The enzymes involved in m6A modification mainly include methyltransferases (writers), demethylases (erasers), and methylated readers (Readers). m6A modification is mainly catalyzed by m6A methyltransferase and removed by m6A demethylase. The modified RNA can be specifically recognized and bound by m6A recognition protein. This protein complex then mediates RNA splicing, maturation, nucleation, degradation, and translation. m6A also alters gene expression and regulates cellular processes such as self-renewal, differentiation, invasion, and apoptosis. An increasing body of evidence indicates that the m6A methylation modification process is closely related to the occurrence of various skin diseases. In this review, we discuss the role of m6A methylation in skin development and skin diseases including psoriasis, melanoma, and cutaneous squamous cell carcinoma.N Among them, N6-methyladenosine (m6A) is the most frequent modification of eukaryotic mRNA with a dynamic and reversible posttranscriptional manner. The modifications of mRNA exist in the 5′ cap and 3′ poly-A regions of eukaryotes. The methylation in the 5′ untranslated region (5′UTR) of mRNA plays an important role in mRNA splicing, editing, stability, degradation, and polyadenylation, while the methylation in the 3′ untranslated region (3′UTR) contributes to mRNA outward transport, translation initiation, and mRNA structural stability maintenance with poly A-binding proteins [6-Methyladenosine (m6A) is enriched near termination codon and 3′UTR and translated near 5′UTR or in long exons [6A methylation regulates cell division and proliferation, death and apoptosis in nervous system development, circadian rhythm, DNA damage response, heat shock response, and tumorigenesis in mammals [6A methylation defect or dysregulation may disrupt gene expression to result in RNA metabolism abnormality and disease.In recent years, RNA methylation-mediated epigenetic epitranscriptome regulation has emerged as a new field of RNA biology. There are several common internal modifications of mRNA including Nng exons . Recent  mammals . Therefo6A methylation modification process is closely related to the occurrence of various dermatological diseases. In this review, we summarized the m6A RNA methylation and the emerging progress in skin diseases including psoriasis, melanoma, squamous cell carcinoma, Merkel cell carcinoma, and atopic dermatitis.Recently, an increasing body of evidence indicates that the m6A methylation requires the participation of three types of regulators. First, m6A methyltransferase with the encoding gene called “Writers” catalyzes the m6A methylation from RNA. Second, m6A demethylase with the encoding gene called “Eraser” removes the m6A methylation in RNA. Third, m6A-methylated reader proteins bind to m6A sites in RNA to perform specific biological functions. Above enzymes play different roles of in dynamic modification of m6A methylation (The dynamic modification of mhylation .6A methyltransferases include methyltransferase-like protein 3 (METTL3), METTL5, METTL14, METTL16, Wilms' tumor 1-associated protein (WTAP), RNA-binding motif protein 15 (RBM15/15B), and zinc finger CCCH-type containing 13 (ZC3H13) [6A installation of methylation is catalyzed by several methyltransferase complexes (MTC). Methyltransferase-like 3 (METTL3) is an S-adenosylmethionine- (SAM-) binding protein, which is the key methyltransferase for m6A methylation and the most important component of m6A MTC. Abnormal expression of METTL3 can change the total methylation level of m6A. As a structural support of METTL3, METTL14 forms a stable methyltransferase complex at a ratio of 1 : 1 to induce m6A RNA [6A modification [6A methylation [6A methylation.RNA mZC3H13) . The m6A m6A RNA . METTL16 m6A RNA . RBM15/1fication . Other p . The m6α-ketoglutarate-dependent dioxygenase family and catalyze m6A demethylation in Fe (II) and α-ketoglutaric acid-dependent manner. They mediate the demethylation process by oxidizing m6A to form N6-hydroxymethyladenosine (hm6A), then converting hm6A to N6-formyladenosine (f6A) and finally converting f6A to adenosine [6A demethylation and is a member of the ALKB family with a highly conserved catalytic domain. ALKBH5 was shown to be an RNA demethylase that can oxidatively reverse m6A modification. Unlike FTO, ALKBH5 with unique crystal structures is a specific demethylase during m6A RNA methylation [6A methyltransferase remain to be verified. The mechanism to maintain dynamic balance with methylation and demethylation is not completely clear.Demethylase mainly includes fat mass and obesity-associated protein (FTO) and AlkB homologue 5(ALKBH5). FTO and ALKBH5 belong to denosine . FTO andhylation . However6, 2′-O-dimethyladenosine (m6Am) with its major target being m6Am in small nuclear RNAs [6Am is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap [6-methylation of m6Am and removed by FTO [6Am is different from that of m6A.It is interesting that FTO has recently been found to preferentially target Near RNAs . m6Am issine cap . A recend by FTO . Thus, t6A-methylated proteins by recruiting eukaryotic translation initiation factor 3 (eIF3) translation initiation complexes. YTHDF2 selectively binds to 3′UTR of m6A and regulates mRNA degradation to affect mRNA stability. YTHDF3 promotes protein synthesis and translation through synergetic effects with YTHDF1 to augment YTHDF2-mediated mRNA degradation [6A sites [6A reader protein mediated by RNA splicing. YTHDC2 interacts with RNA helicase to provide topological regulation of translation and extension [6A recognition. The phenomenon is called the “m6A switch” [6A and enhance mRNA stability and translation in m6A-dependent ways [6A-modified critical sites during methylation.Methylated reader proteins mainly include members of YTH-domain families , heterogeneous ribonucleoproteins , and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs). There is a conservative YTH domain with the properties of single-stranded RNA-binding proteins in members of YTH-domain family. YTHDF1 facilitates the translation of mradation . YTHDF1,6A sites . YTHDC1 xtension . HNRNPC  switch” . In addient ways . These m6A is closely related to skin development and regeneration. The mouse embryonic skin epithelium is initially a layer of pluripotent epithelial progenitor cells that later develop into three tissues consist of epidermis, hair follicles, and sebaceous glands. A research analyzed the potential effect of m6A deficiency on mouse embryonic skin epithelial progenitor by single-cell RNA sequencing and functional studies. It demonstrated that both signaling and canonical translation pathways such as Wnt signal, actin regulators, cell polarity, extracellular matrix- (ECM-) receptor interaction, and NOTCH signal were significantly downregulated after m6A loss [6A methyltransferase impairs the skin's ability to self-renew and heal wounds. The above findings suggested that m6A methylation is indispensable during skin development.Skin with rich blood vessels and a complex structure is the most easily exposed organ to the external environment. Increasing evidence demonstrates that mm6A loss . Immunofm6A loss , 26. Gen6A methylation of Pvt1 as a key step in skin tissue homeostasis and wound repair [6A sites with differential RNA methylation in cashmere fiber growth by methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) and RNA-seq [6A-modified genes were highly expressed in the skin tissue of goats. Keratins, the major components of the epithelial cytoskeleton, are responsible for maintaining the structural stability and integrity of keratinocytes, protecting cells from mechanical damage and affecting the formation of skin accessory organs. Furthermore, GO enrichment analysis showed that methylated genes were mainly involved in keratin filaments and intermediate filaments [6A modification in skin development and growth. It proposes a possible strategy for wound healing via regulating m6A modification.Previous studies have discovered the possible mechanism of epitranscriptome dynamics occur during epidermal differentiation. The study demonstrated that plasmacytoma variant translocation 1 (Pvt1) methylation enhanced the interaction with myelocytomatosis oncogene (MYC) and stabilized MYC protein in epidermal progenitors. It identified md repair . In a re RNA-seq . The resilaments . Therefo6A modification plays an important role in a variety of malignant tumors [6A modification on oncogenes or tumor suppressor genes. m6A reader proteins recognize corresponding markers through a series of biological effects, thereby to upregulate oncogene expression or downregulate tumor suppressor gene expression. On the other hand, the demethylase accelerates tumor progression via tumor-specific oncogenes [6A modification to regulate these genes may be a novel therapeutic strategy for cancer.Many studies have shown that mt tumors , 31. Thencogenes . These o6A RNA methylation was downregulated in arsenic-related human skin lesions. Moreover, FTO removal significantly inhibited arsenic-induced skin tumorigenesis [6A-seq and RNA-seq analysis revealed that neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was the critical target of FTO during m6A RNA methylation, and FTO regulated NEDD4L mRNA stability via IGF2BPs. Another study revealed FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenesis. Analysis of gene expression profiles showed higher expressions of m6A effectors, especially METTL3, in arsenic-exposed groups than low-arsenic-exposed populations. Further experiments found that arsenic promoted m6A methylation by up-regulating METTL3 to increase the secretion of inflammatory factors including interleukin- (IL-) 6, IL-17, and IL-10 in human keratinocytes. Keratin 1 (KRT1) and keratin 10 (KRT10) reflecting skin injury were also significantly increased [6A RNA methylation-mediated DNA damage-binding protein 2 (DDB2) translation and inhibits ultraviolet B- (UVB-) induced skin tumorigenesis. METTL14 knockout suppressed m6A RNA methylation and translation in transcripts of DDB2 [Among them, one group found that FTO was upregulated while migenesis . Furtherncreased . Another of DDB2 . Therefo6A regulators including METTL3, YTHDF1, HNRNPA2B1, FTO, and IGF2BP3 are involved in melanoma (6A demethylase among the m6A regulators. Studies have shown that FTO stimulates melanoma growth. FTO knockout increased m6A methylation of genes including PD-1 (programmed cell death protein 1), CXCR4 (C-X-C motif chemokine receptor), and SOX10 (Sry-related HMG-box-10) in melanoma cells and thereby enhanced the susceptibility to interferon therapy [6A modification to enhance its stability in melanoma. In addition, UCK2 enhanced the migration and invasion ability of melanoma cells by activating the Wnt/β-catenin pathway [6A regulatory factors in tumor samples and normal samples. It was demonstrated that IGF2BP3 promoted proliferation and migration of melanoma cells in vitro. The expression of IGF2BP3 was positively correlated with lymph node metastasis and immune cells infiltrating [Melanoma is a serious and life-threatening skin malignancy . About 7melanoma . Studies therapy . METTL3  pathway . Both YT pathway . A threeltrating . These r6A regulatory proteins such as RBM15, YTHDF1, WTAP, and METTL14 genes in melanoma with a higher degree of genomic variation and a worse prognosis. Then, they identified a subset of molecules with immunological effects based on m6A regulatory proteins [6A methylation, including methylation on writing proteins, methylation reading proteins, and demethylases such as METTL3, YTHDF1, and FTO, play an important role in the development of melanoma. It provides a reliable theoretical basis for the treatment of melanoma and the development of drugs.Some researchers analyzed the gene expression profiles, copy number variation (CNV), and single-nucleotide polymorphism (SNP) data from The Cancer Genome Atlas (TCGA) databases. The study showed that 15 genes were changed on mRNA expression and mproteins . The stu6A-binding protein YTHDF1 expression in skin keratinocytes, even skin mutation and oncogenesis to cause cSCC [Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy, accounting for 20% of nonmelanoma skin cancers. The most important physical factor causing the incidence of cSCC is sunlight ultraviolet rays especially UVB. Excessive sunlight exposure could lead to cell mutation and tumorgenesis. Studies have shown that exposure to UVB and arsenic resulted in upregulation of muse cSCC .6A methyltransferase METTL3 is also a key gene to regulate the occurrence of cSCC genesis. METTL3 was upregulated in cSCC, and METTL3 knockout disrupted the characteristics of stem cells in cSCC. METTL3 deficiency decreased colony-forming ability and suppressed tumorigenicity in vivo by reducing m6A levels and ΔNp63 expression in cSCC. Exogenous ΔNp63 overexpression partially restored the proliferation of METTL3-knockout cSCC cells [The mCC cells .6A RNA methylation in cSCC was not studied as extensively as other related squamous cell carcinomas such as oral squamous cell carcinoma (OSCC), hepatocellular carcinoma (HCC), and gastric cancer (GC). Studies have shown that METTL3 promoted BMI1 translation in OSCC under the cooperation with m6A reader IGF2BP1 and promoted OSCC proliferation and metastasis [6A methylation in cutaneous squamous cell carcinoma.mtastasis . Combinatastasis . The exptastasis . These s6A modifications on small T antigen mRNAs [MCC is a highly aggressive and rare skin cancer . Merkel en mRNAs . The stu6A-binding protein FTO, METTL3, METTL14, and YTHDF1 expression were involved in skin lesion and oncogenesis. FTO promotes oncogenesis via IGF2BPs. METTL3 increases inflammatory factor secretion in human keratinocytes and enhanced ΔNp63 expression in cSCC. METTL14 facilitates GGR to suppress UVB-induced skin tumorigenesis. YTHDF1 activates translation initiation factors to high tumorigenicity in MCC  activation in psoriasis by altering Wnt gene expressions [α/IL-23/Th17 axis is regarded as a key factor in inflammation exacerbation and lesion aggravation in psoriasis, being critical for the expansion of inflammation and aggravation of skin lesions in psoriasis vulgaris [6A methylation in psoriasis patients by MeRIP-Seq and RNA-Seq. The results showed that IL-17A and TNF-α, as two key genes of the TNF-α/IL-23/Th17 axis, are upregulated m6A methylation in skin lesions of psoriasis patients [Psoriasis is a common, complex, and chronic inflammatory disease that severely reduces patients' quality of life. The hallmark of psoriasis is uncontrolled proliferation and differentiation of keratinocytes by persistent inflammation. Previous studies have shown that methylated transcripts were mainly related to the Wnt signaling pathway in skin lesions of psoriasis patients. The Wnt gene family encodes a group of highly conserved secretory signal proteins involved in cell differentiation, proliferation, and immune-mediated inflammatory responses –55. It iressions , 57. Tumvulgaris . One stupatients .6A writer WTAP were significantly increased in psoriasis patients especially in the epidermis. Overexpression of WTAP promoted the proliferation of keratinocytes, which may be possibly related to the upregulation of cyclinA2 and CDK2 [6A may be an important trans-epigenetic modifier to regulate proliferation, differentiation, and inflammation of psoriatic keratinocytes.Additionally, the mRNA and protein levels of mand CDK2 . The abo6A-dependent manner in 2,4-dinitrochlorobenzene- (DNCB-) induced dermatitis. Low levels of MALAT1 promoted ubiquitinated degradation of S100A8/S100A9, thereby prevented p38/mitogen-activated protein kinase phosphorylation and macrophage-mediated inflammation, and finally inhibited M1 macrophage activation [6A RNA methylation in atopic dermatitis proposed a possible role of macrophage-mediated inflammation in disease progression.AD is a common and chronic inflammatory skin disease affecting approximately 10-25% of children and 7-10% of adults. It is characterized by disturbances in epidermal structure and keratinocyte differentiation, as well as excessive T cell-mediated inflammation with a complex genetic condition . The pattivation . The exi6A-tagged mRNAs in a mouse model of bleomycin-induced scleroderma by m6A supratranscriptomic microarrays and m6A RNA immunoprecipitation qPCR. The results showed that there were differences in the m6A methylation of 843 mRNAs and include the methylation and the expression of Hras, Saa1, Ccl3, Ccl9, and Il1b [6A methylation in scleroderma.Scleroderma is an autoimmune connective tissue characterized by organ fibrosis, immune abnormalities, and vascular damage . Althougand Il1b . This st6A modification in CTCL progression. The results showed that METTL3 significantly downregulated CTCL cells both in vivo and in vitro. Furthermore, METTL3 small interfering RNAs and RIP assay indicated that cyclin-dependent kinase inhibitor 2A (CDKN2A) was a key regulator in CTCL, and insufficient methylation blocked the interaction between CDKN2A and m6A reader IGF2BP2, resulting in mRNA degradation [6A in CTCL development and provide a potential biological target for therapy.Cutaneous T-cell lymphomas (CTCL) are a rare type of non-Hodgkin lymphoma characterized by cutaneous infiltration of malignant lymphocytes . Recent radation . This is6A reader protein YTHDC1 interacting with SQSTM1 mRNA was attenuated in keratinocytes during hyperglycemia. In vivo, knockout of endogenous YTHDC1 or SQSTM1 suppressed epidermal autophagy and impaired the biological functions of keratinocytes including promoted apoptosis and delayed wound healing. The results showed that YTHDC1 interacted with ELAV-like RNA-binding protein 1/HuR (ELAVL1) and cooperatively regulated the expression of SQSTM1 [Sequestosome 1 (SQSTM1) is a vital protein marker serving as a selective autophagy receptor . SQSTM1/f SQSTM1 . This st6A modification patterns in hyperplasia scars (HS) and normal skin (NS) tissues by m6A sequencing and RNA sequencing. Several targets of m6A-associated RNA were immunoprecipitated and verified by real-time quantitative PCR. The results showed that 14791 new m6A peaks appeared in the HS samples, while 7835 peaks disappeared. It suggested that the m6A-related genes in HS are associated with fibrosis-related pathways. In addition, they identified differentially expressed mRNA transcripts in HS samples with hypermethylated or hypomethylated m6A peaks [6A epitranscriptome of human HS, which may help to elucidate the possible mechanisms of m6A-mediated regulation of gene expression.One group described m6A peaks . This st6A regulation in skin disease. Only a few studies were published on the mechanisms that control the expressions and activities of m6A “Writers,” “Erasers,” and “Readers” in the skin, especially the “reader.” Previous studies have confirmed that m6A methylase has dual effects of cancer promoting or suppressing. METTL3 serves as an apoptosis driver in high glucose-reliant human lens epithelial cells (HLEC), while WTAP acts as an inhibitor in human natural killer/T cell lymphoma (NKTCL) cell lines [6A methylation in skin tumors and skin inflammatory diseases still needs to be further explored.There is no study to exactly confirm the specific mechanism in dynamic mll lines , 27. Thi6A in RNA can be probed by several methods, including two-dimensional thin layer chromatography [6A dot-blot [6A-seq (m6A RNA immunoprecipitation sequencing), relies on RNA fragmentation but incapable of single-nucleotide resolution detection in methylation sites [6A methylation. Some more effective methods especially in the skin are urgently needed.The total amount of mtography , m6A dotdot-blot , and HPLdot-blot . Howeveron sites . Althoug6A methylation in HeLa cells and had better cellular permeability than MA [6A methylation regulates innate immune response [6A methylation was a crucial regulator of T cell homeostasis. T cells carrying m6A modifying agents may be an effective target for the treatment of autoimmune diseases [There are already some drugs targeting methylations. The nonsteroidal anti-inflammatory agent meclofenamic (MA) has a strong inhibitory effect on FTO and is expected to become a demethylase inhibitor. MA2, an ethylester derivative of MA is hydrolyzed inside the cell to its active form MA. MA2 increased m than MA , 77. Howresponse , 77. It diseases . These s6A is dynamically regulated in many physiological and pathological processes of dermatological diseases. RNA methylation is regulated by methylase, demethylase, and methylation reader proteins, and the process is reversible. RNA methylation is a relatively new field. In recent years, m6A has been prominently discovered in various skin diseases. However, many unknowns still exist in the epitranscriptome modification of RNA. The detection method of m6A methylation needs improvement. The efficacy of methylation-modifying agents needs further clinical studies. Epitranscriptome modification has attracted more and more attention in regulating immune response and immunotherapy. Exploring key epitranscriptome variation sites and developing corresponding immunotherapy drugs have become research hotspots and have good development prospects.Taken together, m


K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7’s ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7 K81S. We examined phenotypic differences between the E7K81S and wild type MmuPV1 in tissue culture and in mice. The E7K81S mutant genome was able to replicate as a nuclear plasmid in tissue culture. In vivo, E7K81S mutant virus caused dysplastic lesions like wild type virus; however, they were smaller in size, arose later, and trended towards being less severe in their grade of neoplastic disease. The E7K81S-induced lesions showed evidence for increased E2F activation and reprogramming of suprabasal cells to support DNA synthesis similar to that seen in lesions induced by wild type virus. One clear molecular difference was that the E7K81S-induced lesions showed an expansion of cells expressing differentiation markers. Our study shows that an intact PTPN14 binding site is important for MmuPV1 E7’s ability to contribute to MmuPV1-induced skin disease.Human papillomaviruses cause human cancers largely through virally encoded E6 and E7 oncogenes. A murine papillomavirus, MmuPV1, causes lesions in laboratory mice that mimic features of human papillomavirus-induced lesions including cancer. In this study, we investigated how the interaction of MmuPV1 E7 with the cellular tumor suppressor protein, PTPN14, contributes to viral pathogenesis using a MmuPV1 E7 mutant defective for interacting with PTPN14, E7 Human papillomaviruses (HPVs) are major human pathogens that cause ~5% of the world’s total cancer burden including anogenital, head and neck, and skin cancers –4. HPVs The ability of mucosal high-risk HPVs to promote carcinogenesis is driven by the virally encoded E5, E6, and E7 oncogenes ,15. The in vivo in the context of a natural infection. The murine papillomavirus (MmuPV1), discovered in 2011, naturally infects laboratory mice . Th. ThK81S < 0.001) . Althougs (2.5X) . Represe animals .K81S causes less severe disease than wild type MmuPV1, mouse ears were collected at the four-month time point and the six-month endpoint post-infection, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned into 5 μm sections, and stained with hematoxylin and eosin (H&E). Histological sections were analyzed by two pathologists (DB and RH) blinded to sample identity and classified as mild or severe dysplasia, carcinoma in situ (CIS), or invasive squamous cell carcinoma (cSCC). While mice infected with wild type MmuPV1 developed CIS, which represents early invasive disease, no mice infected with E7K81S MmuPV1 progressed to this advanced stage of neoplasia by 6 months post-infection. The difference in distribution of disease states in the two populations did not reach statistical significance (p = 0.1) (To test whether MmuPV1 E7p = 0.1) . Our datK81S MmuPV1: MmuPV1 L1 capsid protein expression, MmuPV1 genome amplification, and MmuPV1 gene transcription. To detect L1 protein, we performed immunofluorescence using Tyramide Signal amplification system (TSA) on Formalin-fixed paraffin embedded (FFPE) tissues for the MmuPV1 L1 protein and K14, a marker of basal keratinocytes. L1 capsid protein was produced at comparable levels in the E7K81S MmuPV1-induced lesions and in lesions arising in wild type MmuPV1-infected animals  containing the E4 gene comparable to that observed in wild type MmuPV1-induced lesions  developed warts/lesions at other cutaneous sites . All animals in wild type  and nearly all animals in E7us sites . This deK81S mutant-induced lesions arose later and were smaller at endpoint, we wanted to determine whether the E7K81S mutant MmuPV1 was altered in its ability to increase cellular DNA synthesis or activate E2F. To do so, we assessed the incorporation of bromodeoxyuridine (BrdU) into newly synthesized DNA and expression of MCM7, an E2F-responsive gene, respectively. FFPE tissue slides were subjected to immunohistochemistry using antibodies against MCM7 and BrdU as previously described [K81S mutant-induced lesions showed evidence of increased expression of MCM7 and suprabasal DNA synthesis, similar to lesions caused by wild type MmuPV1 infection  , as well as Keratin 10 (K10) and Involucrin (IVL), which are markers for more differentiated keratinocytes. We observed similar hyperplasia of K14-positive keratinocytes in both the wild type and E7K81S MmuPV1-induced lesions  which impairs E7’s ability to bind PTPN14, we found that this mutant virus was able to cause lesions in nude mice at a similar frequency as wild type MmuPV1 E7 , and horizontal transmission (surrogate marker for infectious virus production) which are markers of the late stage of the viral life cycle  ,56. MmuPPTPN14 is defined as a tumor suppressor because inactivating mutations in PTPN14 are associated with increased risk of some cancers ,58,69. Ain vivo. These findings are consistent with the observation that high-risk HPV E7 proteins inhibit the initial stages of differentiation in 2D cell culture models, dependent on their ability to degrade PTPN14 [K81S virus to determine the importance of this interaction during a MmuPV1 mucosal infection.In our studies, MmuPV1 E7’s ability to bind PTPN14 contributes to MmuPV1’s ability to inhibit epithelial cell differentiation e PTPN14 . In 3D re PTPN14 . Similare PTPN14 . Hence ie PTPN14 ,62. Cutae PTPN14 ,63,80. IWe have previously shown that the cutaneous β-HPV8 and MmuPV1 E6 proteins inhibit keratinocyte differentiation through its interactions with MAML1 as well as SMAD2 and SMAD3, which are critical co-activators of NOTCH and TGF-β signaling, respectively . TherefoNIH 3T3 murine fibroblasts were obtained from ATCC and grown in DMEM supplemented with 10% calf serum. Mouse keratinocytes were isolated from the skin of neonate pups from the FVB/N and C57/Black6 backgrounds. After incubation in phosphate-buffered saline (PBS) containing 10% antibiotics for 2 min, skin pieces were incubated in 0.25% trypsin overnight at 4°C. The epidermis was then separated from the dermis using sterile forceps, minced with a single-edge razor blade, and then stirred for 1 h at 37°C in F-medium to generate a single-cell suspension. The cells were strained using 0.7-mm membrane  and cultured in F-medium containing 10 mM Y-27632 Rho-kinase inhibitor  in the pK81S mutant was PCR amplified with primers containing the K81S mutation and Q5 High Fidelity Polymerase (NEB). The PCR product was treated with DpnI (NEB) for 1 hour, then transformed into 10-Beta competent E. coli cells (NEB).N-terminally and C-terminally Flag/HA tagged MmuPV1 E7 and HPV16 E7 were cloned into pCMV Bam/neo vectors . The MmuNIH3T3 cells were transfected using Lipofectamine 2000 . At 48 hr post-transfection cells were harvested in EBC buffer  supplemented with protease inhibitors (Pierce). Anti-Hemagglutinin  antibodies coupled to agarose beads were used for immunoprecipitations followed by SDS-PAGE and western blot analysis on PVDF membranes. After incubation with appropriate primary and secondary antibodies, blots were visualized by enhanced chemiluminescence, and images captured using a Syngene ChemiXX6 imager with Genesys software version 1.5.5.0. Signals were quantified with Genetools software version 4.03.05.0. Antibodies included PTPN14 (CST #13808), HA (Abcam ab9110), Actin (Millipore MAB1501), pRB (Santa Cruz SC74570), Flag (Sigma F3165), and GFP (Santa Cruz SC-9996). Secondary anti-mouse and anti-rabbit HRP antibodies were from GE Healthcare.https://pave.niaid.nih.gov), aligned by MAFFT FFT-NS-2 (v7.453), and visualized in Jalview. Sequences of PTPN14 orthologs were retrieved from the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene), in which orthologs were calculated using NCBI’s Eukaryotic Genome Annotation pipeline. Sequences were aligned with COBALT and visualized in Jalview. Three-dimensional alignment was performed in PyMOL using the deposited structure 6IWD [E7 protein sequences were retrieved from PaVE .D90A, and E7K81S; the term “quasivirus” is used in the papillomavirus field to describe virus generated in vitro after transfecting in recircularized viral genome with a plasmid that expresses the L1 and L2 proteins in 293 cells) was generated as described before [nu/nu mice using 108 VGE of each virus prep for infected animals and PBS for mocks as previously described [8 VGE with both ears being infected for a total of 2 sites per animal. Lesion development was monitored biweekly for 4–6 months post-infection.MmuPV1 quasivirus . The membrane was then probed with a set of 20 oligos complementary and specific to MmuPV1 that were labeled with γ-32P-ATP. To visualize MmuPV-1 DNA, the washed membrane was exposed to a PhosphorImager screen that was then scanned using a Typhoon . For titering virus prep, 10 μL of virus prep was treated with equal volume virus release buffer  and incubated at 56°C for 1 hr. Samples were subjected to similar analysis as described above starting at gel electrophoresis.8 to 102 using 100-fold dilutions) and cellular DNA concentration (10 μg to .01 μg using 10-fold dilutions) to generate Ct values for viral genome copy number and concentration of cellular DNA. Ct values for specific samples were converted to viral copy number or cellular DNA concentration present within each sample. Viral copy number was normalized to cellular DNA concentration and relative copy number was determined comparing each sample to the average copy number/cellular DNA for WT transfected cells.FVB cells transfected with recircularized viral genome were harvested following selection and outgrowth and DNA was isolated using Qiagen Blood and Tissue kit (69506) following manufacturer’s instructions. Isolated DNA was then used for qPCR analysis using E2 primers (MmuPV1_E2_1 5’-GCCCGAAGACAACACCGCCACG-3’ and MmuPV1_E2_2 5’-CCTCCGCCTCGTCCCCAAATGG-3’) to detect viral genomes and murine 18S primers (18S_1 5’-CGCCGCTAGAGGTGAAATTC-3’ and 18S_2 5’-TTGGCAAATGCTTTCGCTC-3’) to approximate genomic DNA concentration in each reaction . qPCR wath section was stained with Hematoxylin and Eosin (H&E) for histological analysis.One hour prior to euthanasia, infected mice were injected intraperitoneally with 250 μL of 12.5 mg/mL bromodeoxyuridine . Tissues were fixed in 4% paraformaldehyde for 24 hr and then switched to 70% EtOH. Fixed tissues were processed, embedded in paraffin, and 5-μm sections generated. Every 10https://www.protocols.io/view/untitled-protocol-i8cchsw. For immunohistochemistry, slides were deparaffinized and rehydrated as described above. Antigen retrieval was performed on rehydrated sections using citrate buffer (pH 6) for 20 min by heating in a microwave (3 minutes on max and 17 minutes at 70% power). Blocking was performed using 2.5% horse serum in PBS for 1 hr at room temperature (RT). Slides were then treated with primary antibody overnight in a humidified chamber. Vectastain ABC (HRP) kit  was used to detect signal. Following primary antibody treatment, slides were washed and then treated with universal secondary antibody for 1 hr at RT. Following secondary antibody treatment, slides were treated with an equal mix of A and B reagents for 1 hr at RT. Slides were then treated with DAB for signal development and subsequently counterstained with Hematoxylin . Slides were dehydrated using the EtOH concentrations described above but in reverse order, followed by Xylene washes before being mounted. All images were taken using a Zeiss AxioImager M2 microscope using AxioVision software, version 4.8.2. Primary antibodies used include L1 (gift from Chris Buck), K14 , K10 , Involucrin , MCM7 , and BRDU . Secondary antibodies include anti-Rb-AF488  and anti-SA-AF647 . IF images were quantified using ImageJ where areas of positive staining were determined by total area times the percent of the area that had signal. Values for L1, K10, and IVL were normalized to K14 in the analysis. Workflow is provided in S8 FigImmunofluorescence was performed on Formalin-fixed paraffin-embedded tissue sections for Keratin 14 (K14), Keratin 10 (K10), and Involucrin (Ivl). Briefly, sections were deparaffinized in Xylene and rehydrated in 100%, 95%, 70%, and 50% EtOH and then in PBS. Rehydrated sections were then treated with boiling Tris-EDTA pH10 antigen retrieval buffer for 20 mins. Sections were blocked with 5% Milk containing goat serum for 1 hr at RT and then primary antibody added to incubate overnight. Next day, sections were treated with secondary antibody following washes for 1hr using goat-α-rabbit AF488. Sections were then washed and subject to Hoechst stain for 10 mins before mounting. K14 and L1 capsid protein co-staining was performed as previously described using the tyramide-based signal amplification (TSA) method . A detaiTo detect MmuPV1 E4 transcripts in the cytoplasm and MmuPV1 DNA genomes in the nucleus, we used RNAscope 2.5 HD Assay-Brown  and a MmuPV1 E4-specific probe set  according to manufacturer’s instructions as previously described ,51. To dhttps://oncology.wisc.edu/mstat/).All statistical tests were done using MStat software  Amino acid sequence alignment of the C-terminus of several primate, rodent, and bovine papillomavirus E7 proteins. R84, and L91 are two amino acids in HPV18 E7 that make contact with PTPN14 as determined by the HPV18 E7-PTPN14 crystal structure [(B) Amino acid sequence alignment of a segment of the PTP domain of selected primate, rodent, and bovine PTPN14 proteins. F1044, G1055, and E1095 are three amino acids in human PTPN14 that make contact with HPV18 E7 as determined by the HPV18 E7-PTPN14 crystal structure [(C) Additional structure image of MmuPV1 E7 (yellow) and PTPN14 (silver) and how they may interact. Lysine 81 is highlighted in red. (D) Ribbon structures of HPV18 E7 (blue) and MmuPV1 E7 (yellow) overlayed to determine similarity in secondary structure. Lysine 81 in MmuPV1 E7 is highlighted in red.tructure . Identittructure , and the(TIFF)Click here for additional data file.S2 FigExperiment 1 images correspond to the data shown in the left panel of (TIF)Click here for additional data file.S3 Fig(A) Unspliced southern blot showing all conditions run on gel image shown in B) Total RNA was isolated from JB6 mouse epithelial cells 48hrs post-infection with WT MmuPV1, E7D90A, and E7K81S quasivirus preps. cDNA was generated and subjected to RT-PCR to determine if quasivirus prep was infectious using primers that target the E1^E4 viral transcript.(TIF)Click here for additional data file.S4 FigK81S mutation. A representative sequencing result is shown.DNA was isolated from FFPE tissue sections and then subjected to PCR for the MmuPV1 E7 gene. PCR products were sent for sequencing to verify presence of the E7(TIF)Click here for additional data file.S5 FigK81S mutant are shown. Lesions are located within the circle.Representative images of mouse ears for Mock, WT, and E7(TIF)Click here for additional data file.S6 FigFFPE tissue sections subjected to IF analysis for K14 (green) and L1 (orange) where L1 staining is solely detected in K14 negative differentiated cells. All images were taken at 10X magnification. Dashed white lines indicate the basement membrane of epithelial tissues.(TIF)Click here for additional data file.S7 FigK81S-infected animals. All images were taken at 10X magnification. Dashed white lines indicate basement membrane of epithelial tissues.Representative images of FFPE tissue sections subjected to IF analysis using antibodies against K14 (green), K10 (cyan), and IVL (red) for WT- and E7(TIF)Click here for additional data file.S8 FigThe workflow that was used for IF quantification of differentiation markers is shown.(PDF)Click here for additional data file.


Disruption of H3K36 methylation in somatic cells leads to transposon transcription and activation of key genes in the PIWI-interacting RNA pathway together with RNA production from piRNA clusters. Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in  Transposable elements make up a large part of the eukaryotic genome and come in a multitude of variants. They have been shown to drive gene regulation and to exhibit sophisticated ways of selective insertion in the genome , therebyDrosophila melanogaster, three evolutionary conserved proteins methylate H3K36. SET domain containing 2 (Set2) is the only histone methyltransferase to trimethylate H3K36 and Set2 mutants show a 10-fold reduction of H3K36me3, whereas bulk mono- and dimethylated H3K36 remain largely unaffected (Drosophila Nuclear receptor-binding SET domain protein (NSD) but closely related mammalian orthologs  have been shown to mono and dimethylate H3K36  has been extensively studied and is foremost associated with transcriptional activity. However, the role of H3K36 methylation is complex and it has been shown to be a key player in preventing run-away cryptic transcription , DNA repaffected . Absent,affected . Less iste H3K36 . NSD losed H3K36 .D. melanogaster larvae causes increased transcription of transposable elements  also target, yet unknown, non-histone proteins. The loss of Set2 impairs dosage compensation of the male X chromosome independent of H3K36 methylation  and found that the bulk of the transcriptional activation takes place in the pericentromeric regions of all chromosomes and the entire sequenced part of the Y chromosome  on transposon repression, we analysed transcript levels of transposable elements in dissected brains from third instar male larvae with the lysine-to-arginine replacement mutant 2xH3K36R . We haveR larvae . The ∆Hif H3K36M . Neverth2xH3K36K . In our  repeats  and S1     . NSDds46 < 0.05) . The ash < 0.05) . To bettsferases . Analysisferases  comparedtivation . Interesromosome .H3K36 methyltransferase-specific transposon superfamily transcription. Transposon classifications and abundance for histone methyltransferase mutant-specific clusters described in Fig 2D.Table S1. ∆HisC; 12xH3K36R is to a large extent recapitulated in Set21, indicating that disrupted methylation of H3K36 causes increased transposon transcription. We next asked if this activation can be linked to one of the three methylation states. To this end, we analysed ChIP-seq data for mono-, di-, and trimethylated H3K36 from adult fly heads to test if different transposons vary in their H3K36 methylation levels. We observe a significant difference between LINE and LTR compared with simple repeats or genome average where both LINE and LTR transposons have higher relative levels of H3K36me2 but lower levels of H3K36me1 and H3K36me3, whereas simple repeats show a slight but significant elevation of the more ubiquitous H3K36me1       and in S < 0.05) . Similar < 0.05) . In ash1 < 0.05) , which semplates . Corresppe = 5.1 . OverallD. melanogaster are defined as two general types based on their mode of transcription in the germline , proposed to aid the transcription of piRNA source loci via read-through from flanking genes  in ∆HisC; 12xH3K36R showed significant enrichment for genes related to piRNA metabolic processes (Table S2). For Set21, we observe significant up-regulation of AGO3 and aub together with genes encoding the Tudor domain proteins, fs(1)Yb and BoYb (2 fold up-regulation of CG15930 (Tudor domain containing protein 5-like) in ∆HisC; 12xH3K36R. CG15930 has been implicated in the piRNA metabolic process and promoting male sexual identity in the germline . Kots is a germline-specific Tudor domain containing protein essential for maintaining high piRNA levels produced from uni-strand and dual-strand piRNA clusters  in both sexes are: AGO3, piwi, krimp, moon, rhi, fs(1)Yb, papi, egg, qin, SoYb, tud and vas (Table S3). In addition, female larvae also exhibit significant up-regulation (P < 0.05) of: del, BoYb, CG15930, and Kots (Table S3). The only piRNA biosynthesis gene specifically up-regulated in males is squash (Table S3). To summarise, the impact of impaired H3K36 methylation on increased transposable element transcription and transcription of piRNA clusters can be detected in multiple tissues, sexes, and by independent experimental measurements.Finally, we asked if the observed effects on transposon activity, piRNA biosynthesis, and dual-strand cluster activation also occurred outside of eye discs and larval brains. To this end, we reanalysed the results of sequencing nuclear RNA from th sexes . We obseth sexes  with simth sexes , and S1.th sexes  and femath sexes . When loth sexes . However cluster  and, in clusters . This is2 fold change values for differentially transcribed genes related to the PIWI/piRNA biosynthesis machinery in females (left) and males (right). ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001.Differential transcription analysis results for PIWI/piRNA biosynthesis genes in whole larvae. Table showing logTable S3. ∆HisC; 12xH3K36R is already lowered by the induced PIWI/piRNA system. The mechanism to increase the transcription of transposable elements remains elusive but does not depend on DNA methylation because D. melanogaster lacks 5-methylcytosine.We show here that compromised H3K36 methylation, likely through the loss of H3K36me2, causes increased transcription of transposable elements. The activation of transposable elements in turn triggers transcriptional activation of the PIWI machinery followed by transcriptional activation of dual-strand piRNA clusters. As the PIWI/piRNA system has a dampening effect on transposable-element transcription, we speculate that the observed effect seen in D. melanogaster, has evolved to efficiently target runaway transcription of transposons in germ cells. In recent years, however, the transcription of retrotransposons in somatic cells has shown to be tightly connected to cancer progression, neuronal defects, and brain development  also showing increased transposon activity in Set21 . The transposon-activity profile of ash122/ash19011 is the least biased but interestingly has the biggest relative increase in LTR/Copia repeats with 6.25% compared with Set21 (5.03%) and NSDds46 (1.67%) (Table S1). We note that because of multi-mapping of reads, we cannot determine the exact origin of the transcribed transposable elements. However, we show that the bulk of transposable elements originate from intergenic regions and the observed up-regulation of transposable elements is not a result of transcriptional activation or increased transcription of transposon harbouring genes (The clustering of transcribed transposons shows patterns for ng genes .The largely overlapping increase in transcription of repeated elements after knock-out of H3K36-specific methyltransferases (particularly Set2) and H3K36R replacement (replication-dependent H3.2K36R) argues that it is the methylation of H3K36 that keeps transposable elements transcriptionally inactive in somatic cells. However, the histone replacement approach has limitations. First, H3K36R replacement effectively abolishes all lysine modifications including acetylation. It also may affect posttranslational modifications of neighbouring amino acids. Thus, H3K36R is less effectively methylated at lysine 27 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) in vitro . LikewisD. melanogaster  were quantified within Repeatmasker (v.4.0.6) annotations . For ∆HisC; 12xH3K36R, the number of transposons filtered out was 1,694. For Set21, NSDds46, ash122/ash19011, and ∆H3.3B; ∆H3.3A the number of filtered out transposons were 2,354, 3,641, 2,586, and 3,799, respectively. The whole larvae nuclear RNA-seq results are presented unfiltered because of insufficient number of biological and technical replicates. ChIP-seq data were assigned to repeats using Repeatmasker (v.4.0.6) annotations (∆HisC; 12xH3K36R and ∆HisC; 12xH3K36K.The RNA sequencing data were processed as described in otations . For anaotations  alongsidotations . Stringeotations  in SeqMootations  on the t


ERCC5, EXO1, FANCC, NEIL1 and NTHL1 were identified in 5/13 (39%) OC families. Collectively, candidate variants were identified in 7/435 (1.6%) sporadic OC cases and 1/566 (0.2%) sporadic BC cases versus 1/1025 (0.1%) controls. Additional carriers were identified in 6/605 (0.9%) OC cases. Tumour DNA from ERCC5, NEIL1 and NTHL1 variant carriers exhibited loss of the wild-type allele. Carriers of various candidate variants in these genes were identified in 31/937 (3.3%) OC cases of diverse ancestries versus 0-0.004% in cancer-free controls. The strategy of applying a candidate gene approach in a population exhibiting genetic drift identified new candidate OC predisposition variants in DNA repair pathway genes.Not all familial ovarian cancer (OC) cases are explained by pathogenic germline variants in known risk genes. A candidate gene approach involving DNA repair pathway genes was applied to identify rare recurring pathogenic variants in familial OC cases not associated with known OC risk genes from a population exhibiting genetic drift. Whole exome sequencing (WES) data of 15 OC cases from 13 families tested negative for pathogenic variants in known OC risk genes were investigated for candidate variants in 468 DNA repair pathway genes. Filtering and prioritization criteria were applied to WES data to select top candidates for further analyses. Candidates were genotyped in ancestry defined study groups of 214 familial and 998 sporadic OC or breast cancer (BC) cases and 1025 population-matched controls and screened for additional carriers in 605 population-matched OC cases. The candidate genes were also analyzed in WES data from 937 familial or sporadic OC cases of diverse ancestries. Top candidate variants in  BRCA1  pathway  pathway, were rarely implicated in familial and sporadic OC cases in this population  syndrome families having one OC and at least two BC cases in the same familial branch and 435 sporadic OC cases not selected for age at diagnosis with the disease or for family history of any cancers. Genetic data was available from 1025 population-matched controls provided by three independent biobanks as previously described  syndrome families having at least three close relatives with BC within first-, second- or third-degree of relationship from the same familial branch and 563 sporadic BC cases not selected for age at diagnosis with the disease or for family history of any cancers.Targeted analyses of the candidate variants was performed to determine their carrier frequencies , followed by paired-end sequencing on different Illumina HiSeq platforms was performed. Reads were aligned to the human reference genome assembly GRCh37/hg19 using Burrows-Wheeler aligner v0.7.17, followed by PCR deduplication using Picard v2.9.0. Realignment around small insertions and deletions was performed, and germline variants were called using HaplotypeCaller using Genome Analysis Toolkit (GATK) v3.5. Variants were then filtered for base sequencing quality score ≥30 and annotated using Ensembl Variant Effect Predictor (VEP) and GEMINI v0.19.1.For phase I of the study . Variants with minor allele frequency (MAF) >0.005 in gnomAD v2.1.1.  performed at the McGill Genome Center as described previously ; 24, 33.® (® Gold (® SNP Type™(Selected top candidate variants were investigated for carrier frequencies in defined FC study groups (study phase II) comprised of 42 index OC and 33 index BC cases from 75 HBOC families, 139 index BC cases from 139 HBC families, 435 sporadic OC cases and 563 sporadic BC cases Table S1® , Sequeno  in 52 sporadic early-onset FC HGSC cases and in an additional 553 FC OC cases by surveying available genetic data or targeted genotyping of PBL DNA Table S1To investigate evidence for inactivation of candidate genes in cancer cells, we performed loss of heterozygosity (LOH) analysis of tumour DNA from variant carriers. Bi-directional Sanger sequencing of available tumour DNA was performed using customized primers (available upon request) as described above. Extracted DNA from fresh-frozen (FF) or histopathological sections from formalin-fixed paraffin-embedded (FFPE) tumour tissues were provided by the RRCancer biobank for DNA extraction and LOH analysis . Sequencing chromatograms were inspected for loss of the wild-type allele using 4Peaks v1.8. (nucleobytes.com/4peaks/) .https://gnomad.broadinstitute.org). All variants were annotated and subjected to our filtering and prioritizing criteria to identify candidate variants as described above , BRCA2 (NM_000059.4), MLH1 (NM_000249.4), MSH2 (NM_000251.3), MSH6 (NM_000179.3), PMS2 (NM_000535.7), BRIP1 (NM_032043.3), RAD51C (NM_058216.3), RAD51D (NM_001142571.2), PALB2 (NM_024675.4) and ATM (NM_000051.4) based on the National Comprehensive Cancer Network (NCCN) Clinical Practice in Oncology Guidelines 2022 (Version 2.2022) —Genetic/Familial High-Risk Assessment: Breast, Ovarian and Pancreatic (nccn.org/guidelines/category_2). Variants were extracted from the annotated VCF files from carriers and subjected to our filtering and prioritizing criteria as previously described  also harbour rare PVs or LPVs in known OC risk genes (n=11): escribed .We first extracted variants identified in a curated list of 468 DNA repair genes Table S2ALKBH3 c.677A>G; p.Asn226Ser, which was identified in an index case (PT0136) from family F1506, as it has a MAF ≥0.01 in FC controls. We also excluded the missense variant in DNA2 c.836C>T; p.Thr279Ile for further analyses as it was identified in the index case (PT0128) and four unaffected members of the family F694, and was not inherited from the affected mother with the family history of OC and other cancers  from the same family F1528  from family F1288; (2) ATRX c.4377_4379del; p.Glu1464del in one of the index OC case (PT0057) from family F1528 as they were classified as benign in ClinVar and by ACMG guidelines, and the latter as not being harboured by the other index OC case of the same family F1528 (SMARCA2 c.3265C>T in the index OC case (PT0128) from family F694; p.Arg1089Trp; and (4) KMT2C c.6916C>T; p.Pro2306Ser in the index OC case (PT0047) from family F1490 as variants in these genes are associated with non-cancer related syndromes (SMARCA2 are linked with Nicolaides-Baraitser syndrome (MIM: 601358), which is characterized by intellectual disability, seizures, limited to absence of speech ability, short stature, dysmorphic facial features and sparse hair , which is characterized by delayed psychomotor development, variable intellectual disability and mild dysmorphic features  from family F1606 that also harbouring FANCC c.897G>T; p.Arg299Ser as this variant is a plausible and intriguing candidate where FANCC has been reported as a candidate BC predisposing gene  in ClinVar in the context of FA, an autosomal recessive disorder (MIM: 227645) and as LPV by ACMG guidelines. As noted above, FANCC has been associated with BC predisposition in an autosomal dominant mode of inheritance  with BC and melanoma also carried the EXO1 variant, her sibling (PT0180) and maternal female cousin (PT0181) both with BC were not carriers of the EXO1 variant. These observations are interesting given the number of different types of cancer cases on the maternal side of the family. In family F1506, the index carrier harbouring ERCC5 c.2556 A>G also had a remarkable family history of diverse cancer types, whereas the index carrier of NEIL1 c.248G>T; c.1268-1G>T from family F1601 had a cancer family history consistent with HBOC syndrome. The index carrier of FANCC c.897G>T; p.Arg299Ser from family F1606 reported a mother with OC and a father with lung cancer.We genotyped PBL DNA samples from family members of the index carriers where possible to determine if the candidate allele segregated with disease Figure 2NTHL1 c.244C>T; p.Gln82Ter, EXO1 c.1268-1G>T, FANCC c.897G>T; p.Arg299Ser, ERCC5 c.2556A>G; p.Ile852Met and NEIL1 c.248G>T; p.Gly83Asp in different FC OC and BC study groups and population-matched controls, regardless of their carrier status for BRCA1 and BRCA2 PVs (EXO1 variant carriers and 0.5% (2/435) for carriers of each NTHL1, ERCC5 or NEIL1 variants, and one NTHL1 carrier among sporadic BC cases . Carriers were not identified among index cases from HBOC and HBC families. FANCC variant carriers were not identified in any of these FC cancer study groups.We genotyped or surveyed available genetic data of our candidate variants: RCA2 PVs Table S1NTHL1 c.224C>T; p.Gln82Ter was not surprising given the frequency of carriers of this variant = 0.002 in the non-cancer non-Finnish European population in gnomAD v2.1.1  release panel  and NEIL1  variants (NTHL1 and NEIL1 candidate variants.We investigated our variants in 8493 non-cancer SNP array genotyping-based controls from cancer-free FC population see Table S1se panel . HoweverNTHL1 c.244C>T (n=1), EXO1 c.1268-1G>T (n=1), FANCC c.897G>T (n=1) and NEIL1 c.248G>A (n=3)  Table S6BRCA1, BRCA2, RAD51C and RAD51D , FANCC c.897G>T; p.Arg299Ser (n=1), ERCC5 c.2556A>G; p.Ile852Met (n=2) and NEIL1 c.248G>T; p.Gly83Asp (n=3)  Table S7NTHL1 variant; 17 of 516 AUS OC cases (3.3%) harbouring 11 variants in NTHL1 (n=6), NEIL1 (n=3), ERCC5 (n=1) and EXO1 (n=1); 12 of 412 Pan-Cancer – TCGA OC cases (2.9%) harbouring 10 variants in NEIL1 (n=4), EXO1 (n=2), FANCC (n=2), NTHL1 (n=1) and ERCC5 (n=1)  in EXO1; 87/943 (0.09) in FANCC; 85/811 (0.1) in NEIL1 and 47/629 (0.07) in NTHL1. Collectively, candidate variants we identified in cancer cases are comprised of three nonsense, four frameshift, three alternative splicing and nine missense variants. Variants in and NEIL1 (n=3), EXO1 (n=2), NTHL1 (n=2), ERCC5 (n=1) and FANCC (n=1) were LoF variants and classified as PVs or LPVs in ClinVar and/or by ACMG guidelines. The remaining variants were missense predicted to be PV or LPVs by our set of in silico tools. Some of these variants were those already identified in our FC study groups: three of 516 (0.6%) AUS OC cases and two of 412 (0.5%) Pan-Cancer – TCGA OC cases carried NTHL1 c.244C>T; p.Gln82Ter, while one each of 516 (0.2%) AUS OC cases and 412 (0.2%) Pan-Cancer – TCGA OC cases carried NEIL1 c.248G>A; p.Gly83Asp.To determine the relevance of our candidate genes to OC in non-FC populations, we investigated the spectrum and prevalence of rare variants in our candidate genes in genetic data from three defined non-FC study groups Table S1NEIL1 c.159C>T; p.Gly53Gly that was predicted to affect splicing using SpliceAI  and BRCA1 c.5102_5103del; p.Leu1701GlnfsTer14. This BRCA1 variant is one of the most frequently occurring PVs in OC and BC cases from the FC population  (nccn.org/guidelines/category_2) S9. Onlypulation , 41, 116NEIL1 c.248G>A; p.Gly83Asp (PT0314) also harboured a variant in a known OC risk gene RAD51C c.145+1_145+2insC, which was classified as PV by ACMG guidelines  also harboured BRCA2 c.5065_5066insA; p.Ala1689AspfsTer6 and a carrier of EXO1 c.2152C>T; p.Gln718Ter (PT0263) also harboured BRCA2 c.1029del; p.Lys343AsnfsTer6. Both BRCA2 variants have been classified as PVs in the ClinVar database and by ACMG guidelines.Regarding non-FC OC carriers, one of the 17 OC AUS carriers of idelines Table S9ERCC5, EXO1, FANCC, NEIL1 or NTHL1. Genotyping analyses of independently ascertained FC cancer study groups identified multiple carriers with OC harbouring the same variant, which is likely due to common ancestors within the FC population of Quebec   and Xeroderma pigmentosum complementation group G (MIM: 278780), respectively, known to exhibit increased risk to cancer.Our candidate variants were identified by investigating a curated list of 468 known or putative genes involved in different DNA repair pathways.  the BER  and HR   and most 616415) . HomozygNTHL1 c.244C>T; p.Gln82Ter, EXO1 c.1268-1G>T and FANCC c.897G>T; p.Arg299Ser. NTHL1 c.244C>T; p.Gln82Ter has been independently reported in the literature due to its frequency, while FANCC c.897G>T; p.Arg299Ser has been reported only in ClinVar database. The introduction of a termination codon in NTHL1 p.Gln82Ter is predicted to affect NTHL1 protein production, eliciting its classification as PV in ClinVar and by ACMG guidelines. EXO1 c.1268-1G>T and FANCC c.897G>T; p.Arg299Ser were predicted to affect splicing by all of our selected in silico tools. As RNA was not available from carriers of these variants, we were unable to investigate their effect on the gene transcripts. We applied a stringent criteria for prioritizing missense variants using a selected set of high performance in silico prediction tools  where the threshold of predicting missense variants being tolerated is <0.4   , and this association was stronger with the endometrioid subtype    is comparable to that of AUS variant carriers as well as Pan-Cancer – TCGA variant carriers , which in turn is comparable with that of carriers of 8 years)  and the 8 years) , 113. Th8 years)  and thus8 years) . We did ous) see Table S8ERCC5 c.2556A>G, NEIL1 c.248G>T; p.Gly83Asp or NTHL1 c.244C>T; p.Gln82Ter. We also showed loss of the wild-type allele in the tumour DNA from the left and the right ovaries from two NTHL1 c.244C>T carriers with bilateral OC. This suggests the possibility that loss of wild-type allele occurred at an early stage in tumourigenesis  polymerase (PARP) inhibitors . The small sample size in our cases and controls did not allow us to estimate the associated risk with OC of any of the candidate variants to support their candidacy as OC predisposing variants/genes  as such  with OC –147. Thi000s see Table S1ERCC5, EXO1, FANCC, NEIL1 or NTHL1. The genetic analyses of these variants and genes in non-FC OC study groups implicate these genes in other populations. Genetic epidemiology of variant carriers and functional assays to assess the biological impact of variant proteins could elucidate the effect of candidate variants to OC risk.In conclusion, our WES and genetic analyses of 468 genes directly or associated with DNA repair pathways in study groups from a genetically defined population identified candidate variants in patricia.tonin@mcgill.ca. The data from the analyses of investigation of The Ovarian Cancer Association Consortium (OCAC) and The Genome Aggregation Database (gnomAD) are available from each of these data resource banks.The datasets presented in this article are not readily available because sequencing data for familial and sporadic ovarian cancer (OC) cases, CARTaGENE, McGill University-Montreal Neurological Institute (MNI) and Université de Sherbrooke-The Genetics of Glucose Regulation in Gestation and Growth (Gen3G) will be returned to their respective biobanks at the conclusion of our study of OC predisposing genes which is still ongoing. For more information concerning these data contact Patricia N. Tonin at This project has received approval from The McGill University Health Centre (MUHC) REB (MP-37-2019-4783). The patients/participants provided their written informed consent to participate in this study.WA and PT conceived and designed the study. WA performed whole exome sequencing and bioinformatics analyses, conducted, and interpreted the genotyping and Sanger sequencing and wrote the initial drafts of this manuscript. TR performed the bioinformatics pipelines. CF aided in bioinformatics analysis. CS aided in collecting and reviewing all clinical pedigrees with associated information. KO performed imputation and provided required information from CARTaGENE genotyping-based data. DS and IC provided required information from in-house sequencing-based Australian ovarian cancer cases. JB and TP provided required information from The Cancer Genomic Atlas (TCGA) sequencing-based breast and ovarian cancer cases. DS, GR and LB provided required information from in-house sequencing-based control data. A-MM-M, DP, WF and ZH provided the study samples and clinical information. CG oversaw the statistical analyses. JR oversaw the WES analysis and bioinformatics pipeline. PT designed and oversaw all aspects of the study. All authors contributed to the article and approved the submitted version.


Though type 2 diabetes (T2D) has been known as a metabolic disease caused by multiple factors, the etiology remains insufficiently understood. Here, we aimed to figure out whether circulating immune cell profiles causally impact T2D liability.We applied one genome-wide association study (GWAS) summary statistics of blood traits in 563,085 participants from the Blood Cell Consortium and another GWAS of flow cytometric profile of lymphocyte subsets comprising 3,757 Sardinians to identify genetically predicted blood immune cells. We also obtained GWAS summary statistics in 898,130 individuals from the DIAGRAM Consortium to evaluate genetically predicted T2D. We primarily used inverse variance weighted (IVW) and weighted median methods to perform Mendelian randomization analyses and sensitivity analyses to evaluate heterogeneity and pleiotropy.+ T cell and CD4+ CD8dim T cell count were identified with causal effect on T2D susceptibility . No pleiotropy was determined.For circulating blood leukocyte and its subpopulations, the increase of genetically predicted circulating monocyte count was causally correlated with a higher risk of T2D . For lymphocyte subsets, CD8These findings demonstrated that higher circulating monocyte and T-lymphocyte subpopulation predicted increased T2D susceptibility, which confirmed the immunity predisposition for T2D. Our results may have the potential to provide new therapeutic targets for the diagnosis and treatment of T2D. T2D and its complications continue to pose a growing burden worldwide . While Te groups . HoweverMendelian randomization (MR) was initially developed to be an alternative option for randomized controlled trials (RCTs), providing reliable causal evidence between exposures and outcomes through genetic variants . Since g22.1We systematically determined the causal effects of peripheral blood immune cells or lymphocyte subtypes on the risk of T2D using a two-sample MR study. There are three fundamental assumptions that should be followed in the MR study . First, 2.2+ natural killer (NK) cells, NKT cells, CD4 regulatory T cells (Tregs), CD4+ CD8dim T cells, CD8+ T cells, and B cells were derived from the recent GWAS summary statistics including 3,757 Sardinians , which includes 563,085 European ancestry participants . Geneticrdinians . Outcomerdinians . The ave2.3−8) for the blood cell traits from BCX, we used a more stringent association threshold (p < 5 × 10−9) to select genetic instrumental variables (IVs)  based on the linkage disequilibrium (LD) reference panel of the 1000 Genomes Project. Referring to the relatively modest-scale GWAS regarding lymphocyte subpopulations, we applied a p-value cutoff of 5 × 10−8 and a more relaxed clumping threshold (R2 < 0.1 within a 500-kb distance) (−5) as previously reported (Considering the large number of single-nucleotide polymorphisms (SNPs) achieving genome-wide significance (p < 5 × 10es (IVs) . To obtaistance) . Additioreported . We harmreported .2.4http://www.phenoscanner.medschl.cam.ac.uk/) to find out SNPs showing suggestive association (p < 10−5) with those risk factors. Then, we repeated the IVW analysis after removing these SNPs. Since there are multiple tests in this study, we adopted a Bonferroni-corrected significant threshold of 0.05/6. Analyses were implemented by the package TwoSampleMR (version 0.5.6) and RadialMR (version 1.0) in R (version 3.4).As our primary analysis, we used the random-effects inverse variance weighted (IVW) and weighted median (WM) methods, providing an estimate of the effect of the exposure on the outcome when MR assumptions are valid. For a single genetic variant identified, we performed a Wald ratio estimate in our MR studies. As a secondary sensitivity analysis, we applied Cochran’s Q test (p < 0.05) to estimate residual heterogeneity for the IVW model and the MR-Egger intercept test (p < 0.05) to indicate potential pleiotropy on causal estimates. We also performed a radial MR test to identify pleiotropic outliers . We pres33.1After the selection and harmonization of IVs, we utilized 361 SNPs for monocyte cell count, 327 SNPs for white blood cell (WBC) count, 334 SNPs for lymphocyte cell count, 247 SNPs for neutrophil cell count, 296 SNPs for eosinophil cell count, and 104 SNPs for basophil cell count for MR analysis. All SNPs had F statistics above 10, demonstrating their suitability as strong instruments. The harmonized data are presented in The causal effect estimates of peripheral immune cell count on T2D susceptibility are summarized in The results of sensitivity analyses are summarized in A recent summary-level MR study verified 34 causal risk factors for T2D, including 25-hydroxyvitamin D, age at menarche systolic BP, resting heart rate, body fat percentage, birthweight, HDL, and total cholesterol . To excl3.2+ NK cells, NKT cells, CD4 Tregs, CD4+ CD8dim T cells, CD8+ T cells, and B cells. Our results are summarized in + T cell count  and CD4+ CD8dim T cell count  predicted a higher risk of T2D, while genetic liability to lower levels of HLA DR+ NK cell count  may predict a higher risk of T2D. However, no significant association was found between T2D risk and the absolute count of other cell subtypes, such as NKT cells, CD4 Tregs, or B cells. No pleiotropy and heterogeneity were identified in the MR-Egger intercept test and Cochran’s Q test (To test whether lymphocyte subtypes are causally associated with T2D risk, we further carried out MR analysis between T2D and absolute count of lymphocyte subpopulations, comprising HLA DR4The MR analysis has been commonly applied to elucidate the potential causal relationship between risk factors and diseases. In this study, we provide evidence to present the inverse causal association between monocyte count and T2D from a genetic perspective using MR analysis. This is in line with previous results from the INTERCEPT-T2D EU-supported project . However+ macrophage populations can control adipose tissue metabolism plasticity by regulating vascular structure (+CD9+ macrophage populations are related to lipid uptake and are significantly increased in obese populations with increased fat in adipose tissue (It has been known that the pathological characteristics of T2D are due to impaired insulin secretion and sensitivity caused by oxidative stress, endoplasmic reticulum stress, and ectopic lipid deposition in the muscle, liver, and pancreas . All thetructure ; Trem2+Ce tissue . In T2D-e tissue . In summ+ T cell count (T2D is a chronic disease caused by multiple factors. Despite robust studies on the pathogenesis of T2D, the etiology remains unknown. Notably, recent studies have highlighted the significant impact of immunity on T2D incidence, progression, and risk , particull count . However+ NK cells is significantly increased in the peripheral blood of obese patients (+CD8dim T cell, CD8+ T cell, NKT, CD4 Treg, NK, and B cell count, and T2D based on recently published GWAS data using MR analysis. The results showed that the absolute count of CD4+CD8dim and CD8+ T cells may contribute to increased T2D risk, which is consistent with previous reports (+ and CD8+ T cells could facilitate insulin resistance development, probably through modulating vascular regeneration (Recent studies have shown that T cells isolated from the peripheral blood and adipose tissue of T2D patients tend to have a pro-inflammatory phenotype, which can promote the development of insulin resistance . Comparepatients . While Cpatients , 33, the reports , 34. Funneration . However+CD8dim T cell and CD8+ T cell count may predict the risk of T2D. The findings suggest that certain immune cell types and genetic susceptibilities could be used as biomarkers for T2D risk, which could lead to earlier diagnosis and more effective treatment options.Overall, our findings suggest that an increased level of circulating monocyte cell count may contribute to T2D development. While circulating lymphocyte cell count was not causally associated with T2D susceptibility, lymphocyte subtypes including CD4The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.JL: study design, data analysis, and manuscript drafting. QN and AW: data analysis and manuscript revision. YZ and LH: manuscript revision. HW: study design and manuscript revision. All authors contributed to the article and approved the submitted version.


M. tb immunogen and inserted into the FG loop of the HPV16 L1 protein to construct chimeric HPV16L1/Ag85B VLPs. The chimeric HPV16L1/Ag85B VLPs were produced via the Pichia pastoris expression system and purified via discontinuous Optiprep density gradient centrifugation. The humoral and T cell-mediated immune response induced by the chimeric HPV16L1/Ag85B VLP was studied in female C57BL/c mice. We demonstrated that the insertion of the extracellular domain of Ag85B into the FG loop of HPV16L1 did not affect the in vitro stability and self-assembly of the chimeric HPV16L1/Ag85B VLPs. Importantly, it did not interfere with the immunogenicity of Ag85B. We observed that the chimeric HPV16L1/Ag85B VLPs induced higher Ag85B-specific antibody responses and elicited significant Ag85B-specific T cell immune responses in female C57BL/c mice compared with recombinant Ag85B. Our findings provide new insights into the development of novel chimeric HPV16L1/TB VLP-based vaccine platforms for controlling TB infection, which are urgently required in low-income and developing countries.Bacillus Calmette–Guerin (BCG), the only current vaccine against tuberculosis (TB) that is licensed in clinics, successfully protects infants and young children against several TB types, such as TB meningitis and miliary TB, but it is ineffective in protecting adolescents and adults against pulmonary TB. Thus, it is a matter of the utmost urgency to develop an improved and efficient TB vaccine. In this milieu, virus-like particles (VLPs) exhibit excellent characteristics in the field of vaccine development due to their numerous characteristics, including but not limited to their good safety without the risk of infection, their ability to mimic the size and structure of original viruses, and their ability to display foreign antigens on their surface to enhance the immune response. In this study, the HPV16 L1 capsid protein (HPV16L1) acted as a structural vaccine scaffold, and the extracellular domain of Ag85B was selected as the  Mycobacterium tuberculosis (M. tb), remains a threat to the lives of several million people around the world. According to the World Health Organization (WHO), there were about 10.6 million cases infected with TB and 1.6 million deaths from this disease in 2021, including 0.19 million concurrent infections with HIV [Tuberculosis (TB), an ancient infectious disease caused by with HIV . It has with HIV . TherefoStrategies to improve TB vaccines rely heavily on the identification of novel TB antigens involved in protective immunity. Among a dozen of M. tb immunogens identified, members of the antigen 85 (Ag85) complex  play essential and vital roles in the virulence of M. tb via binding to fibronectin and elastin proteins, mycolyltransferase activity, as well as through phagosome maturation prevention in macrophages, and are being utilized as tools in the construction of new vaccines . Of thesAnother strategy to develop TB vaccines is the improvement of vaccine construction methods. Virus-like particles (VLPs) are hollow particles that contain one or more self-assembled structural proteins of a virus but do not contain viral nucleic acids, commonly known as pseudoviral particles . As a neM. tb antigen, Ag85B, and construct the chimeric molecule of HPV-16 L1/Ag85B. Western blotting (WB) confirmed the successful expression of the chimeric HPV16L1/Ag85B in Pichia pastoris. The electron transmission microscopy (TEM) examination results showed that chimeric HPV-16 L1/Ag85B could form particles with a diameter of approximately 50 nm, and the immunogenicity of HPV-16 L1/Ag85B was also evaluated.In this study, HPV-16 L1 VLPs were used as carriers to insert the M. tb antigen, Ag85B, was amplified via PCR using the M. tb H37Rv strain’s genomic DNA as a template. The purified PCR product was then cloned into the pET-28a expression vector, and the recombinant antigen was expressed with a C-terminal His-tag. Recombinant antigen expression in Escherichia coli (E. coli) and Rosetta  was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) and analyzed via WB. The inclusion bodies were denatured via the addition of 0.05 mM TCEP and 4.4% N-lauroylsarcosine and refolded in a universal refolding buffer C7  from the iFOLD Protein Refolding System 1, as described previously [The gene of the eviously . A nickeThe FG loop of HPV16L1 was deleted, and HPV16L1 was modified as follows: The sequence on the left side of the FG loop was first amplified using the paired primers, F1 and R1 , which whttp://www.cbs.dtu.dk/services/TMHMM/, accessed on 1 October 2023) [Pichia pastoris. An initiating ATG codon in a Kozak consensus sequence was required and inserted for proper translation initiation of the gene. The same restriction site, NcoI, was added to both ends of the optimized sequence via PCR. The above fragment was then ligated to the recombinant pBlu vector carrying the modified HPV16L1 with the same restriction, which needed to be dephosphorylated before. The HPV16L1-Ag85B fusion sequence was digested from two restriction sites, BamHI and EcoRI, of the recombinant pBlu vector and cloned into the Pichia expression vector, pPIC3.5K, generating a recombinant pPIC3.5K plasmid construct. The recombinant pPIC3.5K construct was transformed via electroporation into competent E. coli TOP10 cells, and ampicillin-resistant colonies were selected. Plasmid DNA was isolated using a miniprep for sequencing to confirm that ATG was in the proper context for eukaryotic translation initiation. The 5′ and 3′ AOX1 sequencing primers (F3 and R3) were used to sequence the constructs.The transmembrane domain of Ag85B was predicted using TMHMM software (er 2023) , and theHIS4 prior to transformation into Pichia. The linearized construct in pPIC3.5K was transformed into GS115 via electroporation, generating both Mut+ and MutS recombinants. The parent vector linearized in the same manner as the construct was used as a control to confirm the integration via PCR and was used as a control for background for the expression analysis and the quantitative dot blots or Southern blot analysis. Another control was pPIC3.5K containing one copy of the expression cassette. His+ transformants were generated using recombinant pPIC3.5K and screened in vivo to obtain 10 Geneticin®-resistant colonies to test. The colonies with multiple copies were selected to proceed directly to overexpressing the proteins. Cells were harvested after 96 h of incubation at 37° and ultracentrifuged at 136,000× g at 4 °C for 10 min. Pellets resuspended in sorbitol solution with a pH of 5.8–7.2 were lysed with snailase (10 mg/mL) at 37 °C for 1 h, and the supernatants containing HPV16L1/Ag85B VLPs were collected at 136,000× g at 4 °C for 10 min. VLPs were further purified via discontinuous Optiprep density gradient centrifugation (27–33–39%) at 234,000× g at 4 °C for 4 h and detected using SDS-PAGE. A sufficient amount of recombinant pPIC3.5K plasmid DNA was prepared and linearized using the restriction enzyme SalI for insertion at HVP16L1 and HVP16L1/Ag85B VLPs equilibrated with 20 mM Tris-HCl  were layered onto copper TEM grids for 1 min, and negatively stained using 2% uranyl acetate solution for 1 min. The grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a TEM at a magnification of 150,000.6 CFU of BCG intradermally (i.d) and boosted with 20 µg of recombinant Ag85B and HPV16L1/Ag85B VLPs intramuscularly (i.m). In the control groups, the mice were primed with BCG followed by immunization with HPV16L1 VLPs and PBS. Blood samples were collected before priming with BCG and after one or two weeks per immunization and stored at −80 °C for the antibody assay. The mice were sacrificed, and spleen samples were collected two weeks after the final immunization for cytokine analysis. The spleens were crushed and pressed individually through a 70 µm cell strainer  with a 5 mL syringe rubber plunger. The red blood cells were lysed using ammonium chloride–potassium (ACK) lysis buffer  and then resuspended in warm RPMI-1640 .Six- to eight-week-old C57BL/c mice were used in this study, and all experiments were approved by the Institutional Animal Ethics Committee of Navy Medical University. The mice were immunized by priming them with BCG, followed by boosting with vaccine candidates twice at two-week intervals. Purified recombinant Ag85B, HPV16L1, and HPV16L1/Ag85B VLPs were emulsified with a moderate volume of Freund’s incomplete adjuvant and PBS. In the experimental groups, the mice were primed with 102CO3-NaHCO3, pH 9.6), and 100 µL was added to each well of 96-well Immunosorp plates  for incubation overnight at 4 °C. The plates were washed three times with 375 μL PBST  and then blocked in 0.2 mL 3% bovine serum albumin in PBS for 2 h at room temperature. After three washes, the collected serum samples (diluted 1:200 in blocking buffer) were added to each well and incubated at 37 °C for 1 h. After five washes five times, 100 µL of 1:5000-diluted HRP-conjugated anti-human IgG, IgG1, or IgG2 secondary antibody  was added for incubation at 37 °C for a further 1 h. After five washes, the color reaction was developed with 100 µL TMB  substrate solution  and stopped via the addition of 50 µL 2 N H2SO4 . The optical density (OD) was measured and recorded at a wavelength of 450 nm using a microplate reader .An enzyme-linked immunosorbent assay (ELISA) was used for the detection of anti-Ag85B antibodies in the sera of the immunized animals. Briefly, the purified protein derivative (PPD) or the Ag85B protein was adjusted to 2 μg/mL with coating buffer  in RPMI 1640 supplemented with 1% penicillin-streptomycin, 1 mM glutamine, and 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. Triplicate wells were stimulated with GST , LEAF-CD3ε , PPD , and Ag85B antigen . Supernatants were collected after 48 h and IFN-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and IL-10 were quantified using a cytokine ELISA .Splenocytes from the immunized mice were cultured at a density of 1 × 10Statistical analysis was performed using Prism GraphPad software version 8.01 . Statistical analysis was carried out through parametric testing of mean values via one-way analysis of variance (with Tukey’s post hoc testing to identify between-group significance).E. coli is shown in M. tb antigen, Ag85B, amplified via PCR from the H37Rv strain’s genomic DNA template, was cloned into a pET-28a expression plasmid  was then chosen as the structural template to build the HPV16 L1 and modified HPV16 and HPV16L1/Ag85B homology models. Compared with the conformation of HPV16L1, the FG loop was successfully removed with the modified HPV16 and correspondingly replaced by the extracellular domain of Ag85B in the chimeric HPV16L1/Ag85B  at 234,000× antibody C,D. TEM antibody , and it p = 0.0004) and twelve weeks (p = 0.0002). Moreover, it was found that both recombinant Ag85B and HPV16L1/Ag85B VLPs mainly enhanced the anti-Ag85B IgG2a titer, but not the IgG1 titer, which led to a significant increase in the anti-Ag85B IgG2a/IgG1 ratios . Regarding the Ag85B-specific T cell responses, the highest concentration of IFN-γ secretion/106 splenocytes was observed in the HPV16L1/Ag85B VLP immunized mice compared to the mice that received PBS, HPV16L1, or recombinant Ag85B (p = 0.156). IL-2, TNF-α, and IL-10 were also quantified among all groups; however, there were no significant differences between them whether the splenocytes were stimulated with PPD or Ag85B -2, tumor necrosis factor (TNF)-α, and IL-10 quantification. As shown in nt Ag85B A. A signor Ag85B B–D.Pichia pastoris expression system and the discontinuous Optiprep density gradient centrifugation method could be feasible approaches to produce and purify chimeric HPV16L1/Ag85B VLPs; (2) HPV 16 L1 could act as a structural vaccine scaffold, and the insertion of the extracellular domain of Ag85B into the FG loop of HPV16 L1 did not affect the in vitro stability and self-assembly of chimeric HPV16L1/Ag85B VLPs; (3) the sequential Ag85B peptide exposed to the FG loop of chimeric HPV16L1/Ag85B VLPs could be detected by an anti-Ag85B antibody in vitro; and (4) chimeric HPV16L1/Ag85B VLPs could induce Ag85B-specific antibody responses and elicit Ag85B-specific T cell responses in mice. These findings contribute to the development of novel chimeric HPV16L1/Ag85B VLP-based vaccine platforms for controlling TB infection, which are urgently needed in developing and industrialized countries.TB is currently the focus of many vaccine studies as a result of the inability of BCG to end TB. Although a dozen TB vaccine candidates have been developed in clinical stages, novel vaccine construction approaches still deserve to be explored. In this study, we demonstrated that (1) the Pichia pastoris, Saccharomyces cerevisiae, and Saccharomyces polymorphus are the most widely used yeast cells, which can be used to carry out the post-translational modification (PTM) of the expressed proteins to ensure they have the correct conformations and biological activities. In this study, Pichia pastoris was employed to successfully express HPV16L1/Ag85B VLPs, and the products were then purified via the discontinuous Optiprep density gradient centrifugation method. Although chromatographic purification is a common method used to purify VLP proteins, we used gradient centrifugation to achieve a similar purification effect in this study.Several expression platforms, including eukaryotic, prokaryotic, and cell-free systems derived from eukaryotic or prokaryotic expression systems, can be employed for producing VLPs. About 70% of reported VLPs are prepared with eukaryotic systems, which include yeast systems, mammalian cell systems, baculovirus/insect cell (B/IC) systems, and plant systems . Pichia HPV16L1 contains 505 amino acids in its full length and is capable of self-assembly into VLPs; it is also characterized by three virus-facing hinge regions between beta folds: DE, FG, and HI loops. Numerous studies have shown that the insertion of moderate-length foreign sequences into these loops did not influence the formation of VLPs ,32,33. MIn the present study, the augmentation of antigen-specific immune responses via HPV16L1/Ag85B VLPs led to a higher induction of antigen-specific IgG and IgG2a antibodies, and accumulated IFN-γ secretion was also observed in the HPV16L1/Ag85B VLP vaccination group compared with the recombinant Ag85B group, indicating a biased Th1 type induced by the HPV16L1/Ag85B VLPs. However, it is worth noting that the parameters for evaluating an eligible vaccine are too simple, and too much attention is paid to the Th1 immune response effect induced by vaccines. A review study showed that six TB vaccine candidates, namely MVA85A, AERAS-402, H1:IC31, H56:IC31, M72/AS01E, and ID93:GLA-SE, evoked highly similar functional properties of memory T cell responses, indicating a lack of diversity among the available TB vaccine candidates . AlthougM. tb is highly heterogeneous, tens of antigens are required to cover 80% of CD4 T cell responses. However, current vaccine approaches utilizing a few antigens may not induce a sufficient immune response. Conversely, antigens that are poorly recognized during natural infection  may not be fully recognized by the immune system at all during infection, and their role in protection is unclear and worthy of further investigation. The subunit vaccine, M72: AS01E, is composed of unnatural antigens, Rv0125 and Rv1196, combined with the adjuvant AS01. Phase 1 and 2 clinical trials in M. tb-negative populations have shown that M72:AS01E has a relatively high T cell immune induction effect [Most vaccines employ a limited pool of immunologically dominant target antigens, mainly from the secretion of Ag85 and ESAT-6 protein families. In mice, it has been observed that the limitation of antigen availability weakens the Ag85B-induced protective immunity during chronic infection, while the immunity of ESAT-6-specific T cells is restricted as a result of functional exhaustion, highlighting potential challenges in employing these antigens for vaccine development . As the All in all, VLP-based vaccines have gradually become a hotspot in the field of vaccine development due to their numerous characteristics, including but not limited to their good safety without a risk of infection and their abilities to mimic the size and structure of original viruses and to display foreign antigens on their surface to enhance the immune response. To date, some VLP-based vaccines, including those for IV, HBV, HEV, HPV, and malaria, have been successfully licensed in the market , and VLP