(GA)+IL1B+3954(CT), was converted to strong (host 1). Of the remaining four hosts, 2 expressed the strong trait with the homozygous alternate allele, IL10-1082(CC) in the genome, and the other two expressed the weak trait with its homozygous reference allele, IL10-1082(TT), or a homozygous alternative allele of CD14-260(AA) in the genome. In host 3, the presence of COX2+8473(AG) specified a potential replacement of IL10-1082(CC) for a strong phenotype.The strong GCF trait was indicated in hosts 4 through 9 by the alternate IL1B at −511 and +3954 exclusively, or with the addition of a third SNP allele from a second gene. The third allele was the homozygous reference or alternate SNP of IL10-1082, a homozygous alternate SNP of CD14-260, or a heterozygous alternate allele of IL6-1363.IL1B-511 and ILB+3954, alone, or together with an SNP allele of a second gene, separate all strong from all weak GCF responders  and moderate or severe periodontitis [Susceptibility to moderate or severe periodontitis is linked to weak GCF exudation because: (a) Smokers exhibit weak GCF exudation  and signngivalis  and assodontitis ; (c) Unldontitis  which asIL6-1363(T) in their genome, the phenotype of one host with IL1B+3954(CC) was converted from strong to weak GCF exudation, and the one possessing the IL1B+3954(T) was converted from weak to strong. These phenotypic changes suggest specific epistasis [IL6-1363(T) being associated with aggressive (severe) periodontitis [IL6-1363 might provide information as to how this form of epistasis operates in periodontitis [Of the two hosts possessing pistasis , not DNApistasis , a genetdontitis . The strdontitis ; SupplemIL1B+3954(CC)+IL1B-511(GA), here shown as 2 alleles of IL1B connected with a plus sign like the two alleles of IL1A and IL1B that make up the IL1 tooth loss genotype. The conflicted genotype reduces the accuracy with which IL1B+3954(T) alone can associate with periodontitis. Almost 300 hosts were required to break through the obstacles of epistasis and conflicted genotypes before a significant relationship of IL1B+3954(T) with periodontitis was obtained [IL1A and IL1B [IL1B at −511 and +3954 are present in the genome. IL1A-889 is a secondary factor, and not critical for determining GCF exudation trait.Besides epistasis in two hosts, one of these hosts and 3 others possessed a conflicted genotype, obtained , whereasand IL1B . The resIL1B-511, IL10-1082, and CD14-260 in which this function is present in their homozygotic reference (GG) or (TT) alleles (rs16944 and rs1800896). Unlike the other 3 genes, it is specifically the alternate SNPs, (A) allele of IL1B-511 and (C) allele of IL10-1082, that promote strong GCF exudation, efficient bacterial removal from the crevice and mild or no periodontitis  cell-mediated immunity which protects from bacterial invasion of the gingival crevice . We suggdontitis .IL1B-511(A)+IL1B+3954(CC) genotype reported in IL1B-511(GG)+IL1B+3954(CC) genotype in which IL1B-511(GG) produces 25% more IL1β than its alternative (A) allele [IL1B+3954(T)+IL1B-511(A). Conversely, the concurrent presence of all three ‘red’ complex periodontal pathogens and IL1B+3954(T) associated with the greatest IL-1β expression in GCF from periodontitis sites [A related question is whether natural protection from periodontitis can be maintained long enough to permit an EG test in hosts who have reached their early 60s. A 3-week EG experiment used five young hosts aged 20–22 years and five old hosts aged 61 to 65 years, all Caucasians . PI, GI,) allele , or the is sites . P. gingivalis. Within a group of 34 middle-aged, non-smoking US Caucasian patients with periodontitis, P. gingivalis colonization in the deepest periodontal pockets reached almost 50% of total bacteria in 44% of the patients compared with only 12% in the remaining 56% [P. gingivalis colonization in a middle-aged population with moderate periodontitis corresponds to the fraction of weak GCF responders in our original EG study of 16 patients , they digest bacterial products, and protect the host by inducing acquired immunity . If PMNsdontitis .P. gingivalis expresses outer membrane vesicles (OMVs) that spread from periodontitis pockets throughout the body to cause as many as 20 different diseases [P. gingivalis infection may destabilize the atherosclerotic plaques and make them more likely to detach, spread and become trapped in small arteries or veins [diseases . This ordiseases  where thdiseases . In CVD,or veins . The plaor veins .P. gingivalis causing moderate and severe periodontitis [Statins modify this process by inhibiting the enzyme that synthesizes cholesterol , signifidontitis , and itsIL1B-511(GG) displays greater protein expression and greater cytokine activation than its alternate allele, AA, or GA [IL1B-511 individuals, that exclusively differed in the entorhinal-cingulum axis. IL1B-511(GA) polymorphisms modulated the structural covariance strength on the anterior brain and entorhinal-interconnected networks independently of white matter tract integrity. The IL1B-511 GG and GA groups were weaker than AA carriers in covariance strength, and the different network clusters associated with each IL1B-511 GA genotype could predict the cognitive test result [Genetic studies of alleles based on the co-existence of periodontitis and systemic diseases are scarce, but one report utilizes the observation that the presence of the reference allele of A, or GA . When 13t result .P. gingivalis infection than similar individuals from the same population who did not have dementia. How that result would compare with the role of IL1B-511(GA) alone in being able to predict a cognitive test result is uncertain.The prediction of a cognitive test result applies to Chinese patients and may not apply to Hungarian patients in our study. One of our current proposals is to determine whether patients with Alzheimer’s and/or other cognitively impaired diseases in Hungary are more likely to possess the weak GCF trait and greater IL1A-889(A)+IL1B+3954(T) occurs at a frequency of 68% in hosts presenting with at least two of three periodontitis risk factors, smoking, diabetes, or teeth cleaned professionally twice or less per year [The primary procedure for controlling periodontal disease is oral hygiene, self-administered twice daily by toothbrushing and twice yearly by professional scaling, root-planing and prophylaxis. As noted in per year  and theiThe starting point of this article was a preliminary study conducted at Semmelweis University  and concDNA for the present study was obtained from all the participants in that EG study, except a female participant who was abroad. There was no additional recruitment, exposure, follow-up, or repeated data collection. The Appointments for cheek scrapings were made and the scrapings were stored at −70 °C in separate containers until the DNA was extracted, purified and tested for the presence or absence of alleles from each of the genes listed in https://www.thermofisher.com/lu/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/snp-genotyping-taqman-assays.html, accessed on 3 April 2023).We used the TaqMan assay described by ThermoFisher Scientific  to genotype the SNP alleles at each locus in each of the 7 genes listed in https://www.socscistatistics.com/,accessed on 3 June 2022).Strong and weak responses used biofilm lysine concentration (µg/mg biofilm) as the independent variable and GCF response (µL/min) as the outcome. A quadratic polynomial regression model  was employed. The significance of the GCF response (main effect) and its interactions with lysine content and lysine content-squared (independent variables) were determined using an F-ratio test with 3 degrees of freedom to compare the sum of squares explained by the two independent terms (Lys and Lys-squared) relative to the sum of squares error (legend to Devices, kits, and methods for determining increased susceptibility to and treatment and prevention of periodontitis, Alzheimer’s disease, and other conditions. PCT/US22/33045. Received 10 June 2022, Inventor, Martin Levine.