Background: Assessment of human papillomavirus (HPV) type-specific viral load (VL) is a valid tool for determining the etiology of HPV-related skin tumors, especially when more than one HPV type is detected within one lesion.Methods: The causative HPV type was determined in 185 fresh-frozen tissue specimens of histologically confirmed common warts (CWs) collected from 121 immunocompetent patients. All tissues were tested using the type-specific quantitative real-time polymerase chain reactions (PCR) for the most common wart-associated Alpha-PV (HPV2/27/57) and Mu-PV types (HPV1/63/204). The presence of 23 additional low-risk HPVs was evaluated using a conventional wide-spectrum PCR.Results: HPV DNA was detected in 176/185 (95.1%) CWs and multiple HPV types in 71/185 (38.4%) lesions. Using the VL approach and a robust cutoff of one viral copy/cell established in this study, HPV2/27/57 were determined as causative agents in 41/53 (77.3%) and 53/71 (74.7%) CWs with single and multiple HPVs, respectively.Conclusions: CWs are mostly etiologically associated with HPV2/27/57 and only rarely with HPV1. In the majority of CWs containing multiple HPVs, a single HPV type was present in high concentration, indicating etiological association. No significant differences in VLs of lesion-causing HPV types in CWs containing single or multiple HPVs were found. Cutaneous warts are ubiquitous benign skin tumors   types belonging to genera To the best of our knowledge, only seven small-scale HPV prevalence studies performed on a total of 129 histologically confirmed CW tissue samples have been published in peer-reviewed literature to date  Gross e, with HP1, 1.1 × 104, and 4.4 × 104 viral copies/cell, respectively, suggesting an etiological role of these HPVs in a subset of CWs.Köhler et al.  suggesteThe aim of this study was to accurately determine the prevalence of several HPV types in by far the largest collection of fresh-frozen tissue samples of histologically confirmed CWs and to reliably assess the causative HPV based on estimation of type-specific HPV VLs using the robust cutoff value established in this study, especially in the most challenging warts containing multiple HPV types within one lesion.The study included 185 histologically confirmed fresh-frozen CW tissue samples prospectively collected from 121 immunocompetent patients, older than 5 years, referred to a single dermatology outpatient clinic in Slovenia between February 2014 and April 2016.The majority of patients  were older than 12 years, with a mean age of 29.3 years (standard deviation (SD) 16.4, median 25 years, range 5–78 years); 66/121 (54.5%) patients were males.n = 131), lower extremities including dorsal sides of feet (n = 26), upper extremities (n = 14), and head and neck region (n = 14). Mean duration of warts was 16 months , with a mean wart diameter of 8.4 mm .A single wart was collected from 76/121 (62.8%) patients and multiple warts from 45/121 (37.2%) patients: two, three, and five warts were collected from 28/121 (23.2%), 16/121 (13.2%), and 1/121 (0.8%) patients, respectively. Warts were collected from hands including wrists .Alpha-PVs (LR-HPVs): HPV2, 3, 6, 7, 10, 11, 13, 27, 28, 29, 32, 40, 42, 43, 44, 55, 57, 74, 77, 91, 94, 117, and 125 etiologically associated with various mucosal and cutaneous warts. Furthermore, in all samples, the determination of potential Mu-PV  infection was performed using three type-specific quantitative RT-PCRs, as described previously , following protocol for nucleic acid purification from mammalian tissues, and spectrophotometrically quantified using the NanoDrop 2000c spectrophotometer . The integrity of extracted DNA was verified by real-time polymerase chain reaction (RT-PCR) amplification of a 268-bp fragment of human beta-globin gene, as described previously  and HPV63 . Based on the clear bimodal distribution of estimated HPV VLs in 53 randomly selected CWs containing a single HPV, ranging from 5.0 × 10−4 to 7.1 × 106 copies/cell  CWs, and such an HPV was securely assigned as the causative HPV. In 4/71 (5.6%) warts, two HPVs present in the same lesion had high VLs and were both co-assigned as potentially causative HPVs (HPV1+27 and HPV1+57 in two CWs each). The causative HPV could not be reliably determined in 15/71 (21.1%) CWs containing multiple HPVs due to the fact that: (i) the VLs of all HPVs detected within the lesion were far <1 viral copy/cell (six samples), or (ii) in addition to targeted HPVs with minute VLs, the CW also contained additional HPVs , for which we were unable to measure VLs due to the lack of appropriate methods , and others were collected from legs , arms , and head . The most frequent causative HPV in all four anatomical regions was HPV57 and caused all four CWs collected from the head. Interestingly, all four CWs samples with co-assigned causative HPVs (HPV1+27 and HPV1+57 in two CWs each) were collected from hands.Among 45 patients with multiple CWs sampled, complete concordance of HPVs detected was found in only 14 (31.1%). However, at least one common HPV was present across all warts obtained from the same individual in 40/45 (88.9%) patients. In addition, in all 21 patients in whom multiple warts were available and HPV VLs could be estimated in all CWs sampled, 100% agreement across assigned causative HPVs was found.Although a broad range of HPV types and a relatively high frequency of multiple HPVs have previously been reported in CWs' tissue samples . Multiple HPVs were detected in 71/185 (38.4%) CWs, which is substantially higher in comparison to the majority of similar studies , with the most frequent combinations being HPV1+27 and HPV1+57, in line with the findings of some previous studies , which is in line with similar studies, in which HPV positivity ranged from 64.3 to 100%  , and significantly higher median VLs of HPV2/27/57 were observed in comparison to HPV1/63/204 , irrespective of single or multiple HPVs present in CWs. This phenomenon could be a consequence of different phylogenetic backgrounds of the species Alphapapillomavirus 4 and the genus Mu-PV  that could be used to reliably differentiate between causative and bystander HPV types in CWs, especially in challenging cases such as CWs containing multiple HPVs, have not been established yet. Based on the VLs measured in 53 randomly selected CWs containing a single HPV type, and clear bimodal distribution of the VLs obtained, in this study the cutoff value for causal determination was set at one copy/cell. Using this approach, HPV57 has been identified as the most important CW-related HPV causing a significant proportion of CWs . Three HPV types (HPV2/27/57) were determined to be causative HPVs in three-quarters of CWs irrespective of whether CWs contained single or multiple HPVs, whereas HPV1 was assigned as the causative HPV in ~4% of CWs, again irrespective of single/multiple HPV status. Similarly, a previous study performed on nine samples determined HPV2/27/57 and HPV1 to be causative HPVs in 6/9 (66.6%) and 1/9 (11.1%) CWs, respectively  CWs, we assigned two co-causative HPVs. In these CWs, we most probably had colliding wart lesions, each associated with an individual causative HPV, as recently demonstrated in cervical intraepithelial neoplasia lesions using laser capture micro-dissection and HPV genotyping, under the “one virus, one lesion” concept  CWs, and, in one-fifth of CWs containing either single or multiple HPVs, the causative HPV remained undetermined, suggesting that these CWs might be etiologically attributed to HPV types from other species such as Alphapapillomavirus 2 or 8, Gamma-PVs, Nu-PVs, or other novel, still-unidentified HPV types. Although the cumulative prevalence of HPVs from species Alphapapillomavirus 2 and 8  was relatively low  and their VLs were not estimated in this study, these HPV types could potentially be considered as the causative HPV of warts in question because HPV1 and 63, co-detected in these CWs, were present in minute quantities. In previous studies performed on histologically confirmed CWs, the reported detection rate of HPV4/60/65 (genus Gamma-PV) and HPV41 (genus Nu-PV) varied between 0 and 100%  as potential causative HPVs of nine warts, due to the lack of appropriate methods we were unable to measure the VLs of these HPVs, and consequently these CWs remain without a reliably assigned causative HPV.The main limitation of this study is potential selection bias because our cohort might not be fully representative of the general population due to enrollment of patients referred to the tertiary level of dermatological care, possibly due to more treatment-resistant warts or clinically atypical lesions. In addition, considering the benign nature of CWs and potential adverse events of the biopsy, including pain, allergic reactions to local anesthetics, and scarring  as causative HPVs of three-quarters of CWs irrespective of whether CWs contained single or multiple HPVs. This study provides further evidence for informed decisions about selecting the most appropriate targets for future vaccine(s) against cutaneous HPV-related benign tumors.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Republic of Slovenia (consent number 63/10/13). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MP, KF, JM, and VB designed the study. VB and KF organized the database and performed the statistical analysis. LH and KF performed laboratory testing. RK and BL performed histological assessment. VB drafted the first version of the manuscript. LH, KF, and MP drafted individual sections of the manuscript. MP produced the final version of the manuscript. All authors revised manuscript, read it, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Age-related macular degeneration (AMD) is a blinding disease caused by multiple factors and is the primary cause of vision loss in the elderly. The morbidity of AMD increases every year. Currently, there is no effective treatment option for AMD. Intravitreal injection of antivascular endothelial growth factor (anti-VEGF) is currently the most widely used therapy, but it only aims at neovascularization, which is an intermediate pathological phenomenon of wet AMD, not at the etiological treatment. Anti-VEGF therapy can only temporarily delay the degeneration process of wet AMD, and AMD is easy to relapse after drug withdrawal. Therefore, it is urgent to deepen our understanding of the pathophysiological processes underlying AMD and to identify integrated or new strategies for AMD prevention and treatment. Recent studies have found that autophagy dysfunction in retinal pigment epithelial (RPE) cells, cellular senescence, and abnormal immune-inflammatory responses play key roles in the pathogenesis of AMD. For many age-related diseases, the main focus is currently the clearing of senescent cells (SNCs) as an antiaging treatment, thereby delaying diseases. However, in AMD, there is no relevant antiaging application. This review will discuss the pathogenesis of AMD and how interactions among RPE autophagy dysfunction, cellular senescence, and abnormal immune-inflammatory responses are involved in AMD, and it will summarize the three antiaging strategies that have been developed, with the aim of providing important information for the integrated prevention and treatment of AMD and laying the ground work for the application of antiaging strategies in AMD treatment. AMD is the leading cause of visual impairment among the elderly in western countries. Although AMD usually does not lead to complete blindness, it can result in the severe loss of central vision. A study estimated that, by 2020, 196 million people will be afflicted with AMD worldwide, increasing to 288 million people by 2040. As a result, the cost of AMD is predicted to increase to $59 billion over the next 20 years , aging [There are two major proteolytic systems that are responsible for maintaining cellular function: the proteasomal and lysosomal systems. Both systems remove irreversibly damaged proteins and recycle amino acids for protein synthesis , 7. The ΔRPE mice and 45% of the Atg7ΔRPE mice aged 8 to 24 months old. In addition, the degeneration severity increased with age while the POS thickness decreased. Early AMD-like RPE defects were found in all the Atg5ΔRPE and Atg7ΔRPE mice starting at 13 months, including uneven RPE thickness, RPE hypertrophy/hypotrophy, pigmentary irregularities, choroidal neovascularization, and necrosis [trans retinol (ROL) to synthesize the chromophore 11-cis retinal (RAL), which is then shuttled across the interphotoreceptor matrix to POS by the interphotoreceptor retinoid-binding protein (IRBP). Within the POS, 11-cis RAL is bound to G protein-coupled receptors (opsins) to form a light-sensitive visual pigment. Under light stimulation, 11-cis RAL transforms into an all-trans configuration, altering the three-dimensional structure of the opsin protein and activating the phototransduction signaling cascade. All-trans RAL then releases from the opsin protein, transforms into all-trans ROL, and is transported back to the RPE to be recycled back into 11-cis RAL. The Atg5ΔRPE mice showed abnormal POS degradation and decreased visual cycle activity [cis-RAL content was normal in Atg7ΔRPE mice, and only abnormal RPE homeostasis was observed [Studies have shown that, compared with those of the normal population, the RPE cells of AMD patients demonstrate increased numbers of autophagosomes, decreased LC3 II/I concentrations, decreased autophagy flow, and increased vulnerability to oxidative stress, indicating that autophagy dysfunction in RPE cells is involved in AMD . The RB1necrosis . The visactivity  while thobserved . During observed .Lipofuscin is a kind of photosensitizer and spontaneously oxidative substance, which can increase mitochondrial stress and irreversibly inhibit lysosomal protease activity following light irradiation, leading to RPE cell damage. Once formed, lipofuscin cannot be degraded by proteasomal or lysosomal enzymes or be transferred out of cells by extracellular secretion . The accOxidative stress, one of the pathogenic factors of AMD, can mediate reactions to DNA damage, alter autophagy levels, and regulate cellular senescence . OxidatiINK4A-pRB and p53-p21CIP1/WAF1 pathways are primarily involved in the mechanism of cellular senescence  activity; (3) the accumulation of DNA damage foci; (4) the accumulation of senescence-associated heterochromatic foci (SAHF) and other chromatin modifications; (5) chromosomal instability; (6) the induction of SASP; and (7) the altered expression of senescence-related genes  [Cellular senescence can be divided into two types: replicative senescence (RS) and stress-induced premature senescence (SIPS) , 33. Recnfection , 30, 31.kinases) , 34.Cellular senescence is one of the pathogenic factors underlying AMD. The senescence-accelerated OXYS rat is an animal model of AMD that can spontaneously undergo an AMD-like retinopathy, including RPE degeneration, loss of photoreceptors, and the decreased expression of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEGF) , 36. ChoTelomere shortening of RPE cells is also one of the characteristics of AMD. Late passage RPE cells from primary culture demonstrated a reduced capacity for cellular division, which could be caused by telomere shortening . A2E cou2O2), tert-butyl hydroperoxide (TBHP), and light and can result in the premature aging of RPE cells, which is characterized by increases in ROS and SA-β-GAL activities, higher expression levels of p53, p21WAF1/CIP1, p16INK4A, and SASP factors, the accumulation of p-γH2AX foci and 8-OHdG DNA damage lesions, mitochondrial dysfunction, increased VEGF, and decreased CFH [2O2-mediated RPE senescence through the p38 pathway [CIP1/WAF1 levels [α, a transcription regulator, is involved in mitochondrial metabolism and is associated with many age-related diseases. PCG1α protects RPE cells from oxidative stress by upregulating antioxidant enzymes and DDR and is regulated by AMPK and SIRT1 during the process of posttranscriptional modification and activation [Long-term and chronic oxidative stress, which are pathogenic factors of AMD, can be generated by cigarettes, hydrogen peroxide  immCD36) is enriched in both atherosclerotic plaques and on SNC membranes, serving as a critical participant in macrophage recognition. This receptor produces a surface accessible phagocytic “eat me signal” to facilitate the recognition of SNCs and oxidized lipoproteins as part of its immune surveillance function [NK cells are a component of the innate immune system. One of the receptors responsible for NK cell activation, the NKG2D receptor, has been implicated in the interaction between NK cells and SNCs during tumorigenesis, tumor therapy, and tissue injury. The NKG2D receptor recognizes the ligands MICA/B and ULBP1-6 on the surface of SNCs to recruit NK cells for immune surveillance regulation. For example, NK cells mediate the clearance of SNCs during liver fibrosis . Intercefunction . In addifunction . Ipilimufunction . In addifunction .CIP1/WAF1, clearing senescent fat precursor cells. Q clears senescent human endothelial cells and mouse bone marrow mesenchymal stem cells (BM-MSCs). D+Q can downregulate p21CIP1/WAF1, BCL-Xl, and PAI-2 and effectively clear senescent fibroblasts (MEFs) [INK4 and the SASP components MCP-1, IL-6, MMP-2, and TGF-β [INK4A. An aging BubR1H/H mouse model containing INK-ATTAC lines was established, which showed shortened lifetime, lordosis, cataracts, and the aggregation of p16INK4A-positive cells. AP20187, a synthetic drug that induces apoptosis through cell membrane dimerization, was given to the BubR1H/H mice. AP20187 activated INK-ATTAC, which aided the accurate identification of p16INK4A-positive SNCs and effectively cleared them while not affecting normal cells, reducing the senescent phenotypes of adipose tissue, skeletal muscle, and the eye [Dasatinib (D), a tyrosine kinase inhibitor, can inhibit cell replication and migration and induce apoptosis . Quercets (MEFs) . Bleomycnd TGF-β . Transplnd TGF-β . The appnd TGF-β , inhibitnd TGF-β , and uprnd TGF-β , 60. ABTnd TGF-β , 28, 53.nd TGF-β . In anot the eye .κB and reduces SASP component levels by inhibiting the phosphorylation of IκB and IKKα/β [β [SASP inhibitors include rapamycin, metformin, and JAK1/2 inhibitors. Rapamycin reduces the secretome of inflammatory factors in SNCs by inhibiting mTOR1 , 53, plad IKKα/β . JAK is d IKKα/β . Using sd IKKα/β , 65. UBXKKα/β [β . The intKKα/β [β .INK4A-positive cells by senolytic drugs has the following problems: (1) not all senescent cells necessarily have increased p16INK4A expression; (2) not every cell with substantial p16INK4A expression is senescent; (3) targeting aging mechanisms can phenocopy the effects of genetic or pharmacological SNC clearance without actually affecting SNCs; and (4) hypothetically, the genetic clearance of p16INK4A-positive cells could have the same effects on a particular downstream phenotype as a drug that affects that downstream phenotype directly, without affecting truly senescent p16INK4A-positive cells. To determine whether senolytic drugs actually cause the alleviation of senescence-associated phenotypes due to SNC clearance requires following a modified set of Koch's postulates, which are the following: (1) individual SNCs or transplanted SNCs must have a senescent phenotype; (2) the clearance of SNCs genetically or pharmacologically must be associated with the alleviation of the phenotype; and (3) the effects on the phenotype should persist even after the drug has been removed [Among the three aging-therapy strategies, senolysis holds the most therapeutic promise for two reasons. First, the permanent removal of SNCs leads to the durable abolishment of deleterious SASP components. Second, once SNCs are eliminated, there is no risk of tumorigenic “escape” from senescence, which may be possible if SNCs are permitted to linger indefinitely . However removed .SNC clearance can alleviate the further senescence and tissue damage of surrounding cells, thereby delaying disease progression. However, SNC clearance methods are not universal and depend on the types of cells and diseases, which complicates the treatment prospects . At presInflammation is the body's response to cell and tissue damage and occurs through a series of processes that are designed for the eventual clearance of pathogens and the repair of damaged tissue. Acute inflammation is a short-term process that involves leukocyte infiltration, the removal of the trigger, and tissue repair. Chronic inflammation is a prolonged response that can result in tissue injury or destruction if the inciting trigger is not neutralized . Inflammβ and IL-18 through a two-step process. Classically, the first step, referred to as inflammasome priming, involves the NF-κB-mediated synthesis of the inactive precursors pro-IL-1β and pro-IL-18 in response to the recognition of a specific ligand by its corresponding PRR and the upregulation of inflammasome components, including NLRP3. A second signal is required for NLRP3 oligomerization, the recruitment of ASC and procaspase-1, and the subsequent cleavage of procaspase-1 into its active form, leading to the processing of pro-IL-1β and pro-IL-18 and eventually to the release of the mature cytokines IL-1β and IL-18 [β, and IL-18 can be detected in the photoreceptor and RPE cells of AMD patients [−/− mice [The body recognizes exogenous pathogens and endogenous risk factors through pattern recognition receptors (PRRs) that sense microbes through conserved molecular structures called pathogen-associated molecular patterns (PAMPs). PPRs include the Toll-like receptors (TLRs), the nucleotide-binding oligomerization- (NOD-) like receptors (NLRs), the RIG-I-like receptors (RLRs), the C-type lectin receptors, and advanced glycosylation end product (AGE) receptors (RAGE) . When thnd IL-18 . High expatients . Mitochopatients . Laser-i−/− mice . However−/− mice . The stuAlu is the most abundant transposable element, which is transcribed into Alu RNAs, and the accumulation of Alu RNAs has been confirmed to be related to AMD . Alu RNAβ-protein (Aβ). In the central nervous system, the accumulation of Aβ is associated with the activation of neurodegenerative and inflammatory pathways. In the ocular system, Aβ upregulates IL-1β, IL-18, and TNF-α in RPE cells. The intravitreal injection of Aβ can activate inflammation [γ, while other proinflammatory cytokines, such as IL-8, MCP-1, and IP10, were downregulated, suggesting a that parainflammation state occurred under AGE stimulation [In addition to RAGE, some substances that are secreted by dead cells and damaged tissues are also receptors for AGEs, including amyloid ammation . AGEs acmulation . Parainfmulation . One expmulation . All of The complement system is part of the host innate immune system that enables many essential functions, including the following: 1) the opsonization and lysis of microorganisms, (2) the recruitment of inflammatory cells, (3) the removal of dead cells, (4) the regulation of antibody production, and (5) the removal of immune complexes. There are three classic complement pathways: the classical pathway, the mannan-binding lectin (MBL) pathway, and the alternative pathway. All of these pathways ultimately lead to the formation of the cytolytic MAC . The com the opso−/−) mice, MCs and drusen-like deposits accumulated subretinally with age [−/−/CCL2−/− (double knockout) mice, AMD-like retinal lesions developed, characterized by abnormal RPE cells, drusen, photoreceptor atrophy, and choroidal neovascularization [high, IL-23high, and IL-10low phenotype, while the M2 macrophages are relatively anti-inflammatory with IL-12low, IL-23low, and IL-10high phenotype. In addition, CXCL9, CXCL10, and CXCL11 represent M1 chemokines, and CCL17 and CCL22 represent M2 chemokines [κB and JAK-STAT pathways [γδT, and ILC promoted experimental intraocular neovascularization during laser-induced CNV in mice. Additionally, there was a greater increase in the expression levels of IL-17RC in the blood of siblings with AMD compared to that in the blood of their respective siblings without AMD, and increased levels of IL-17RC can cause damage to photoreceptors [Immune cells in a normal retina include microglial cells (MCs), macrophages, and dendritic cells. MCs play roles in neuronal homeostasis and immune surveillance, which are normally absent from the outer retina but can infiltrate into the subretinal space and become activated during aging and AMD, likely to support the RPE cells and clear age-related debris. However, MCs may also induce oxidative stress and promote further degeneration. Two chemokines, CX3CL1 and CCL2, and their respective receptors, CX3CR1 and CCR2, play important functions for the recruitment of macrophages/microglia to tissue lesions . In CX3Cwith age . In CX3Crization . Macrophemokines . CXCL11 pathways . IL-17 heceptors , 87. TheκB pathway. It appears that the role Nrf2 plays in autophagy, especially through interactions with p62, is strongly dependent on the cellular context as there are many reports suggesting that this protein acts differently depending on the cellular state [Autophagy dysfunction, cellular senescence, and abnormal immune-inflammatory responses interact with each other. Autophagy dysfunction accompanied by lipofuscin accumulation and ROS increases, can activate inflammatory reactions, further promoting long-term and chronic cascade inflammation and accelerating RPE cell senescence . Nrf2, aar state . Aging car state . The admar state . All of β activated SIRT6, autophagy, and inflammation. Silencing SIRT6 led to the decreased expression levels of Beclin1, ATG5, and LC3. Using 3-MA to inhibit autophagy mediated by Aβ led to decreased levels of IL-1β, IL-6, IL-8, IL-12b, NLRP3, and TNF-α [β, IL-2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. In contrast, rapamycin augmented A2E-mediated autophagy and attenuated the protein expression of inflammation-associated and angiogenic factors [SIRT6 and autophagic markers are upregulated in the RPE cells of aged mice. Intravitreal injections of And TNF-α , indicat factors , indicat factors –91, thus factors . Therefoβ-positive cells, SAHFs, and cell cycle arrest at the G1 phase, indicating that SIPS and increased autophagy occurred simultaneously. However, the inhibition of autophagy with 3-MA promoted SIPS whereas inducing autophagy with rapamycin attenuated SIPS [With the depletion of glutathione (GSH) from RPE cells, increased autophagy and SIPS activation were apparent, as reflected by increased LC3 expression levels, autophagic vacuoles, and autophagic flux and an increased percentage of SA-ted SIPS .In summary, autophagy dysfunction, cellular senescence, and abnormal immune-inflammatory responses interact with each other and jointly participate in and promote AMD.AMD is a blinding disease caused by genetic and environmental factors. The roles of autophagy dysfunction in RPE cells, cellular senescence, and abnormal immune-inflammatory responses have been recognized in AMD. The relationships among these three processes can be described as both stimulating and restrictive. Autophagy dysfunction in RPE cells leads to clearance system abnormalities. Cellular senescence leads to cell dysfunction and the promotion of senescence among neighboring cells. Abnormal immune-inflammatory responses lead to chronic retinal inflammation. Autophagy dysfunction can accelerate the senescence of RPE cells, while either promoting or inhibiting inflammation. In conclusion, if improved autophagy, alleviated cellular senescence, and the inhibition of abnormal retinal immune-inflammation responses can be achieved simultaneously, it may be possible to delay the progress of AMD and to obtain better clinical efficacy. At present, these three antiaging strategies have achieved good results when applied to atherosclerosis, pulmonary fibrosis, and osteoarthritis. Although there is currently no relevant application of these strategies for AMD, the use of antiaging strategies for AMD prevention and treatment is expected to achieve a new breakthrough in the future.


Influenza A viruses (IAVs) possess a great zoonotic potential as they are able to infect different avian and mammalian animal hosts, from which they can be transmitted to humans. This is based on the ability of IAV to gradually change their genome by mutation or even reassemble their genome segments during co-infection of the host cell with different IAV strains, resulting in a high genetic diversity. Variants of circulating or newly emerging IAVs continue to trigger global health threats annually for both humans and animals. Here, we provide an introduction on IAVs, highlighting the mechanisms of viral evolution, the host spectrum, and the animal/human interface. Pathogenicity determinants of IAVs in mammals, with special emphasis on newly emerging IAVs with pandemic potential, are discussed. Finally, an overview is provided on various approaches for the prevention of human IAV infections. Orthomyxoviridae. Both human IAV and IBV trigger seasonal global epidemics, while human infections with ICV are less frequent and generally cause mild illness without reported epidemics. IDV mostly infect cattle and are not yet known to infect humans [Influenza is a contagious respiratory disease caused by influenza viruses (IVs). IVs are categorized antigenically based on the variations of the nucleoprotein (NP) into four genera: influenza A viruses (IAV), influenza B viruses (IBV), influenza C viruses (ICV), and influenza D viruses (IDV) . Togethet humans .IVs have a segmented, negative sense, single-stranded (-ss) viral RNA (vRNA) genome. Both IAV and IBV contain eight vRNA segments, while ICV and IDV contain only seven segments . The eigEach segment encodes at least one structural protein: polymerase basic protein 2 , polymerase basic protein 1 , polymerase acidic protein , hemagglutinin , nucleoprotein , neuraminidase , matrix proteins , and non-structural proteins (NS1 and NS2 or nuclear export protein (NEP), segment 8) .The viral envelope contains two viral transmembrane glycoproteins; hemagglutinin  and neuraminidase NA, ~17%). The HA protein forms a trimer, which protrudes approximately 13.5 nm from the viral surface and has a rod-like shape, while the NA protein forms a tetramer with mushroom-like shape ,5. In ad%. The HAAs a complex genomic entity with unique structure and function among RNA viruses, the vRNP of IAVs is composed of the RNA-dependent RNA polymerase (RdRp) subunits  as a globular head linked to rod-shaped structures of the vRNAs folded on the NP protein. A breakthrough technology that allowed detailed comprehension of the vRNP structure/function, to deduce the mechanisms of RNA replication and transcription, the intracellular trafficking of the viral genome, selective packaging of the vRNPs and viral gene reassortment, was the development of the in vitro reconstitution of recombinant vRNPs that represent efficient replicons. This gave also rise to the development of minigenome assays and different reverse genetic systems for IAV to study the impact of specific adaptive genetic changes and viral segments reassortment of zoonotic potential ,37,38 F.Unlike influenza B, C, and D viruses, IAV are subtyped according to the antigenicity of the surface glycoproteins into 16 HA- and 9 NA-subtypes. Except for bat-origin influenza-like viruses H17N10 and H18N11, all IAV subtypes were isolated initially from avian hosts  39,40,4,42,43,44Humans are susceptible to infection with influenza A, B, and C viruses. A limited number of IAV subtypes are circulating in humans such as the H1N1 and H3N2 subtypes leading to seasonal infections or occasional pandemics ,46,47,48AIV are typed according to their pathogenicity in chickens into low pathogenic (LP) and highly pathogenic (HP) strains. LPAIVs are maintained in wild aquatic birds almost without developing severe clinical signs of the disease. The clinical signs in domestic poultry induced by LPAIVs include a body weight reduction and/or a slight drop in egg production in layers poultry . In contIAV evolve mainly by two mechanisms: (1) through accumulation of point mutations due to the lack of a proof-reading function of the RdRp, leading to aa changes (referred to as antigenic drift) A and (2)Unlike epidemics, pandemics  can spreFurthermore, AIVs circulated in different bird species for decades and caused dramatic outbreaks . In 1997As already noted, the HPAIV H5N1 strain was documented for the first time to have crossed the animal-to-human barrier in 1997, causing zoonotic fatal infections. By genetic analysis of the 1997 HPAIV H5N1, it was revealed that the six internal protein-encoding segments  were derived from co-circulating avian H9N2 viruses ,95 Figu. The preFrom January 2003 to May 2018, 860 human HPAIV H5N1 infections  were reported worldwide. As evidenced by the low incidence rate compared to the H7N9 infections see , H7N9, tIn 2003, clade 1 strains were first detected in northern Vietnam. Recently, clade 1 viruses are phylogenetically designated as clades 1.1.1 and 1.1.2 ,103. In Currently, out of the more than 30 previously reported genetic clades, clades 1.1.2 (Cambodia and Vietnam), 2.1.3.2b (Indonesia), 2.2.1.2 (Egypt), 2.3.2.1c , 2.3.4.2 (China), and 7.2 (China and Vietnam) are circulating in domestic poultry .reassortant A-HPAIV H5N6” represents the prototypic HPAI H5N6 strain, which led to the first fatal H5N6 infection in humans. Independently, “reassortant B-HPAIV H5N6” concurrently resulted from reassortment between H5N8 (clade 2.3.4.4), and H6N6 viruses. Subsequently, the “reassortant B-HPAIV H5N6” was subjected to a single reassortment event by which it acquired the six internal genes of a co-circulating influenza A/H9N2 strain resulting in “reassortant C-HPAI H5N6”  without prior adaptation, the H6-type viruses were not detected in humans until May 2013 , when a First evidence of human infections with H7N2 (LPAIV) and H7N3 (LPAIV) was based on positive retrospective serologic analysis of workers involved in the poultry outbreaks in the United States  and Italy in 2002 and 2003, respectively . The H7NIn February 2018, the Chinese National Health and Family Planning Commission (NHFPC) announced the first non-fatal human infection of H7N4 in Jiangsu Province. The patient was a 68-year-old woman who acquired the infection after exposure to poultry in a live bird market .Mild human infections with HPAIV H7N7 “conjunctivitis” was reported in 1959, 1977, and 1981 . The mosIn 2013, zoonotic infections with LPAIV H7N9 were first reported in China . The humAs indicated, the highest number of human H7N9 infections has been reported during the fifth epidemic wave, with a geographic range expansion within Chinese provinces . DespiteY280-like lineage, represented by A/duck/Hong Kong/Y280/97 (Y280-like), A/Chicken/Beijing/1/94 (BJ94-like), and A/Chicken/Hong Kong/G9/97 (G9-like); (2) G1-lineage represented by A/quail/Hong Kong/G1/97-like (G1-like), and (3) Korean lineage represented by A/Duck/Hong Kong/Y439 (Y439-like) and A/chicken/Korea/38349-p96323/96 (Korean-like) [Since the mid-1980s, the LPAIV H9N2 circulates extensively worldwide in poultry resulting in high genetic diversity ,130. Basan-like) ,131,132.Since the mid-1990s, the BJ/94-, G9-, and G1-like H9N2 viruses are predominantly circulating in chickens and quails in China. Since 2010, the G1-like lineage demonstrated a widespread distribution and prevalence throughout Asia, the Middle East, North Africa, and Europe ,133,134.H9N2 viruses are predominantly isolated from domestic poultry and live-bird markets, which were proven to be risk factors for zoonotic transmission of AIVs from birds to humans ,136. TheAlthough outbreaks of H10N7 are uncommon, this virus can sporadically cross the species barrier to mammals including humans. Human infections with H10N7 were occasionally reported from Egypt (2004) and Australia (2010) . Recent In late 2013, a fatal human infection with LPAIV H10N8 was identified in China . NotablyAlthough the infection of humans with zoonotic influenza viruses is less frequent than infections with seasonal influenza viruses, there is a global concern that these zoonotic viruses may acquire mutations in animals or humans that favour the efficient animal-to-human or sustained human-to-human transmission. Poultry and pigs are the major sources of human infections with IAVs. The portal of entry for human infections is mostly through the conjunctiva , nasal and mucosal membranes , or probably swimming in contaminated pools. Eating of well-cooked meat is not a source of human infections with IAVs until now ,149,150.Generally, the human infections with AIV were found to be limited to individuals with intensive direct contact with the infected animals and few numbers of family clusters without sustainable human-to-human transmission were reported ,152,153.The pathogenicity of IAV is a complex and multigenic trait, which is affected by several viral and host factors. Nowadays, the accessibility of sequencing data from a large number of viral strains together with the availability of different reverse genetics (RG) approaches for IAV ,162,163,Amino acid (aa) substitutions in the subunits of the RdRp have been reported to alter the replication, pathogenicity and host range of IAV in mammals and poultry . The preIn PB1, most avian IAV carry asparagine at position 375, whereas most human IAV including the pandemic 1918-H1N1, 1975-H2N2 and 1968-H3N2, contain a serine at this position . TherefoIn the PA protein, 97I was shown to enhance the virulence of influenza virus A/Aquatic bird/Korea/W81/05 (H5N2) in mice rather than in chicken and to promote efficient replication in vitro in mammalian rather than avian cell lines . In addiSeveral studies have indicated an important role of the NP protein in viral replication and pathogenicity. It is now known that the cellular antiviral-acting Mx1 (mouse) or its human ortholog MxA interact with the viral NP and thereby inhibit IAV replication ,191. TheThe classical nuclear import of vRNP is primarily regulated by adaptor importin-α proteins, which subsequently link the nuclear localization signals (NLSs) of imported vRNP molecules to importin-β and in turn mediate their nuclear localization ,199. TheThe fourth segment of the IAV genome encodes for the hemagglutinin (HA), the major trimeric surface glycoprotein. It is synthesized as a single polypeptide precursor (HA0). To activate the HA fusion peptide that mediates fusion between the viral envelope and the host cell membrane, HA0 needs to be proteolytically cleaved into the two subunits, HA1 and HA2, that are held together via a disulphide bond ,209,210.The HA plays a key role in the replication and adaptation of IAV to new hosts  because The receptor binding residues of IAV are located in the HA head domain . SeveralTo date, the molecular determinants of efficient replication, transmission and virulence for the seasonal IAV infection in mammals have been intensively studied. Several aa substitutions were found to be connected to changes in the receptor preference of HA in subtype specific manner such as N154S, N182K, Q192R, Q222L, S223N, G224S, Q226L, S227N, L129V + A134V, G139R + N182K, Q192R + S223N, Q222L + G224S (H5-subtype), E190D, D22G (H1-subtype), G225D (H1- and H9-subtypes), and G228S  . For insFurthermore, airborne transmission of AIV among humans is of special concern. In 2012, Imai et al. have shown that the introduction of 4 mutations (N158D/N224K/Q226L/T318I) into H5-HA via serial-passaging of the HPAIV H5N1 in ferrets changed the receptor binding preference towards α2,6-SA and enabled airborne virus transmission between ferrets . NeverthConsistently, Q226L, located in the HA receptor binding domain, contributed to the adaptation of a H10N7 AIV to seals/mammals by improving a binding affinity to the human-like α2,6-SA receptor . RecentlThe neuraminidase (NA), encoded by the 6th segment of the IAV genome, forms a tetrameric spike protein with enzymatic activity. Since NA mediates the enzymatic removal of SA from the surface of infected host cells , a balanThe non-structural protein 1 (NS1) is small, multifunctional protein (mostly 230 aa/26 kDa) that is encoded by the eighth vRNA segment and translated from the unspliced viral mRNA of the NS segment. The NS1 protein exerts different activities in infected cells to promote efficient viral replication and virulence. NS1 interferes with type I IFN production as well as with IFN-inducing factors. In addition, it plays a positive regulatory role in RdRp activity and the nuclear export and translation of viral mRNAs ,242. In Beside the IFN antagonistic function of the NS1 protein, the PDZ-ligand binding motif (PBM) at the C-terminal end of NS1, commonly ESEV, KSEV, and RSEV, was shown to enhance the pathogenicity of 1918 H1N1 and HPAIV H5N1(KSEV) in mice ,256. DifIn addition to gradual aa changes in the NS1 protein sequence, genetic shift or reassortment of the NS segment harbouring the NS1 gene extends the host range and tissue tropism, impairs the cellular immune response and alters the replication and pathogenicity of the reassortant virus in vitro and occasionally in vivo ,246,259.Vaccination is still the primary defence line and the most effective method to combat IAV infection  and woulAnimal vaccination can reduce the chance of human infections with AIV with zoonotic potential. Nevertheless, AIV infections occur even in vaccinated birds and pigs and are frequently associated to vaccines, which demonstrated clear antigenic mismatches ,267,268,Luckily, the human-to-human spread of H5N1 and H7N9 strains is yet not well established, which reduces the urgency to recommend a regular annual vaccination against both strains. Nevertheless, some countries maintain national stockpiles of pre-pandemic vaccines against AIVs  to control infections if similar viruses should transmit from human-to-human ,275. An Should a zoonotic AIV against which vaccines have not yet been generated infect and spread in the human population antivirals will be urgently needed. The momentarily available antiviral medications to control IAV infection are optimally prescribed within 48 h of symptom onset for patients admitted to the hospital with influenza-like illness and individuals at high risk of developing influenza-related complications. Several essential preventive and therapeutic strategies could help to combat epidemic and pandemic infection with zoonotic strains, including the stockpiling of anti-influenza drugs . CurrentThe matrix protein 2 (M2) forms a homotetrameric integral membrane protein (97 aa). The M2 protein exerts an important proton channel activity that mediates the uncoating process through the acidification of the interior of the virion. This dissociates the M1 protein from the vRNPs mediating the release and the nuclear import of the vRNPs after HA-mediated fusion between viral and endosomal membrane . Two oraThe sialidase activity of NA is exerted by specific and highly conserved aa residues (active sites). These aa residues are differentiated into 8 catalytic- and 11 framework residues  287,288288,289. Regarding non-seasonal AIVs, the limited surveillance of the NAI sensitivity of circulating viruses, especially for the H5N1- and H7N9-strains, remains a problem. So far, only a few epidemiological/antiviral studies have described H5N1 and H7N9 subtypes with reduced oseltamivir sensitivity, found with low prevalence in both poultry and humans in different geographical localities ,299,300.The effect of antiviral drug resistance relies primarily on the viral replication fitness of the drug-resistant variant versus the drug-sensitive strain. This was shown in studies that investigated the relevance of specific aa residues (known to confer NAI resistance) for neuraminidase activity, viral replication fitness, transmissibility and virulence in vitro and in vivo. However the results were not consistent and were dependent on the subtypes studied  and their particular genetic differences ,303,304.Despite that NAIs are presently effective in controlling infections by seasonal and zoonotic IAV strains, the emergence and global spread of NAIs-resistant IAV variants is a concern, especially in areas of high level of NAI use like Japan . ConsequAs mentioned before, during the viral entry process, the pH decrease within the endosome leads to an irreversible conformational change in the HA2 subunit activating the fusion peptide, which interacts with the endosomal membrane to initiate the fusion process. Subsequently, the vRNPs are released into the cytoplasm and are then transported into the nucleus to initiate viral genome replication/transcription . TherefoThe RdRp complex comprises conserved and independently folded subdomains with defined functionalities in viral transcription and replication. To date, T-705 (Favipiravir), a purine pseudobase inhibitor of RdRp, is licensed in Japan for the treatment and prophylaxis of pandemic influenza A/H1N1 . RibavirDue to the expansion of drug-resistance to M2-blockers and NAIs, new therapeutic strategies have been developed targeting the cellular components such as sialic acid receptors, posttranslational processing of the viral proteins, or essential proviral intracellular signaling cascades. DAS181 (Fludase) is a recombinant sialidase, which is designed to be applied by inhalation to restrict the binding of influenza viruses to the host cell by removing sialic acid receptors from glycan structures on the human airway epithelia .Nitazoxanide, an orally active anti-parasitic drug, controls IAV infection by impairing the trafficking of the HA from the endoplasmic reticulum (ER) to the Golgi and inhibiting HA maturation by blocking HA terminal glycosylation. It also stimulates antiviral cellular innate immunity via interferon induction ,278.d,l-lysine-acetylsalicylate-glycine (LASAG), an anti-inflammatory derivative of Aspirin, resulted in a significantly faster alleviation of influenza symptoms, more rapid decline in viral shedding, and decreased risk of viral spread [Currently, further inhibitors are under investigation in late-phase clinical trials: (i) VX-787, an orally active inhibitor of PB2; (ii) S-033188, an orally active PA inhibitor; and (iii) AVI-7100, intravenously active, small interfering antisense RNA constructs, targeting influenza M1/M2 translation ,278,287.l spread . LASAG al spread . The in l spread ,315.Furthermore, five monoclonal antibodies that efficiently neutralize invading IAV are under evaluation in clinical trials. These mAbs target either the globular head or the highly conserved stem region of the viral HA, or the conserved ectodomain of the M2 (M2e); namely CR6261, CR8020, MEDI8852, MHAA4549A, and VIS-410 ,278.More than 50% of all human viral infections can be transmitted from animals. The One Health approach is based on the fact that animal, human and environmental healths are interrelated. A hundred years ago in 1918–1919, mankind was confronted with the first documented influenza pandemic of the 20th century, representing a devastating global occasion of influenza virus infections. Since then, influenza viruses showed a high genetic and antigenic diversity either by gradual acquisition of particular changes (antigenic drift) important for the adaptation to a new host or by monogenic/polygenic reassortment events (antigenic shift). This wide diversity of IAVs permitted them to adapt to different avian and mammalian host species including humans. In recent years, new AIVs emerged that have evolved independently to cross animal/human interface and have caused mild to fatal complications in human. Despite the massive screening for new antiviral drugs against influenza viruses, limited numbers of FDA-licensed or conditionally/regionally licensed anti-influenza drugs are available as a preventive strategy. Therefore, a better understanding of the genesis of newly emerging IAVs, improved surveillance efforts and intensified research to monitor and understand these foes are mandatory. Virtually, the One Health concept, a collaborative effort of different specialized experts across animal, human and environmental health to ameliorate animal and human health, must be implemented to fight against the high-consequence zoonotic AIVs.


Panax notoginseng and ginseng in traditional Chinese medicine. Its pharmacological effects exert remarkable neurotrophic and neuroprotective effects in the central nervous system. To explore and summarize the protective effects and mechanisms of ginsenoside Rg1 against cerebral ischemia and reperfusion injury, we conducted this review, in which we searched the PubMed database to obtain and organize studies concerning the pharmacological effects and mechanisms of ginsenoside Rg1 against cerebral ischemia and reperfusion injury. This study provides a valuable reference and clues for the development of new agents to combat ischemic stroke. Our summarized review and analysis show that the pharmacological effects of and mechanisms underlying ginsenoside Rg1 activity against cerebral ischemia and reperfusion injury mainly involve 4 sets of mechanisms: anti-oxidant activity and associated apoptosis via the Akt, Nrf2/HO-1, PPARγ/HO-1, extracellular regulated protein kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) pathways  and the caspase-3/ROCK1/MLC pathway; anti-inflammatory and immune stimulatory-related activities that involve apoptosis or necrosis via MAPK pathways (the JNK1/2 + ERK1/2 and PPARγ/HO-1 pathways), endoplasmic reticulum stress (ERS), high mobility group protein1 (HMGB1)-induced TLR2/4/9 and receptor for advanced glycation end products (RAGE) pathways, and the activation of NF-κB; neurological cell cycle, proliferation, differentiation, and regeneration via the MAPK pathways ; and energy metabolism and the regulation of cellular ATP levels, the blood-brain barrier and other effects via N-methyl-D-aspartic acid (NMDA) receptors, ERS, and AMP/AMPK-GLUT pathways. Collectively, these mechanisms result in significant neuroprotective effects against cerebral ischemic injury. These findings will be valuable in that they should further promote the development of candidate drugs and provide more information to support the application of previous findings in stroke clinical trials.Cerebral ischemia-reperfusion is a complicated pathological process. The injury and cascade reactions caused by cerebral ischemia and reperfusion are characterized by high mortality, high recurrence, and high disability. However, only a limited number of antithrombotic drugs, such as recombinant tissue plasminogen activator (r-TPA), aspirin, and heparin, are currently available for ischemic stroke, and its safety concerns is inevitable which associated with reperfusion injury and hemorrhage. Therefore, it is necessary to further explore and examine some potential neuroprotective agents with treatment for cerebral ischemia and reperfusion injury to reduce safety concerns caused by antithrombotic drugs in ischemic stroke. Ginseng Rg1 (G-Rg1) is a saponin composed of natural active ingredients and derived from the roots or stems of  Nearly 6 million people die from stroke each year, and it is estimated that the lifetime risk for stroke is 8% to 10%. Ischemic stroke accounts for 85% of all strokes, while hemorrhagic stroke accounts for 15% ,3. Addit factors ,5,6,7,8. factors ,5. In pa factors , and it Panax notoginseng (Burk) F. H. Chen and Panax ginseng C. A. Mey are two commonly used Chinese medicinal herbs, the roots or stems of which have been used for the treatment of cardiovascular disease in many Asian countries for several hundred years [P. notoginseng, P. ginseng, and their extracts, panax notoginseng saponins (PNS) and ginseng total saponins (GTS), exert multiple pharmacological activities, such as anti-inflammatory [ed years . Pharmacammatory , anti-oxammatory ,13, platammatory .P. notoginseng and P. ginseng that is obtained via an extraction and purification processes  levels.As shown in rocesses  and chem ginseng ,17,18, 4oginseng . These ds system ,27,28,29s system  and antis system ,33,34,35s system ,37. Varis system ,39, inclHowever, to date, no systematic review has been conducted to assess the protective effects of and mechanisms underlying how G-Rg1 combats cerebral ischemia/reperfusion injury (CI/RI). A systematic review of all evidence available from animal experiments preceding clinical trials would provide an adequate interpretation of the limitations and potential of novel treatment strategies. Moreover, while various candidate drugs have failed to treat cerebral ischemia, those studies have prompted series of suggestions that could improve the likelihood of successful translation. Among these is that if a systematic review and analysis of preclinical studies of alternative active ingredients was to be carried out, it would likely promote candidate drug development and provide more information from the previous literature that could be used as a bridge into clinical trials of stroke. Therefore, in the present study, we conducted a systematic review of all available animal studies to evaluate the preclinical evidence related to G-Rg1 in experimental CI/RI studies.https://www.ncbi.nlm.nih.gov/pubmed/?term = ((ginsenoside ± Rg1%5BTitle%2FAbstract%5D) ± AND ± ischemia)). This allowed us to organize and analyze the literature concerning the pharmacological effects and mechanisms of ginsenoside Rg1 against CI/RI, which will be valuable for further promoting candidate drug development and providing more citation-based information that can be applied in clinical trials of stroke.To explore and summarize the protective effects and relevant mechanisms of ginsenoside Rg1 against CI/RI, we conducted this review by searching the “PubMed” database via using “Ginsenoside Rg1” and “Ischemia” as search terms to obtain the literature concerning animal experiments in latest 10 years (2−), hydrogen peroxide (H2O2), nitric oxide (NO), and hydroxyl radicals (OH-) [Compared with other organs in the human body, brain tissues and neurons are more oxygen-consuming and more prone to produce higher levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), and unsaturated fatty acids than can be rapidly oxidized, whereas the anti-free radical system of nervous tissues are relatively weaker, indicating that neurons are more sensitive to oxidative stress than is observed in other tissues. Active radicals include superoxide anion (Ols (OH-) ,41. Ischls (OH-) ,44,45,46ls (OH-) ,49,50,51First, various animal models of cerebral ischemia have been tested, with results demonstrating that G-Rg1 may exert its neuroprotective activities on CI/RI by regulating oxidative stress ,53,54,552O2-induced SH-SY5Y cell injured models [2O2-treated PC12 cell models, and human vascular endothelial EA.hy926 cells [2O2-induced cell apoptosis in cortical pyramidal cells, NSCs, SH-SY5Y, or PC12 cells; markedly elevated cell viability and survival rates in various cells  treated with OGD or H2O2; diminished the amount of ROS production and the levels of MDA, NO, leaked LDH and intracellular Ca2+ [2O2-induced injuries in mouse cultured astrocytes were prevented by G-Rg1 because it inhibited ROS production, intracellular Ca2+ overload, and the loss of mitochondrial membrane potential (MMP) [Second, as shown in d models , H2O2-tr26 cells . Comparelar Ca2+ ,63; incrlar Ca2+ ,60,61,63al (MMP) ,64, indi2O2-induced PC12 cells [2O2-induced SH-SY5Y model of cell injury [In summary, the main mechanisms by which G-Rg1 exerts its significant neuroprotective effects in cerebral ischemic injury are closely associated with anti-oxidation and the inhibition of apoptosis. Previously published studies have confirmed that G-Rg1 attenuates OGD-induced oxidative stress and regulates the Nrf2/HO-1 pathways in modeled animals and cells ,56 and m12 cells , OGD-tre12 cells , and an l injury . Hence, l injury .Immunity and inflammation are key elements that contribute to the pathobiology of stroke, and CI/RI and the secondary damage it causes to brain tissues are closely associated with immunity and inflammation responses. Increasing evidence has indicated that an inflammatory response is involved in all stages of ischemia-reperfusion injury ,69. OnceCoincidentally, multiple in vitro and in vivo experiments have shown that G-Rg1 may reduce ischemia-reperfusion injury by inhibiting necrosis and apoptosis associated with anti-inflammatory activity ,76,77,78Moreover, in in vitro experiments, treatment with G-Rg1 was demonstrated to significantly relieve the inflammatory response that occurred after hypoxic injury by reducing multiple indicators of inflammation in an OGD-induced cortical neurons model ; by incrThe studies evaluated here  demonstrIt is well known that the MAPK/ERK pathway is involved in cell proliferation, differentiation, senescence, and apoptosis, and MAPK/p-38 is activated by various inflammatory extracellular mediators, whereas JNK isoforms are strongly activated during various cellular stress responses. These findings indicate that in cortical neurons, the phosphorylated (activated) status of p38 and ERK-1/2 is upregulated in the absence of oxygen, whereas in rat hippocampal slices, MAPK (p38) and MAPK (ERK1/2) and the phosphorylation status of ERK-1 are upregulated in response to hypoxia, but existing tests found this upward trend of phosphorylation was downregulated by G-Rg1 ,85,86,87In addition to its roles in the regulation of oxidative stress and inflammation, G-Rg1 also could regulate cytokine expression, the cell cycle, cell proliferation, cell differentiation, and cell apoptosis after stroke by activating PI3K-Akt/mTOR, mitogen-activated protein kinases (MAPKs) and ERS regulatory signaling pathways, as confirmed by the results of various types of testing models, such as the modified Rice–Vannucci model , hypoxiaIn HIBD-induced animals, G-Rg1 treatment enhanced the neural survival rate, reduced neurological impairment and pathologic damage ,91, clea+ concentrations and consequential cytotoxic cerebral edema and nerve damage [In general, abundant blood must be supplied to the brain, with the cerebral blood flow accounting for 20% of cardiac blood output at rest. In cerebral ischemia, the blood flow to the brain is reduced or blocked, and the ischemic brain tissue area cannot obtain sufficient blood oxygen. Additionally, brain nerve tissue can only obtain energy through anaerobic sugar glycolysis, which reduces ATP production, increases adenosine diphosphate (ADP) production, and increases the ADP/ATP ratio, resulting in a lack of nutrients required for brain tissue metabolism, damage to mitochondria and other organelles, and further aggravation of brain tissue damage in ischemic areas . Furthere damage . After Ge damage , and mite damage . The mec2+ influx, calcium overload, ROS aggregation, mitochondrial injury, brain barrier permeability, and cytotoxic brain edema, all of which result in cell necrosis and apoptosis. Nevertheless, G-Rg1 treatment restored the intracellular calcium overload in neurons, reduced the excitatory Glu toxicity induced by CI/RI by reducing the contents of Glu and Asp in serum [Moreover, glutamate (Glu) is one of the most widely distributed excitatory amino acids (EAAs) in the central nervous system. Under ischemia and hypoxia conditions, Glu is released in large quantities, resulting in the activation of Glu receptors, and causes irreversible damage or even death of nerve cells via two major mechanisms, including N-methyl-D-aspartate (NMDA) receptor-mediated delayed injury of nerve cells and non-NMDA receptor-mediated neuronal injury in the acute phase of ischemia ,7,8; thein serum , decreasin serum , enhancein serum , and bloin serum . These pCerebral ischemia-reperfusion is a complicated pathological process. The damage and cascade of reactions caused by ischemia and reperfusion in brain tissues are related to decreased blood flow, ischemic-induced energy metabolism disorder, oxidative stress, inflammatory stress, cytokine damage, excitatory toxicity by glutamate, intracellular calcium overload, NO synthesis, and many other factors ,6,7,100,Panax notoginseng, and its pharmacological effects are remarkable in that it exerts neurotrophic and neuroprotective effects on the central nervous system. In our review, we summarize the protective effects of G-Rg1 against CI/RI in addition to the mechanisms underlying these effects. The results of our analysis show that 4 main mechanisms are involved , is a traditional stem extract of ginseng and involved : anti-oxFirst, G-Rg1 can upregulate the anti-oxidant capacity of SOD, MPO, GSH-Px, and CAT, while simultaneously downregulating oxidative free radicals, such as ROS, RNS, and OH; it can also inhibit ischemic nerve damage and associated apoptotic effects  caused by oxidative stress and induced via the Akt, Nrf2/HO-1, PPARγ/HO-1, ERK, p38 and JNK MAPK pathways, the mitochondrial apoptosis pathway and the caspase-3/ROCK1/MLC pathway, thereby providing significant neuroprotective effects against cerebral ischemic injury.Second, G-Rg1 can downregulate harmful inflammatory cytokines, such as IL-6, IL-1β, TNF-α, ICAM-1, and MMP-9, at both the protein and mRNA levels; upregulate anti-inflammatory factors regulated by NF-κB (p50 and p65) and IKK; inhibit the levels of PPARγ, Bax, CC-3, and CC-9 at both the protein and mRNA levels; and inhibit HMGB1 and the associated necrotic and apoptotic effects caused by oxidative stress  by regulating MAPK pathways, such as the JNK1/2, ERK1/2, and JAK1/STAT1 pathways, in addition to ERS, the HMGB1-induced TLR2/4/9 and RAGE pathways, and activate NF-κB, resulting in significant neuroprotective effects against cerebral ischemic injury. Third, G-Rg1 can increase the levels of cytokines that promote cell proliferation and differentiation, such as HIF-1α, EPO, VEGF, BDNF, and NGF, at both the protein and mRNA levels; promote angiogenesis and induce neurogenesis by regulating MAPK pathways, such as the JNK1/2 and ERK1/2, PI3K-Akt/mTOR, PKB/Akt, and HIF-1α/VEGF pathways; and affect ERS, resulting in significant neuroprotective effects against cerebral ischemic injury. However, the specific regulatory mechanisms that are affected in neurons and during angiogenesis remain unclear.Finally, G-Rg1 can upregulate the energy metabolism capacity of Na-K-ATPase in addition to iNOS activity, ATP, AMP, total adenine nucleotides (TANs), and energy charge (EC); downregulate the free radical contents of Glu and asparaginic acid (Asp), modulated (inhibited) the influx of extracellular calcium and the release of intracellular calcium as well as nNOS activity; enhance the expression of GLUT3 and the activation of AMPKα1/2; and inhibit ischemic nerve damage and its associated apoptotic effects  via its effects on NMDA receptors, ERS, and the AMP/AMPK-GLUT pathways.Panax notoginseng and ginseng have been comprehensively elaborated, a situation that is more conducive to the development and utilization of Panax notoginseng and ginseng. These findings provide ideas for research into the pharmacological actions and mechanisms of other active constituents, a reference for the rational clinical use of drugs, and scientific protection of resource utilization.In summary, ginseng Rg1 is a tetracyclic triterpenoid derivative derived from natural medicinal plants that has significant and representative pharmacological activities. Additionally, in this overview, we show that GR promotes anti-ischemic stroke via its links to multiple pathways and its multitarget effects, as shown in However, many of the actions and mechanisms of G-Rg1 remain unknown. These include its ability to regulate inflammation after the I/R activation of keratinocytes, and few studies have explored its effects on neurogenesis and autophagy regulation in brain nerve cells . At the


Illuminating the comprehensive lipid profiles after dietary supplementation of polyunsaturated fatty acids (PUFAs) is crucial to revealing the tissue distribution of PUFAs in living organisms, as well as to providing novel insights into lipid metabolism. Here, we performed lipidomic analyses on mouse plasma and nine tissues, including the liver, kidney, brain, white adipose, heart, lung, small intestine, skeletal muscle, and spleen, with the dietary intake conditions of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) as the ethyl ester form. We incorporated targeted and untargeted approaches for profiling oxylipins and complex lipids such as glycerol (phospho) lipids, sphingolipids, and sterols, respectively, which led to the characterization of 1026 lipid molecules from the mouse tissues. The lipidomic analysis indicated that the intake of PUFAs strongly impacted the lipid profiles of metabolic organs such as the liver and kidney, while causing less impact on the brain. Moreover, we revealed a unique lipid modulation in most tissues, where phospholipids containing linoleic acid were significantly decreased in mice on the ARA-supplemented diet, and bis(monoacylglycero)phosphate (BMP) selectively incorporated DHA over ARA and EPA. We comprehensively studied the lipid profiles after dietary intake of PUFAs, which gives insight into lipid metabolism and nutrition research on PUFA supplementation. Polyunsaturated fatty acids (PUFAs) are essential nutrients that have a range of biological effects such as on brain function, cardiovascular disease, obesity, cancer, and bone health in humans ,2, and aThe lipid profiles in tissues are affected by different balances of dietary PUFAs ,6,7,8,9.After dietary intake, PUFAs are actively incorporated into cells as acyl chains of membrane phospholipids and other lipid classes, such as triacylglycerol and cholesteryl esters. Intake of ARA, EPA, and DHA affects several physiological functions, such as membrane scaffold formation to harmonize biomolecule interactions , energy Liquid chromatography coupled with high resolution tandem mass spectrometry (LC-HR-MS/MS) and triple quadruple mass spectrometry (LC-QqQ/MS) are popular techniques for untargeted and targeted lipidomic analysis, respectively ,14. LC-QIn this study, we investigated the lipid profiles in plasma and tissues including the liver, kidney, white adipose, skeletal muscle, heart, small intestine, lung, brain, and spleen after dietary intake of the ethyl ester form of ARA (ARA-E), EPA (EPA-E) or DHA (DHA-E) in mice. In total, 1026 molecular species of lipid were identified, of which 915 and 111 were annotated in untargeted and targeted analyses, respectively. 4-d4 and PGE2-d4 were obtained from Cayman Chemical . Highly purified EPA ethyl ester (EPA-E) (> 98%), DHA ethyl ester (DHA-E) (> 97%), and ethyl arachidonate (ARA-E) (> 99%) were obtained from Nippon Suisan Kaisha, Ltd. , Harima foods, Inc.  and NuChek Prep, Inc. , respectively. All solvents of LC/MS grade were obtained from Wako .ARA-d8, 15-HETE-d8, LTBw/w) (EPA-fed group), 1% DHA-E (w/w) (DHA-fed group), or 1% ARA-E (w/w) (ARA-fed group) for 2 weeks. Animals were dissected under isoflurane anesthesia, after which organs, such as the liver, kidney, white adipose, skeletal muscle, heart, small intestine, lung, brain, and spleen, were collected and plasma was isolated. For the lipidome analyses, four biological replicates (n = 4) were used for liver tissue, and five biological replicates (n = 5) were used for the other tissues and plasma. Two of the white adipose samples from the EPA-fed group and one of the skeletal muscle samples from the ARA-fed group in were excluded from the targeted lipidomics results owing to LC-MS analysis failure. All animal experiments were carried out in accordance with the guidelines for the use and care of laboratory animals of Mochida Pharmaceutical, and were approved by the Institutional Animal Care and Use Committee (identification code: PMS14-041).Male C57BL/6J mice  were purchased at 10 weeks of age, and housed under controlled temperature and lighting (12 h light/dark cycle) with free access to water and a controlled diet . The fatty acid composition and other nutritional factors for fish-meal-free F1 are described in Standard enzymatic methods were used to determine plasma total cholesterol (TC) and triglyceride (TAG) with commercially available kits purchased from Wako Pure Chemical Industries Ltd. Total fatty acids in plasma were analyzed by gas chromatography . C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, C20:5, C22:5, and C22:6 levels were determined using the standard substances: palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, dihomo-γ-linoleic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid, respectively.13C3 , 0.5 µM phosphatidyl glycerol (17:0/14:1) , 0.5 µM lyso phosphatidylcholine (17:1) , 5 µM acylcarnitine (18:0)-d3 , 25 µM palmitic acid-d3 , 25 µM stearic acid-d3 , 5 µM cholic acid-d4 , and 0.5 µM Cer/Sph mixture I  (v/v/v for volume ratio containing 5 mM ammonium acetate and 10 nM EDTA) and mobile phase B (100% isopropanol containing 5 mM ammonium acetate and 10 nM EDTA), and the composition was produced by mixing those solvents. LC gradient consisted of holding solvent (A/B: 100/0) for 1 min, then linearly converting to solvent (A/B: 60/40) for 4 min, linearly converting to solvent (A/B:36/64) for 2.5 min and holding for 4.5 min, then linearly converting to solvent (A/B: 17.5/82.5) for 0.5 min, linearly converting to solvent (A/B: 15/85) for 8.5 min, and linearly converting to solvent (A/B: 5/95) for 1 min followed by returning to solvent (A/B: 100/0) and holding for 5 min for re-equilibration. The flow rate and column temperature were set to 0.3 mL/min and 45 °C, respectively. Data dependent MS/MS acquisition mode was applied as previously described +, [M+NH4]+, [M+Na]+, [M+C2H3N+H]+, [M+H-H2O]+, [M+C3H8O+H]+, [M+C2H3N +Na]+, [M-C6H10O5+H]+, [2M+H]+, [2M+NH4]+, [2M+Na]+ in positive ion mode and [M-H]−, [M-H2O-H]−, [M+CH3COO]−, [M-C6H10O5-H]−, [2M-H]−, and [2M+CH3COO]− in negative ion mode. All lipid classes supported in the MS-DIAL version were used for the targeted lipids. The annotation was performed using the in silico spectral library of lipids, and the false positive annotations were manually curated by mass spectrometry experts. The principal component analysis and circus plots were executed in the R language with the packages of “prcomp” and “OmicCircos”. Our untargeted lipidomic data were discussed in terms of relative quantification, defined by lipidomic standard initiative (https://lipidomics-standards-initiative.org/), among dietary conditions in each tissue. All of the mass spectrometry data are available at RIKEN DROP Met .The targeted analysis data was analyzed using MultiQuant software . The chromatogram peaks were manually curated. The untargeted analysis data was analyzed using MS-DIAL software ,18 versiw/w) ARA-EE, EPA-EE, or DHA-EE for 2 weeks significantly decreased plasma cholesterol and decreased triacylglycerol levels as compared to the levels of the control group , and total fatty acids in mouse plasma. Dietary intake of 1% . According to the PCA score plots of the targeted lipidomic analysis, the oxylipins and their related molecules of plasma and tissues, except for the brain, were significantly affected by the dietary intake of PUFAs . FurtherThe levels of EPA and EPA-derived oxylipins were in fact lower than those of ARA and DHA in plasma and all tissues , while tThe ARA-, EPA-, and DHA-derived epoxides, which are known as CYP metabolites, were enriched in metabolic tissues such as the liver, kidney, and white adipose tissue. ARA-derived epoxides activate various signaling pathways, and elicit functional responses such as vasorelaxation and anti-inflammation . EPA-derThe ω3 oxylipins of EPA were increased following the dietary intake of DHA, while the amounts of ARA-derived oxylipins were decreased by ω3 PUFA dietary intake. Similar observations have also been reported in several other studies ,7,28. InWe used circus plots to display the result of untargeted lipidomics  for the A few differences in lipid profiles were observed in the mouse brain: free fatty acids (FA) of ARA, EPA, and DHA were affected by dietary intake . MoreoveO-alkenyl or O-alkyl chains were enriched in the kidney, spleen, small intestine, muscle, heart, and brain  [Untargeted lipidomics also revealed unique lipid features in the other tissues. The diacylglycerol (DAG) and TAG lipids were the most abundant species in the white adipose tissue, as has previously been described , and thend brain , while te (AGPS) .http://ds.biogps.org/?dataset=GSE10246&gene=225010). Importantly, these results indicate the importance of untargeted lipidomics, where unexpected and novel insights can be discovered in a data-driven manner.In addition, we found that the levels of phospholipids containing LA (LA-PLs) were substantially decreased after the dietary intake of ARA a, and prIn this study, we applied lipidomics techniques to monitor the comprehensive lipid profiles of mouse plasma and tissues, and their changes following the dietary intake of different PUFAs: ARA, EPA, and DHA. Importantly, we examined the effect of dietary supplementation of pure ARA, EPA, or DHA instead of dietary oils such as fish, linseed, and sunflower oils, and investigated the plasma and nine organs to better understand the common and/or differential lipid modulations in tissues. Mouse tissues, except for the brain, effectively incorporated the dietary PUFAs into glycerolipids and glycerophospholipids, and tissue levels of free fatty acids and oxylipins were well correlated with dietary PUFA intakes. It should be noted that our lipidomics data provided the lipidome result from “bulk” cells summing the heterogeneous nature in each tissue, and the results mainly reflect the lipid profiles of the major cell type or the major part of tissue. As for the brain, Arnold et al. reported similar results  when theARA, EPA, and DHA supplementation induced various changes in lipid profiles. Dietary PUFA intake substantially increased the levels of free PUFAs and oxylipins, as well as the incorporation of PUFAs in phospholipids and triglycerides. In contrast, this side-by-side study revealed the unique features of ARA, EPA, and DHA metabolism. The basal amount of EPA was much lower than that of ARA and DHA in the plasma and tissues. This may have been because EPA easily undergoes degradation and/or conversion in tissues ,44. DietIn addition, dietary DHA was selectively incorporated into a unique phospholipid BMP. BMP is known as an acyl chain positional isomer of phosphatidylglycerol (PG), in which one acyl moiety is incorporated into the glycerol polar head, and the structures can be distinguished by positive ion mode MS/MS spectra, while the MS/MS spectra are the same in negative ion mode c. The biPCA analysis  and circThe comprehensive lipidomics approach revealed many findings about dietary PUFA metabolism. Our untargeted/targeted lipidomics data incorporating 1026 unique lipid molecules  are poweThis experimental design to investigate the PUFA metabolisms is expandable for further nutrient researches, and we believe that this study is a fundamental step towards clarification of the effects of dietary PUFA intakes in mice. Our lipidomics study offers a comprehensive picture of dietary PUFA metabolism in different tissues, and could provide an opportunity for data-driven hypotheses and biological insights into the molecular mechanisms of how different PUFA balances affect human and livestock health and disease.


PAX1 DNA methylation testing was approved as an adjunct to cytology, in Taiwan, and is now undergoing registration trials in China. However, the function of PAX1 in cancer biology remains largely unknown. Here, we show that PAX1 inhibits malignant phenotypes upon oncogenic stress. Specifically, PAX1 expression inhibited the phosphorylation of multiple kinases, after challenges with oncogenic growth factors such as EGF and IL-6. Analogously, PAX1 activated a panel of phosphatases, including DUSP1, 5, and 6, and inhibited EGF/MAPK signaling. PAX1 also interacted with SET1B, increasing histone H3K4 methylation and DNA demethylation of numerous phosphatase-encoding genes. Furthermore, hypermethylated PAX1 associated with poor prognosis in cervical cancer. Taken together, this study reveals, for the first time, the functional relevance of PAX1 in cancer biology, and further supports the prospect of targeting multifold oncogenic kinase cascades, which jointly contribute to multiresistance, via epigenetic reactivation of PAX1.DNA methylation alteration, such as global hypomethylation and localized hypermethylation, within the promoters of tumor suppressor genes, is an important risk factor in cervical cancer. The potential use of DNA methylation detection, in cervical cancer screening or triage of mildly abnormal cytology, has recently been demonstrated. In particular,  Over 90% of deaths from cervical cancer occur in developing countries. However, even in developed countries, 13,240 and 62,356 annual new cases occur in the U.S and Southeast Asia, respectively2.Cervical cancer is the fourth-most common female cancer, and the fourth overall cause of cancer death, in the world. In 2018, there were an estimated 569,847 worldwide new cases, and 311,365 deaths3. While most HPV infections are transient, few cells remain persistently infected. However, HPV persistent infection is insufficient for cancer formation, as the cervical epithelia must accumulate other genetic and epigenetic changes to become fully malignant4. To that end, genetic alterations, such as karotypic abnormalities 7, amplification or activating mutations of oncogenes such as PIK3CA and EGFR, and inactivating mutations of tumor suppressor genes, such as PTEN, are all common contributors to cervical cancer11.The causative agent of cervical cancer is the human papillomavirus (HPV)12. During the past decade, genome-wide approaches, such as microarray and next-generation sequencing (NGS), have more comprehensively identified differentially methylated genes in cancers. Moreover, several clinical trials are examining DNA methylation as a biomarker for cancer detection, including cervical and colon cancer14, with a methylation test for the advanced disease being approved in 2016 by the U.S. Food and Drug Administration (FDA)15.In addition to genetic events, epigenetic alterations, such as DNA methylation, play a critical role in cancer development and progression. A hallmark of cancer cells is a global decrease in DNA methylation, and localized hypermethylation of the promoters of specific tumor suppressor genes18. Cytology-based cervical cancer screening has been used for nearly a century, albeit with unsatisfactory sensitivity and a 20% false positive rate19. And, while a quadravalent vaccine against HPVs 5, 11, 16, and 18 has reduced infection by those strains, it is minimally effective against other strains, and is little available in developing nations3. To more accurately detect cervical cancer, DNA methylation, as an adjunct for cytology or human papilloma virus (HPV) testing, is becoming the standard for molecular cervical cancer screening20. The methylation of the paired box-1 (PAX1) gene in cervical cancer was firstly reported in 200816. Serial validation studies performed worldwide have demonstrated its potential in cervical cancer molecular screening24. In 2016, the Taiwanese FDA approved the application of PAX1 methylation as an adjunct of conventional cytology in cervical cancer screening25. Despite this diagnostic association with cervical cancer, however, there remains a gap in the mechanistic understanding of the precise role of PAX1 in disease etiology.Similar to the above, DNA methylation as a biomarker for cervical cancer early detection is also promisingPAX gene family is named for the paired box DNA-binding domain that is critical for tissue development and cellular differentiation in embryos26. In cancer, however, the function of PAX genes is controversial and has not been well characterized27. Four PAX family subgroups exist, based on their structural domains: paired domain, octapeptide, and homeodomain26. PAX1 and PAX9 belong to subgroup I, which has a DNA-binding paired domain without a homeodomain, and these show functional redundancy during embryogenesis28. PAX9 is also amplified and highly expressed in lung cancer, and its knockdown reduced lung cancer formation in a xenograft study, supporting a role in oncogenesis29. On the contrary, PAX1 was aberrantly methylated, and downregulated, in cervical, oral, and ovarian cancer, suggesting that PAX1 is a tumor suppressor gene31. Nevertheless, the mechanism underlying the role of the PAX gene family in cancer biology has not been elucidated. Here, we present the first comprehensive, functional investigation of the mechanistic role of PAX1 in cancer.The in situ to invasive cancer. Consequently, we overexpressed PAX1 in two cervical cancer cell lines, HeLa and SiHa, and found that it did not significantly inhibit malignant phenotypes, including proliferation, migration, and invasion, in vitro, and tumor growth, in vivo , strongest in high-grade squamous intraepithelial lesion (HSIL), but weak in invasive cancer cells Fig. . These divo Fig. . Howeverion Fig. . Analogoion Fig. . Moreoveion Fig. . Togethe34. Furthermore, IL-6 expression has been correlated with poor prognosis in cervical cancer35. In this context, we observed PAX1 to inhibit IL6-mediated activation of several oncogenic signaling kinases, such as AMPK, P70 S6 kinase, AKT, β-catenin, and PRAS40 , and the SRC pathway  Fig. . In addiS40 Fig. .Figure 317, we tested whether PTPRR is a transcriptional target of PAX1. Those results demonstrated that PTPRR was indeed upregulated upon PAX1 expression, especially in the presence of EGF , without changing H3K9 trimethylation (a repressive modification), within the PTPRR, DUSP1, DUSP5, and DUSP6 promoters  of the non-tumor specimens and tumors were 0.05 and 0.58, respectively . In addition, hypermethylated PAX1 associated with poor progression-free survival (PFS), and poor overall survival (OS), in patients with cervical squamous cell carcinoma  concentration in cervix45, suggesting the indirect effects of HPV infection on epigenomics. Furthermore, the methylation of PAX1 was increased in the keratinocyte after immortalized by the full-length HPV1646. Together with the present study, the infection of HPV may cause the promoter methylation of PAX1 and result in the reduction of phosphatases’ expression. Therefore, hypermethylated PAX1 may derepress the oncogenic kinase cascade, thus promoting cancer progression.Our previous study demonstrated that the methylation of 48, while conversely, PAX3, PAX7, and PAX6 were reported to be oncogenic in embryonal rhabdomyosarcomas and retinoblastoma50. However, these reports were all based only on correlations between expression and malignant phenotypes, and the underlying mechanisms were not investigated. Although PAX1 is known to participate in neuronal development51, there have been no reports of a possible role in cancer biology. Previous studies regarding the PAX family and chromatin modifiers (histone deacetylases (HDACs) and histone lysine methyltransferases (HMTs)), in developmental biology, revealed interactions between PAX2 and MLL2/3 , in embryonic kidney cell lines, PAX5 and HMTs/HDACs, in mouse B cell development, and PAX7 and HMTs, in mouse myogenesis55. In cancer, PAX-interacting proteins remain largely unknown. Here, we demonstrate that PAX1 interacts with SET1B, but neither MLL1–MLL4 nor SET1A. Further, the cooperation between PAX1 and the SET1B histone H3K4 methyltransferase complex c activated the phosphatome and suppressed kinase signaling. These findings represent the first indication of PAX1-modulated epigenetic regulation of the kinase–phosphatase loop in cancer biology.Previously, the PAX family was reported to participate in development, but its role in cancer has been little studied. It was suggested that PAX2 and PAX5 play tumor suppressor roles in ovarian cancer and leukemia56. Additionally, compensatory oncogenic kinase cascades, such as the MAPK57, PI3K/AKT58, and SRC/AKT59 pathways, may render TKIs ineffective. In that regard, our study revealed that PAX1-activated phosphatases can inhibit multiple MAPK pathways and the SRC kinase. Consequently, reactivation of DNA methylation-silenced PAX1 may be a strategy to inhibit multiple oncogenic kinase pathways, which could reverse multi-pathway resistance to antineoplastic TKI drugs.In addition, resistance to targeted therapy in cancer is a notorious problem. For example, tyrosine kinase inhibitors (TKIs), designed to target mutant EGFR, have shown dramatic therapeutic efficacy in lung cancer; however, patients often develop resistance to therapyPAX1 expression, however, through DNA methylation, may disrupt this balance, leading to cancer development, supporting the further study of PAX1 DNA methylation as a biomarker for cervical cancer detection. Moreover, reactivation of PAX1 may overcome the failure of current target therapies targeting single kinases in cervical cancer treatment. We strongly contend that such epigenetic therapies, and consequent reactivation of tumor suppressors, are a promising strategy for overcoming the insidious problem of cancer chemo- and targeted therapy multiresistance.In conclusion, PAX1 is responsible for maintaining an on-and-off homeostasis between kinases and phosphatases, within the cervical epithelium. Loss of in situ (n = 21) and invasive carcinoma (n = 46). A standard protocol for immunohistochemistry was used with rabbit polyclonal anti-human PAX1 antibody . All tissue microarray slides were examined and scored by pathologists, according to the summation of the percentage of area stained multiplied by the stain intensity. All patients were diagnosed, treated, and had their tissues banked at the Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan. The final diagnosis was made by tissue-proven pathology rather than cytology except for controls. Exclusion criteria included pregnancy, chronic or acute systemic viral infections (except HPV infection), a history of cervical neoplasia, skin or genital warts, an immunocompromised state, the presence of other cancers, or a history of surgery to the uterine cervix. This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Tri-Service General Hospital. All of the patients signed informed consent forms before study.Paraffin-embedded cervical tissues of Chinese patients were retrieved from the Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taiwan. The tissue microarrays comprised histologically normal squamous epithelium (n = 19), mild dysplasia (n = 15), carcinoma 2 atmosphere for 4–5 h, after which the medium was removed and replaced with fresh culture medium. Cells were selected by antibiotic (G418) after 2 days in culture. Control cells were transfected with pCMV-Tag 2B vector only. The concentration of EGF (Thermo Fisher Scientific) in culture medium in this study is 50 ng/mL.The human cervical cancer cell lines HeLa and SiHa were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 medium containing 10% (w/v) fetal calf serum, penicillin at 100 U/ml, streptomycin at 100 μg/ml, and L-glutamine at 2 mmol/l . PAX1 (NM_006192) was constructed by inserting a full-length cDNA product into a pCMV-Tag 2B vector. The construct was then transfected into HeLa and SiHa cells with Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM I reduced-serum medium (Thermo Fisher Scientific) at 37 °C in a 5% CO43. Briefly, cells were seeded in 96-well plates at a density of 2000 cells/well. On days 0, 1, 2, 3, and 4, culture medium was replaced by fresh medium containing MTS reagent (100 µl medium plus 20 µl MTS reagent/well). After incubation at 37 °C for 2 hours, the absorbance at 490 nm was measured. Experiments were repeated four times.Cell proliferation was measured using MTS assay (Promega) as described in our previous study43. Cell migration was assessed using the scratch wound-healing assay. Cells were seeded into 6-well dishes until confluent and then wounded using a pipette tip. The migration area was photographed at 0 and 24 h. Cell migration was quantified by measuring the migrated area using ImageJ. Cell invasion was measured in the Transwell system with Matrigel (BD Bioscience). 2000 cells were seeded in the upper chamber in culture medium without serum. The same medium with serum was added to the lower chamber. Cells that had permeated the Matrigel and migrated to the bottom of the insert were fixed with methanol and stained with Giemsa’s azur eosin methylene blue solution (Merck) after 24 h. The cells in each chamber were photographed and counted.Cell migration assay and invasion assays were described in detail in our earlier study43. Total RNA was isolated by Qiagen RNeasy kit with DNase I digestion (Qiagen) and reverse-transcribed to cDNA using the Super Script III first-strand synthesis system for RT-PCR with Oligo(dT) as the primer (Thermo Fisher Scientific). qPCR was performed by Roche SYBR Green Real-Time PCR System (Roche). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. The comparative threshold cycles (Ct) method was used for relative quantification. All values are expressed as mean ± SD. The primers used in this study are shown in Supplementary Table RNA extraction, cDNA synthesis, and quantitative real-time PCR were performed as described in our earlier studies43. In brief, genomic DNA was extracted with the QIAmp DNA Mini Kit (Qiagen) and bisulfite-modified by CpGenome Fast DNA Modification Kit (Millipore). qMS-PCR was performed using SYBR Green Real-Time PCR System (Roche). Internal control gene was the type II collagen (COL2A). Samples with a crossing point-PCR-cycle (Cp) value for COL2A greater than 36 were defined as failure to detect. The DNA methylation level was assessed as the methylation index (M-index), 10 000 × 2([Cp of COL2A] − [Cp of gene]. The primers used in this study are shown in Supplementary Table qMS-PCR was performed as described by in our former studyChromatin immunoprecipitation (ChIP) assays were performed according to the protocol from Millipore (EZ-Magna ChIP G Chromatin Immunoprecipitation Kit). DNA was eluted with 100 ul elution buffer, and 4 ul was used per Q-PCR analysis. Quantitative PCR was performed using RT² SYBR Green qPCR Master Mixes (Qiagen) in a LightCycler 480 Real-Time PCR System (Roche). The antibodies used in the ChIP-PCR analysis were anti-Flag ; anti-trimethyl-Histone H3K4 (07-473), anti-trimethyl-Histone H3K9 (07-442) and anti-mouse IgG (12-371) were from Merck Millipore. The primers used in this study are shown in Supplementary Table 43. The antibodies used in the immunoblot analysis were: anti-phospho-ERK 1/2 (9106), anti-ERK 1/2 (9102) from Cell Signaling; anti-Set1A (sc-515590), anti-Set1B (sc-248564), anti-MLL1 (sc-377274), anti-MLL2 (sc-292359), anti-MLL4 (sc-517017), anti-TET2 (sc-136926), anti-TET3 (sc-139186) from Santa Cruz; anti-TET1 (GT1462) from GeneTex. Kinome was analyzed by Human Phospho-Kinase Array Kit  according to the manufacturer’s instructions. Cells used for kinome analysis were serum starvated for 16 h and treated with medium with EGF or IL-6 for 15 min.Cells used for immunoblot were serum-starvated for 16 h and treated with medium with EGF for 0, 5, 15 and 30 min. The process of immunoblot were described in detail in our earlier study6 cells from each stable line were resuspended in 0.1 ml PBS and injected subcutaneously into both flanks of each mouse. The mice were sacrificed at day 30. Tumors were removed from the mice and weighed. The protocol for this animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Defense Medical Centre, Taipei, Taiwan. All animal procedures and animal care were performed according to institutional animal research guidelines.Six-week-old nude mice were used in the tumorigenicity analysis. 10p-values > 0.05 in all of the samples. The average of signal  of each probe was calculated from duplication of expression data. Differentially expressed genes were identified as having a ≥1.25-fold change in the average expression in the PAX1-1 set and PAX1-2 set compare to EGF set .Total RNA samples were isolated and the absorbance ratio of A260/A280 and A260/A230 analyzed by Nanodrop 2000 (Thermo Scientific) should bigger than 1.8 and 1.0, respectively. The further qualification, the ratio of 28S/18S and RNA quality indicator (RQI) was analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies). Samples with ratio of 28S/18S is bigger than 1 and RQI is bigger than 7 were send to the core service unit  for whole-genome expression analysis. We measured gene expression profiles using Illumina HumanHT-12 v4 Expression Beadchip . The data had been deposited in the Gene Expression Omnibus (GEO) database (GSE102986). After quantile normalization, we removed the probes with detecting p-values < 0.05 were considered to be statistically significant. Methylomics profiles of cervical cancer cases, obtained from The Cancer Genome Atlas (TCGA), were based on data generated using Human Methylation 450 BeadChips (Illumina). The results were downloaded from the Broad Institute GDAC Firehose (http://gdac.broadinstitute.org/), and used in compliance with TCGA’s data usage policy.The Mann-Whitney U test, two-tailed, was used to compare data groups for cell proliferation, migration, invasion, and tumor formation. Standard deviations were used for error bars and various comparisons. Kaplan-Meier analysis and log-rank tests were used to calculate survival, and to compare differences between curves for progression-free survival (PFS) and overall survival (OS). Supplemental dataSupplemental Table S2