Cellular senescence represents a state of irreversible cell cycle arrest occurring naturally or in response to exogenous stressors. Following the initial arrest, progressive phenotypic changes define conditions of cellular senescence. Understanding molecular mechanisms that drive senescence can help to recognize the importance of such pathways in lung health and disease. There is increasing interest in the role of cellular senescence in conditions such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) in the context of understanding pathophysiology and identification of novel therapies. Herein, we discuss the current knowledge of molecular mechanisms and mitochondrial dysfunction regulating different aspects of cellular senescence-related to chronic lung diseases to develop rational strategies for modulating the senescent cell phenotype in the lung for therapeutic benefit. Cellular senescence is characterized by a permanent cell-cycle arrest triggered by various stimuli, including DNA damage to telomere shortening, genomic instability, epigenetic alterations, loss of proteostasis, and mitochondrial dysfunction  1]. Des. Des1]. Senescent cells are thought to have beneficial effects on repairing injured tissue and maintaining organismal integrity. The role of senescent cells in tumor suppression is also recognized. Under normal conditions, senescent cell burden is limited by removing excessive senescent cells via the immune system. However, with aging, impairment of the immune response results in accumulation of senescent cells that can exacerbate their effects leading to detrimental consequences, i.e., diseases of aging. Furthermore, there is now increasing evidence for different senescent cell phenotypes such that a shift towards detrimental, pro-inflammatory, pro-fibrotic senescent cells and SASP can occur with aging, contributing to disease.Compared to other organ systems where senescence, SASP and contributions to aging and diseases of aging have been substantially explored, there is relatively less data on the aging lung, and senescent cells in aging-associated lung diseases such as COPD, pulmonary fibrosis, and even asthma. Senescent cells do accumulate in aging lungs and can exacerbate lung diseases ,5,6 Fig. HoweverOne factor relevant to cellular senescence and aging that may be of importance to the lung is cellular stress, which promotes mitochondrial dysfunction, including mitochondrial oxidative stress, mitochondrial DNA (mtDNA) mutation, imbalance in mitochondrial fission and fusion, and alterations in mitochondrial respiration . MitochoIn this review, we discuss mechanisms of cellular senescence relevant to different aspects of the structure and function of aging lungs and to lung diseases, focusing on COPD and IPF. We review the influence of mitochondrial dysfunction in the context of cellular senescence and lung diseases. Finally, we summarize promising methods currently used to target senescent cells as a potential therapy to improve healthspan in the context of normal aging lung, and counteract lung diseases associated with aging. We appreciate that cellular senescence and SASP signaling is complex, and likely cell- and context-dependent. Accordingly, a review of these topics is necessarily brief and perhaps simple, but is relevant to the specific topic of aging lung and associated diseases. Cellular senescence was originally described by Hayflick and Moorhead , where tAmong cellular stressors, telomere shortening is now recognized as a hallmark of aging and senescence. Telomeres contain a repetitive nucleotide sequence of complementary double-strand DNA (5′-AGGGT-3′ and 3′-TCCCA-5) and wind up with a tail of a single-stranded DNA (5′-TTAGGG-3′) ,20,21. TSenescent cells are thought to support physiological functions during embryonic and postnatal development, tissue regeneration, and wound healing ,27,28,29CIP1 and p16Ink4a-Rb [1/S transitional phase distinguishing it from the quiescent phase, G0 [CIP1, to result in permanent arrest in the cell cycle [Cellular senescence is regulated by two signaling pathways that interact but are also independent: p53-p21Ink4a-Rb . Permanehase, G0 ,34,35,36ll cycle ,34,35,36In the nuclei, DDR foci originate in response to DNA double-strand breaks (DSB). A subnuclear focus and accumulation of DDR proteins such as p53-binding protein 1 (53BP1), histone variant H2AX phosphorylated at serine-139 residue (γ-H2AX), and Ataxia Telangiectasia Mutated (ATM) in the vicinity of chromosomal DNA double-strand reflect early molecular events of cellular responses to DSB ,37,38. DCIP1, which in turn inhibits cyclin-dependent kinases-2 (CDK2) [CIP1/Rb results in a permanent arrest in the cell cycle [ATM and ATR stabilize p53 by activating cyclin-dependent kinase inhibitor p212 (CDK2) ,40,43,462 (CDK2) ,31,43,47ll cycle ,31,35,43INK4a-ARF-INK4b locus [INK4a and INK4b locus encode for two cyclin-dependent kinase inhibitors, p16ink4a and p15ink4b, while ARF is associated with p14ARF in humans (p19ARF in mice) [INK4/ARF activates cyclin-dependent kinase inhibitor p16ink4a that selectively inhibits cyclin-dependent kinases-4 (CDK4) and cyclin-dependent kinases-6 (CDK6) [ink4a is meditated by the downstream signaling of p53-p21CIP1 [CIP1 upregulates earlier than p16 ink4a [ink4a appear to demonstrate a non-linear functional relationship. Another tumor suppressor that influences key roles during cessation of cell division is the 4b locus ,46,48,49in mice) ,46,48,496 (CDK6) ,31,32,436 (CDK6) ,51. Alth-p21CIP1 , it is b16 ink4a , giving Accumulation of senescent cells can be recognized by utilizing various methods in vitro and in vivo. For instance, upregulation of the transcriptional factors p53, p21, and p16, and SASP elements such as IL-6 and IL-8, are well-validated markers ,32,33,41Given the clinical impact of aging per se, and that of aging-associated lung diseases, it is important to identify biomarkers and signaling pathways in the context of senescence and its contributions to COPD  and IPF CIP1, p16ink4a, a robust release of SASP, positive staining for SA-βgal, TAF, and upregulation of anti-apoptotic signaling networks [Lung tissues from patients with COPD and IPF show hallmarks of senescent cells ,35,63,64networks ,67,68,69networks . In ROS-networks ,26.COPD is a major healthcare issue with a high morbidity and mortality rate . COPD isStudies have shown that endothelial colony-forming cells (ECFC) derived from COPD patients have increased expression of SA-βgal, p16, p21, and γ-H2AX compared to ECFC isolated from control group patients . In addiTobacco smoke can trigger cellular senescence via oxidative stress-mediated DNA damage. Conversely, targeting p16-positive cells can inhibit tobacco smoke-induced emphysema in mouse models . FurtherMultiple senescence signaling pathways may be involved in COPD, and activated in patients with a history of tobacco smoking and/or E-cigarette vaping ,63,80,81Another signaling pathway that plays a role in cellular senescence and COPD is p38 MAPK ,93. IncrOverall, these data provide evidence of senescence in COPD, and the potential involvement of multiple signaling pathways that could contribute to at least the inflammatory aspects of this disease. Of note, these signaling pathways are also well known to contribute to cell proliferation and remodeling and may thus be relevant to these aspects of COPD as well.IPF is a life-threatening chronic lung disease with poor prognosis and survival. IPF is characterized by scarred lungs associated with hyperproduction of ECM proteins ,97. In tMitochondria are essential in eukaryotic cells for maintaining cellular homeostasis and function. Mitochondria regulate numerous cellular activities such as metabolism, replication, differentiation, senescence, and apoptosis . MitochoMitochondrial dysfunction can contribute to cellular senescence. For instance, gradual alterations of mitochondrial DNA (mtDNA mutations), variations in mitochondrial fission and fusion, elevated mitochondrial ROS production, and changes related to mitochondrial morphology  can all play a role ,103,104.Induction of senescence via disruption of mitochondrial function results in a distinctive SASP portfolio compared to senescence induced by genotoxic stress . This miOverproduction of mitochondrial ROS is also an important player that causes DNA damage and results in DDR. The circle of ROS-DNA damage involves phosphorylation of DDR kinase ATM and Akt . ATM actPatients with COPD show changes in proteins that influence oxidative stress. PTEN-induced protein kinase-1 (PINK1), a mitochondrial stress protein marker that accumulates on the outer membrane of damaged mitochondria, is found to be elevated in COPD ,113. On Unlike the nuclear genome, the mitochondrial genome is a ~16.6 kb circular DNA molecule encoding subunits of polypeptides ,106,121.Emerging evidence illustrates that alterations of mtDNA are associated with electron transport efficiency. These changes are due to mutations in encoded subunits of polypeptides making up mitochondrial respiratory complexes that serve as primary sources of ROS. Depletion of mtDNA has been associated with premature aging and multiple chronic diseases . Using aInk4a positive cells extends lifespan by decreasing growth hormone signals has helped to excite the field regarding the therapeutic potential of targeting senescent cells [Demonstrating that suppressing the accumulation of p16nt cells . Subsequnt cells . The ident cells ,132. Sennt cells ,68,132. nt cells . Human tnt cells . Recentlnt cells .Another approach to eliminating senescent cells is targeting the higher mitochondrial potential in senescent cells . MitochoBesides senolytics, strategies to develop drugs that target signaling pathways critical to senescent cells have investigated using senostatic (senomorphic) drugs ,127. UnlFurthermore, the widely prescribed and FDA-approved anti-diabetic drug metformin shows promise in the context of senescence ,151,152.Cellular senescence is a hallmark of aging lungs and aging-associated lung diseases. While senescent cells have beneficial roles, with aging, an enhanced senescent cell burden and a pro-inflammatory and pro-fibrotic SASP can contribute to pathophysiology of diseases such as COPD, IPF, and even asthma. Senescence can be activated by multiple upstream mechanisms, and conversely can involve multiple, interactive downstream pathways. SASP effects on naïve cells can be cell and context dependent with multiple effects on remodeling relevant to lung disease. Thus, there is substantial enthusiasm in exploring the use of senolytics and senostatics in eliminating senescent cells or modulating SASP effects towards therapy for lung diseases. Here, what remains to be understood is the differences in senescent cell and SASP phenotypes in different diseases, further complicated by likely cell-type differences in senescence in the lung. Appreciating that aging may differentially influence different cell types in the lung, the contribution of senescence remains to be understood in a cell-specific fashion. Thus, understanding the relative roles of resident cells such as bronchial and alveolar epithelium, smooth muscle and fibroblasts in senescence and its downstream impact is critical. In this regard, the relative roles of different senescence pathways may also show cell dependent variability that remains to be understood. Thus, modulation of senescence as therapy may be a reality for multiple aging-associated lung diseases; the potential for future research is high.


DYRK1A gene, which encodes dual-specificity tyrosine-phosphorylation-regulated kinase 1A. Here, we report a case of MRD7 that was diagnosed using Face2Gene and whole-exome sequencing (WES). A 22-year-old man presented with microcephaly, intellectual disability, slender body, long slender fingers, and facial dysmorphisms. He was previously diagnosed with Cornelia de Lange syndrome (CdLS) at four years of age. However, his CdLS clinical diagnostic score was low at 22 years of age. The Face2Gene application introduced several candidate diseases including MRD7. Finally, by utilizing WES and Sanger sequencing analysis of cloned cDNA, we identified a novel heterozygous duplication variant ) in the DYRK1A gene, which introduces a premature stop codon. This report provides more information about the phenotypic spectrum of a young adult patient with MRD7. Face2Gene helped us introduce candidate diseases of the patient. Registering further genetically confirmed cases with MRD7 will improve the accuracy of the diagnostic recommendations in Face2Gene. Moreover, WES is a powerful tool for diagnosing rare genetic diseases, such as MRD7.Intellectual developmental disorder, autosomal dominant 7  is a rare disease characterized by microcephaly, intellectual disability, speech delay, feeding difficulties, and facial dysmorphisms. This disorder is caused by pathogenic/likely pathogenic variants of the  DYRK1A gene, which encodes dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), are responsible for MRD7. DYRK1A has a serine/threonine kinase with numerous proteins involved in cell proliferation, neuronal morphogenesis, synaptogenesis, and synaptic function, and it is essential for brain development and function . All samples were diluted and pooled for multiplexed sequencing on NovaSeq 6000 . Bioinformatic analysis was performed by cBioinformatics, Inc. . Reads were aligned to the human genome assembly GRCh38/hg38 using Burrows-Wheeler Aligner Enrichment (v0.7.17) -13. FiltDYRK1A gene , Clinvar (http://www.ncbi.nlm.nih.gov/clinvar), iJGVD (https://jmorp.megabank.tohoku.ac.jp/ijgvd/), and HGMD (http://www.hgmd.cf.ac.uk/ac/index.php). This variant was classified as being pathogenic according to the American College of Medical Genetics and Genomics criteria [NIPBL, SMC1A, SMC3, RAD21, HDAC8, and ANKRD11), AS (UBE3A), WKOS (SIN3A), and RTS (CREBBP and EP300).As a result of the abovementioned analysis, the patient showed a novel heterozygous duplication variant ) in the coding region of exon 7 of the criteria . In thisDYRK1A gene, i.e., c.848dup, p.(Asn283Lysfs*6) in our patient and diagnosed him with MRD7. The variants associated with MRD7 are widely distributed in the DYRK1A gene [DYRK1A gene, and affecting its catalytic function [DYRK1A gene in MRD7 are heterozygous, which leads to haploinsufficiency for the kinase activity.We identified a novel de novo heterozygous variant in the , p.Asn28Lysfs*6 i, p.Asn28Lysfs*6 ifunction . The varOur patient had thick eyebrows and long eyelashes, similar to a patient with CdLS, but he had a slender body, long slender fingers, and friendly behavior, unlike patients with CdLS .In the preliminary step of performing WES, we used the Face2Gene application, which introduced the following candidate diseases; AS, WKOS, RTS, and MRD7. These diseases and CdLS mostly have autosomal dominant inheritance, caused by de novo pathogenic variants, and share the following phenotypes: intellectual disability, facial dysmorphisms, speech delay/absence, behavior issues, microcephaly, feeding difficulty, sleep disorder, seizure, eye involvements such as strabismus, and skeletal anomalies Table  1,9,16--16-18.Contrarily, each disease has characteristic dysmorphic facial features Table .MRD7 has facial features such as prominent ears, deep-set eyes, upslanted palpebral fissures, a short nose with a broad nasal tip, and retrognathia. Given that the reported prevalence of MRD7 is less than 1/1,000,000 , which iIn addition to the typical dysmorphic symptoms of MRD7, our patient presented with distinct long slender fingers Figure . To dateDYRK1A gene was identified in our patient. Although WES remains expensive and its use in clinical practice is limited, its accessibility has expanded in recent years. Therefore, we believe that WES is a powerful and recommended tool for diagnosing rare genetic diseases from the initial steps.Finally, by utilizing WES, variant of the DYRK1A gene by utilizing WES. Face2Gene helped introduce candidate diseases for the patient. Registering further genetically confirmed cases with MRD7 will improve the accuracy of diagnostic recommendations in Face2Gene. WES is a powerful tool for diagnosing rare genetic diseases, such as MRD7.This report provides more information about the phenotypic spectrum of a young adult patient with MRD7. We identified a novel variant in the


PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti–PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti–PD-1 antibody treatment improved the health span of podocytes. Administering the same anti–PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2).  The US population is aging, and the number of Americans aged 65 and older will more than double over the next 40 years (US Census Bureau); Eurostat forecasts that 28% of Europeans will be older than 65 years by 2060. As life expectancy increases, the impact of advanced age on kidney health and function is becoming an increasingly important medical and socioeconomic factor. Glomerular filtration rate (GFR) declines after age 40 by 0.8%–1.0% per year , which i3 in young kidneys to less than 100 per 100 cm3 by 70–80 years of age. In fact, increased age is independently associated with both absolute and relative podocyte depletion  and aged (n = 15) mice, in which mRNA expression was assayed by quantitative PCR performed on podocyte fractions sorted by magnetic-activated cell sorting . PD-1 increased 6.7-fold , PD-L1 increased 2.3-fold , and PD-L2 increased 17.3-fold  in aged podocytes. In situ hybridization confirmed the increase in PD-1 mRNA in aged glomeruli  and clinical parameters for glomerular aging and function. PDCD1 expression in humans increased with age  . Uninjected, 4-month-old mice were used as young age controls. Treatments had no major impact on mortality, body weight, or kidney function as measured by urinary albumin/creatinine ratios, blood urea nitrogen, serum creatinine, and blood soluble urokinase plasminogen activator receptor  once weekly for a total of 8 weeks , which was partially restored in the age-matched mice injected with aPD1ab (P = 0.0315)  . This wa 0.0315)    and the  0.0315) , 18  . Both we 0.0315) . To asse 0.0315)  was perf 0.0315) .We next analyzed the other resident glomerular cell types. PECs marked by the expression of PAX8 were decreased with age, but in contrast to podocytes, their numbers were not restored by aPD1ab treatment . However+ nuclei × 106 μm3, P < 0.0001), but was unchanged with aPD1ab  (8 integrin), was increased in aged IgG2a control mice compared with young mice, a trend that was also not changed by aPD1ab . GEN density, identified by nuclear staining for the ETS transcription factor ERG , 37, was= 0.902) , A–D. Ye= 0.902) –40, was = 0.902) , E–G. Mey aPD1ab , H–K. Fi1ab mice , L–O.Together, these data demonstrate that interrupting PD-1 signaling can partially reverse the glomerular aging phenotype and improve podocyte life span with respect to cell number, function, and ultrastructure. While it did not impact the numbers or life span of the other glomerular cell types, interfering did have beneficial impacts on the age-dependent activation of PECs and GENs and injury to the tubular epithelium.PD-1 and its ligands. In podocytes, aPD1ab did not alter the mRNA expression of PD-1 and PD-L1 measured by quantitative reverse transcriptase PCR, but lowered PD-L2  did not change in aged mouse podocytes (not shown). Finally, consistent with at least partial suppression of the PD-1 pathway in vivo, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that several downstream targets of the PD-1 signaling pathway were reduced by aPD1ab, including Cd4, Cd28, Ptpn6 (Shp1), Ptpn11 (Shp2), Pik3cd (Pi3k), Rasgfp1, and Nfat . Similarly, of the upregulated transcripts, about a third  were downregulated in aged versus young podocytes (P = 1 × 10–32). Six hundred forty-nine genes were upregulated in aged aPD1ab-injected podocytes compared with aged IgG2-injected podocytes. Together, these data suggest that more than 40% of the genes regulated by PD-1 are part of the natural podocyte aging process.Based on these data and to obtain a better understanding of the mechanism(s) responsible for the reversal of glomerular aging by aPD1ab treatment, we performed mRNA-Seq analyses from podocyte and non-podocyte cell fractions from each individual mouse from each of the groups . Principal component analysis showed excellent clustering of both podocytes and non-podocyte cell fractions in the individual treatment groups . A totalActn4, Cdkn1, Col4a, Fat1, Lamb2, Nphs1, Nphs2, Podxl, and Synpo) was reduced in aged IgG2a-injected mice, but significantly upregulated upon injection with aPD1ab (Nphs1) and synaptopodin (Synpo)  .Vegfa mRNA levels were 4.2-fold lower in aged podocytes than in young podocytes, but were 3.4-fold higher in podocytes from aged aPD1ab-injected mice , Osr2 (7-fold), and Foxc2 (2-fold). The restoration of the podocyte gene regulatory network was further underscored by a VIPER  analysis, which identifies transcription factor activities based on the expression of their downstream targets , the transcriptional activity of 4  was increased while 6 were decreased  upon aPD1ab injection.Finally, we investigated the transcription factor drivers of these expression changes in podocytes. Compared with control aged IgG2a-injected mice, several podocyte transcription factors were expressed at significantly higher levels in aged mice injected with aPD1ab. These included  targets , 42. As Taken together, these data show that the expression levels of canonical podocyte genes and the function and transcriptional regulation required for their health span are partially restored with aPD1ab.P < 0.0001 vs. control) and reduced upon aPD1ab injection   in aged control mice and reduced in the aPD1ab-injected group. Interestingly, we also observed changes in pyroptosis, another form of podocyte death (The ectopic expression of PD-1 in podocytes in vitro induced high levels of apoptosis that were in part reduced by inhibition of caspase-3 . Moreove 0.0001) . This wate death .P < 0.0001 vs. young) and was completely restored in aPD1ab-injected aged mice (P = 0.001 vs. aged control)  and autophagy , 46. Thecontrol) . TogetheWe have recently reported that the podocyte aging phenotype is caused by changes in autocrine and paracrine signaling molecules . Thus, wNext, we performed a gene set enrichment analysis of the mRNA-Seq data to obtain a deeper understanding of the molecular pathways altered upon aPD1ab treatment. The podocytes from mice injected with aPD1ab displayed decreases in T cell activation, positive regulation of protein kinase activity, mitotic nuclear division, DNA repair, and calcium ion transport and marked increases in many metabolic pathways, such as oxidative phosphorylation, amino acid metabolism, organic acid transport, mitochondrial translation, and glucose metabolism , A and BPD-1 inhibitors are part of the wider group of immune checkpoint inhibitors (ICPIs), and their use is connected with the occurrence of acute kidney injury (AKI) and, in particular, tubulointerstitial nephritis , 48. WhiInterference with PD-1 signaling in mice is a systemic treatment, and thus its effects on cellular aging would not be restricted to the glomerulus. Indeed, tubular epithelial cell senescence assessed by SA-β-gal staining and immunostaining for the senescent proteins p16 and p19 was lowered upon aPD1ab injections . In lineFinally, by extending our analysis to the liver, we observed a similar anti-aging effect outside of the kidney. PD-1 protein was present in the liver of aged mice, but not young mice . MoreovePDCD1 mRNA expression in human microdissected glomeruli from patients undergoing indication kidney biopsies enrolled in the Nephrotic Syndrome Study Network (NEPTUNE). PDCD1 expression was significantly higher in patients with nephrotic-range proteinuria versus sub-nephrotic-range proteinuria  Aged mouse and human kidneys displayed higher levels of PD-1 immunostaining in epithelial cells , but not in glomerular mesangial and endothelial cells. (b) Ectopic expression of PD-1 in cultured podocytes triggered an apoptotic response that was partially caspase-3 dependent. (c) Interfering with PD-1 signaling in mice using a neutralizing anti–PD-1 antibody reduced senescence markers in the kidney glomerulus, tubular epithelial cells, and the tubulointerstitium as well as the liver. (d) While interfering with PD-1 signaling reversed some aspects of aging, it did not reverse all effects. (e) Finally, blocking PD-1 signaling improved podocyte life span in an experimental model of FSGS.Many tumors evade being detected by suppressing T cell immune responses upon activation of negative regulatory pathways called immune checkpoints. The latter include programmed cell death protein 1 (PD-1) and its ligands, PD-L1 and PD-L2. The development of checkpoint inhibitors for the PD-1 pathway restores T cell recognition and killing of cancer cells , 31, 54.Nphs1, Nphs2, Synpo, and Vegfa) or signaling networks , but entire podocyte gene regulatory networks as illustrated by the VIPER analysis. Likewise, PD-1 inhibition improved the senescent and inflammatory phenotypes present in aged podocytes, which improved podocyte health span. Restoring these probably has a long-lasting effect above simply preventing podocyte death. In this regard, the observation that aPD1ab injections restored normal metabolic pathways such as oxidative phosphorylation, glycolysis, and lipid metabolism further supports the pro–health span effects of PD-1 signaling inhibition. There may also be other consequences that improve the health of podocytes. For example, we have recently reported that aging podocytes exhibit not only autocrine loops, but also paracrine loops, in which podocytes signal to other cell types in the glomerulus . These data are consistent with the interpretation that the anti–PD-1 antibody improves rather than harms the tubulointerstitium. Similarly, with respect to the glomerulus, individual case reports of glomerulopathies as a complication of anti–PD-1 immunotherapy exist , these glomeruli undergo rapid global glomerulosclerosis due to massive podocyte catastrophe  were not changed upon aging either. This is in agreement with several reports showing that the majority of mouse strains do not show appreciable microalbuminuria, nor do they develop proteinuria as they age –75. In fastrophe , 81. Yetastrophe .In summary, the present study shows that the PD-1 pathway is increased in aged mouse podocytes and in aged human glomeruli, in podocytes of young mice and humans with FSGS, and in aged mouse livers. Increased expression of the PD-1 signaling pathway in human glomeruli predicts poor human kidney outcomes. Mechanistically, increased PD-1 levels in podocytes cause apoptotic loss (shortened podocyte life span) and reduce their health span. These events and glomerulosclerosis are partially reversed by inhibition of PD-1. These results are consistent with PD-1 being a critical mechanism contributing to glomerular damage in the aged kidney and following kidney injury.https://yuliangwang.shinyapps.io/podocyte_PD1_experiment/The methods are described in detail in JWP, DGE, MB, OW, JCV, and SJS designed research studies. JWP, NK, YW, DGE, YZ, UT, CJL, CO, CP, HSP, and SA conducted experiments. JWP, NK, YW, DGE, YZ, UT, CJL, CO, CP, HSP, JCV, and SA acquired data. JWP, NK, YW, DGE, CO, MB, OW, SJS, CP, HSP, JCV, and SA analyzed data. JWP, NK, DGE, CO, MB, OW, and SJS wrote the manuscript. AC and SA provided material. SA performed clinicopathologic evaluation of human kidney biopsies.


TLR4, NOD2, CXCL8, and OAS2. Moreover, the alternative splicing (AS) pattern of transcriptional sequence differences between healthy cows and subclinical mastitis cows suggested a molecular mechanism of mastitis resistance and susceptibility. A total of 2,212 differential alternative splicing (DAS) events, corresponding to 1,621 unique DAS genes, were identified in both groups and significantly enriched in immune and inflammatory pathways. Of these, 29 DAS genes were subject to regulation by 32 alternative splicing SNPs, showing diverse and specific splicing patterns and events. It is hypothesized that the PIK3C2B and PPRPF8 splice variants associated with AS SNPs (rs42705933 and rs133847062) may be risk factors for susceptibility to bovine subclinical mastitis. Altogether, these key blood markers associated with resistance to subclinical mastitis and SNPs associated with alternative splicing of genes provide the basis for genetic breeding for resistance to subclinical mastitis in cows.Subclinical bovine mastitis is a pathogenic infection of the breast characterized by a marked decrease in milk production and quality. As it has no obvious clinical symptoms, diagnosis and treatment are challenging. Therefore, searching for biomarkers in cows’ peripheral white blood cells is valuable for preventing and treating subclinical mastitis. Thus, in this study, the transcriptome of peripheral blood from healthy and subclinical mastitis cows was characterized to find the regulatory signatures of bovine subclinical mastitis using RNA-seq. A total of 287 differentially expressed genes (DEGs) and 70 differentially expressed lncRNAs (DELs) were detected, and 37 DELs were documented near known Quantitative Trait Loci (QTL) associated with the mastitis of cows. Bioinformatic analysis indicated that lncRNAs MSTRG25101.2, MSTRG.56327.1, and MSTRG.18968.1, which are adjacent to the SCS QTL and SCC QTL, may be candidate lncRNAs that influence the pathogenesis of mastitis in cows by up-regulating the expression of genes  Staphylococcus aureus (S. aureus), Streptococcus agalactiae, and Mycoplasma spp as well as environmental pathogens such as Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes are the main causes of mastitis in cattle  was most likely responsible for chondrodysplasia during early fetal development in cows. Similarly, the SNP g.5880C > T, SNP g.18174A > G, and SNP g.10766T > C might affect the binding with splicing-related factors, subsequently causing the production of aberrant splice variant HMGB3-TV1 . A further amount of blood was used for genotyping at GGP Bull 150K BeadChip .A Sysmex K-4500 automated hematology analyzer  was used to test 24 hematological parameters, including White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (HGB), Red blood cell-specific volume (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet count (PLT), Neutrophil Ratio (NETU%), Neutrophil count (NETU#), Lymphocyte Ratio (LYMPH%), Lymphocyte count (LYMPH#), Monocyte Ratio (MONO%). Monocyte count (MONO #), Eosinophil Ratio (EO%), Eosinophil count (EO#), Basophil Ratio (BASO%), Basophil count (BASO#), Platelet Distribution Width (PDW), Mean Platelet Volume (MPV), Red cell distribution width - stand error (RDW-SD), Red blood cell distribution width - coefficient of variation (RDW- CV), Platelet-Large Cell Ratio (P-LCR), Platelet cubic metric distribution width (PCT).Total RNA of peripheral white blood cells for each cow was extracted with the TRIzol reagent  following the manufacturer’s procedures. The concentration of RNA was estimated using the NanoDrop 2000  and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2,100 system was used to assess RNA integrity. The RNA quality was checked for contamination and degradation by a 1% agarose gel. Then, qualified RNA was used to construct RNA-seq libraries. Finally, cDNA libraries were sequenced with 150 bp paired-end reads from Illumina NovaSeq 6,000 platform .The quality of the raw data was evaluated with FastQC version 0.11.8. The clean data were obtained by using Trimmomatic version 0.38 to filter out reads with adapter sequences and low-quality reads from the raw reads . SpecifiIn the current study, strict filtering conditions were set on the annotated transcripts to obtain novel lncRNA transcripts. Firstly, transcripts shorter than 200bp and less than two exons were discarded. Then, transcripts with the class codes “i" , “u" , and “x"  were preserved. Finally, protein-coding potential predictions were performed using the Coding-Non-Coding Index (CNCI) , ORF Lenp-value < 0.05 and log2 (fold change) > 1.5.Transcripts were quantified using HTSeq-counts version 2.0.1 and results were expressed as read counts . Subsequcis- and trans-target genes of lncRNAs. Protein-coding genes near the transcript positions of lncRNAs (100k upstream and downstream) were selected as cis-target genes of lncRNAs with the BEDTools version2.1.2  and the effects of all SNPs were annotated by ensemble Variant Effect Predictor (VEP) to screen for SNPs associated with alternative splicing. Subsequently, SNPs were localized to target genes based on positional information to identify candidate SNPs associated with alternative splice genes.http://kobas.cbi.pku.edu.cn/kobas3/).Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and gene ontology (GO) analysis of DEGs, target genes of DELs, and DAS genes were performed on the KOBAS 3.0 online website  and Student’s RNA-seq was performed on six cDNA libraries using the Illumina NovaSeq 6,000 platform. The total reads mapped to the bovine genome have been summarized in The identification of novel lncRNAs transcripts was followed by a series of criteria. Upon transcript filtering and protein-coding potential prediction, 5,510, 5,664, 5,296, and 5,165 lncRNAs transcripts were predicted by CPC, CNCI, LGC, and PLEK, respectively . Of partIn the peripheral blood of healthy cows and subclinical mastitis cows, a total of 6,365 lncRNAs were screened, including 1,941 known lncRNAs transcripts. These transcripts were widely distributed in all chromosomes of the bovine genome, and the highest number of transcripts was on chromosome five . lncRNAsp-value < 0.05, log(fold change) > 1.5)  and 70 differentially expressed lncRNAs (DELs) were identified between the two groups , which contained 55 molecular compositions (CC), 190 biological processes (BP), and 56 molecular functions (MF). Furthermore, KEGG results of DEGs showed 17 enriched signaling pathways   . Among t < 0.05) . The gen pathway . Notablyegulated  in subclDCK, ARGE, VNN2, and OAS2 were significantly correlated with White Blood Cell (WBC) count (109/L)  (GBP6 and TBXAS1 were significantly correlated with the Monocyte Ratio (MONO %) in peripheral blood   . Similar < 0.05) .cis-target genes and 124 trans-target genes were predicted   . Most of pathway . Notably pathway .In this study, the functions of DELs were further annotated by comparing the genomic positions of DELs and QTL-related regions in Cattle QTLdb. The results showed that 245 DELs were located adjacent to 481 QTLs in Cattle QTLdb . These Qcis-and trans-target genes. In this study, five signaling pathways were shared by DEGs and DELs, including NOD-like receptor, S. aureus infection, cell adhesion, B cell receptor, and IL-17 signaling pathways, involving 18 DEGs, 37 DELs, and 7 DEGs. As shown in OAS1Y, GBP4, OAS2, FCAR, SELPLG, DCK, and ICAM3. This result implied the important role of lncRNAs in bovine subclinical mastitis.Previous studies have shown that lncRNAs may impact disease resistance of livestock and poultry by regulating the expression levels of their (AS) events were found between healthy and subclinical mastitis cows (FDR < 0.05), including 2,212 differential alternative splicing (DAS) events. Five patterns of AS consist of exon skipping (ES), retained intron (RI), mutually exclusive exon (MXE), alternative 5′ splice site (A5SS), and alternative 3’ splice site (A3SS). A total of 2,212 events were segregated into 82.59% ES events, 11.66% MXE events, 2.44% A3SS and A5SS events, and 3.30% RI events  on all SNPs, 51.97% of the SNPs were intronic variants, 34.00% were intergenic variants, and only 0.02% of the variants were associated with alternative splicing , which iPIK3C2B had the genotype AA in healthy cows and AG in subclinical mastitis cows. Furthermore, SNP rs133847062 on chromosome 19 at 22,777,382bp of gene PRPF8 had the genotype AC in healthy cows and AA in subclinical mastitis cows . As shown in S. aureus infection pathway were also significantly enriched in the available studies of bovine mammary epithelial (BME) cells  cells , suggestNOD2, CXCL8, OAS2, and TLR4 were differentially expressed in the blood transcriptome of subclinical mastitis cows and involved in the NOD-like receptor, Toll-like receptor pathway and were identified as key candidates for this study. TLR4 and NOD2, as key PRRs, and their recognition of MAMP have been shown to be a key event in the development of mammary inflammation  . These Pammation . CXCL8 iME cells , induces markers .SLC40A1 (SLC11A2 (ACSL6, GCLC (ACSL5, CYBB (VDAC3 (, and FTH1 (SLC40A1 was up-regulated in subclinical mastitis cows compared to healthy cows (SLC40A1, ACSL6, GCLC, ACSL5, CYBB, VDAC3, and FTH1 were also DAS genes. Of these, the SLC40A1 presented SE and RI events and the FTH1 showed four alternative splicing patterns, including SE and RI, MXE, and A3SS. It is hypothesized that alternative splicing may influence the development of subclinical mastitis by altering the alternative spliced pattern of genes associated with ferroptosis.Ferroptosis is a regulatory form of iron-dependent cell death , and is SLC40A1 , SLC11A2(SLC11A2 , ACSL6, L6, GCLC , ACSL5, L5, CYBB , VDAC3 (B (VDAC3 , and FTHand FTH1  were invand FTH1 . Among tthy cows . MoreoveS. aureus (RNA splicing is an important regulatory mechanism that links trait-associated variants and complex traits. Alternative splicing generates diverse transcripts with significant roles in disease resistance  and the . aureus . ConsequJAK2 were dramatically associated with SCC and SCS, and SNP rs43046497 on the intron nine of the gene STAT5A was significantly linked to IL-6 (PIK3C2B and PRPF8 (PIK3C2B and PRPF8 were simultaneously regulated at the transcriptional level, indicating independent regulation of genes by alternative splicing, while the PPI interaction network showed that these two alternative splice genes interacted with subclinical mastitis-related genes at the protein level (Mutations affected by pre-mRNA splicing account for more than 15% of disease-causing mutations . Previou to IL-6 . These r to IL-6 , of whicnd PRPF8 . Notablyin level .PIK3C2B belongs to class II of the PI3Ks family and performs a pivotal role in the control of membrane trafficking and intracellular signaling (PRPF8 is critical for breast cell survival and has potential prognostic value in breast cancer (PIK3C2B and PRPF8 to exhibit exon skipping, which in turn affects the resistance of cows to subclinical mastitis, but the detailed regulatory mechanism remains to be verified in subsequent future studies.Among them, ignaling . The splt cancer . What ist cancer , which at cancer , and thet cancer . TherefoTLR4, NOD2, CXCL8, and OAS2 are key components involved in the host immune response to bovine mastitis and that the lncRNAs MSTRG25101.2, MSTRG.56327.1, and MSTRG.18968.1 adjacent to the SCC QTL and SCS QTL may potentially regulate expression of these candidate genes. In addition, characterization of peripheral white blood cell transcriptome alternative splicing events indicated that specific types of alternative splicing events appear to be associated with mastitis resistance in cows and that single nucleotide polymorphisms in the DAS genes PIK3C2B and PPRF8 may be risk factors for mastitis susceptibility in cows. The genes and lncRNAs highlighted in this study could serve as expression biomarkers for mastitis that can be used for genetic improvement of dairy cattle for resilience to mastitis. In conclusion, our study provides a basis for further investigation of the molecular mechanisms of resistance and susceptibility to subclinical mastitis in cows.Our data suggest that genes


Myeloid Sarcoma with monocytic differentiation is rare and quite likely is missed by surgical pathologists. However it is frequently misdiagnosed because of its non-specific imaging and histological pattern.FLT3 and PTPRB, which are associated with myeloid sarcoma, and in TP53, CD44, CD19, LTK, NOTCH2, and CNTN2, which are associated with lymphohematopoietic tumors and poorly differentiated cancers.We report the case of a 64-year-old woman with gastric primary myeloid sarcoma with monocytic differentiatio. Upper endoscopy revealed a neoplastic growth at the junction of the lesser curvature and gastric antrum. Except for a slightly increased peripheral monocyte count, no abnormalities were found on hematological and bone-marrow examination. Gastroscopic biopsy showed poorly differentiated atypical large cells with visible nucleoli and nuclear fission. Immunohistochemistry showed positive CD34, CD4, CD43, and CD56 expression, and weakly positive lysozyme expression. Immune markers for poorly differentiated adenocarcinoma, malignant melanoma, and lymphohematopoietic-system tumors were negative. The final diagnosis was myeloid sarcoma with monocytic differentiation. Chemotherapy did not shrink the tumor, so, radical surgery was performed. Although the tumor morphology did not change postoperatively, the immunophenotype did. CD68 and lysozyme expression (tumor tissue markers) changed from negative and weakly positive to strongly positive, AE1/3 expression  changed from negative to positive, and CD34, CD4, CD43, and CD56 expression (common in naive hematopoietic cell-derived tumors) was greatly attenuated. Exome sequencing revealed missense mutations in FLT3 gene mutations. We hope that the above results will improve our understanding of this rare tumor.We diagnosed myeloid sarcoma with monocytic differentiation after excluding poorly differentiated adenocarcinoma, common lymphohematopoietic-system tumors, epithelioid sarcoma, and malignant melanoma. We identified that the immunophenotypic of patient had alterations after chemotherapy, and The online version contains supplementary material available at 10.1186/s13000-023-01311-1. 1. We used exome sequencing to detect and report the genetic changes in a case of extramedullary gastric primary myeloid sarcom with monocytic differentiationa.2. Immunophenotypic alteration of tumor cells occurred after chemotherapy, suggesting that chemotherapy drugs induced tumor differentiation.The online version contains supplementary material available at 10.1186/s13000-023-01311-1. FLT3 and PTPRB genes, which are associated with myeloid sarcoma; missense mutations in LTK (associated with poorly differentiated cancer), NOTCH2 (associated with diffuse large B-cell tumor), and CNTN2 (associated with T-cell lymphoma); and a frameshift deletion mutation in the oncogene TP53. We also reviewed the relevant literature from 1990 to 2021, and identified 10 cases of myeloid sarcoma with monocytic differentiation. We have summarized their clinicopathological characteristics to improve our understanding of this disease.Myeloid sarcoma most commonly consists of myeloblasts with or without features of promyelocytic or neutrophilic maturation, some of cases displays myelomonocytic or pure monoblastic morphology . MyeloidA 64-year-old woman presented with epigastric discomfort and erratic reflux since 2 months. Upper endoscopy revealed a 2.5 × 3.0 cm mass at the junction of the lesser curvature and gastric antrum Fig. a. ComputEndoscopic biopsy at low magnification showed the tumor cells poorly differentiated, atypical, large-cell lamellar and striated infiltrates. Under high magnification, tumor cells showed slightly basophilic cytoplasm, and some cells showed obvious nucleoli and nuclear fission. Some tumor cells had large nuclei, and some nuclei were deviated, resembling plasma cells Fig. a–h. SomeThe patient began treatment with the DA regimen. After 2 months of treatment, repeat gastroscopy showed that the tumor mass was unchanged Fig. c and d. MED23, PTPRB, ERG, PDE4DIP, FAT1, GRIN2A, CNOT1, WNK1, SH2B3, TJP2, MET, ANK3, and NKX3–1. Annotation of the screening results revealed that the PTPRB gene (associated with angiosarcoma) had a missense mutation. Among genes with missense mutations, 96 differential genes were screened out. These genes were involved in 30 significant pathways according to KEGG pathway-enrichment analysis  using SAMtools Table . In totaMYC gene, which is associated with cancer pathways, was located on chromosome 8. The oncogene TP53 was located in the affected segment on chromosome 17. Finally, we compared somatic mutations with known driver genes in the database, and screened out the following possible driver genes in the tumor sample: TP53, WHSC1, and SMARCA4 , LTK (associated with poorly differentiated adenocarcinoma), NOTCH2 (associated with diffuse large B-cell tumorigenesis), and CNTN2 , or myelodysplastic syndromes (MDSs) .FIP1L1-PDGFRA rearrangement, and the other patient had an MLL gene rearrangement .


Formyl peptide receptors, abbreviated as FPRs in humans, are G-protein coupled receptors (GPCRs) mainly found in mammalian leukocytes. However, they are also expressed in cell types crucial for homeostatic brain regulation, including microglia and blood–brain barrier endothelial cells. Thus, the roles of these immune-associated receptors are extensive, from governing cellular adhesion and directed migration through chemotaxis, to granule release and superoxide formation, to phagocytosis and efferocytosis. In this review, we will describe the similarities and differences between the two principal pro-inflammatory and anti-inflammatory FPRs, FPR1 and FPR2, and the evidence for their importance in the development of neuroinflammatory disease, alongside their potential as therapeutic targets. The formyl peptide receptors (FPRs) are seven-pass, transmembrane G-protein coupled receptors (GPCRs) crucially involved in the inflammatory response. Although their roles in the response to infection and sterile peripheral inflammation have been extensively studied, their function in the central nervous system (CNS) and neuroinflammatory responses has only gradually become apparent ,2,3,4,5.Inflammation is a complex biological process essential in responding to both tissue injury and infection. Ideally, it is a protective process, including the involvement of both cells from the immune system and the vascular endothelium, as well as a vast array of molecular mediators . It servOne of the primary triggers of inflammation is infection, wherein an acute insult activates both the initial innate and later adaptive arms of the immune system. The initial innate response, characterized by a broad and non-specific pro-inflammatory signaling cascade, involves the release of inflammatory mediators including cytokines, chemokines, and reactive oxygen species (ROS). This response facilitates the activation and movement of immune cells such as neutrophils and monocytes in the periphery or microglia in the CNS towards the injury site, wherein pathogen killing commences. The innate immune system can also activate a more precise adaptive response, wherein T and B cells undergo clonal selection, responding to a specific antigen. Their activation triggers the engagement and specific targeting of both adaptive and innate effectors, including natural killer cells and neutrophils . This duFollowing insult removal, innate cells transition towards pro-resolving phenotypes, which are responsible for the degradation of cellular debris and apoptotic cells, including effete immune cells, in tandem with supporting tissue repair. Upon successful transition, inflammation will begin to subside. However, if this transition fails, chronic inflammation can result—a response associated with the development of many human diseases ,9,10.The formyl peptide receptors, abbreviated as FPRs in humans, are pattern recognition receptors (PRRs) with central roles in host defense and inflammation ,11,12,13N-formylated peptides such as N-formylmethionine (fMet), produced through the degradation of both bacteria and mitochondria [N-formyl peptides, including the potent FPR1 agonist and chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (fMLF), led to the conclusion that FPRs act as PRRs [N-formyl peptides, but also non-formyl peptides of both microbial and host origin, synthetic small molecules, and eicosanoid lipids . Of these receptors, FPR1 and FPR2 share a particularly high overall gene sequence homology, with some overlapping functionality [FPR gene family on chromosome 17A3.2, denoted as ‘Fprs’. Despite the discrepancy in receptor numbers across the two species, several receptors share similar functionality, including FPR1/Fpr1, both of which are known to respond to host infection [FPR2 as well, although murine functionality is encoded by two receptors which work in synergy to carry out comparable functions: Fpr2/3 [There are three genes which encode for human FPRs: nfection  and regunfection ,60,61. T: Fpr2/3 ,63. High4, alongside pathogenic ligands such as serum amyloid A and β-amyloid (Aβ). In terms of the latter, cryo-electron microscopy recently helped elucidate the interaction between Aβ1-42 and FPR2 [For many years, the crystalline structure of these receptors remained elusive. Instead, structure simulation and molecular modeling , computeand FPR2 . HoweverIn summary, identification of novel receptor binding pockets for both pro-resolution and disease associated ligands may prove crucial for the future development of improved FPR associated therapeutics.In contrast to their extensive analysis in the periphery, the role of these receptors in the CNS has only recently begun to be addressed ,73,74. IPRRs are a crucial first-line defense system expressed in innate immune cells, inducing an immune response to injury or infection. Activation of PRRs by pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) result in the upregulation of inflammatory mediators which act synergistically to help eliminate the cause of damage . While TE. coli in vitro, and reduces neutrophil recruitment in vivo, a response associated with increased infection-induced mortality in mice [Conventionally, FPR1 ligands tend to be pro-inflammatory in effect, with activation of this receptor contributing to the induction of inflammatory responses ,94. With in mice ,99. In aN-formyl peptides post-hemorrhage, suggesting a feedback association between the agonists and their receptor [Fpr1 knockout mice, where the acute CNS inflammatory profile was reduced, with final validation in wild-type mice treated with the Fpr1 antagonist T-0080, resulting in improved neurological outcomes and reduced oedema [There is evidence that FPR1 may be important in sterile inflammatory responses within the CNS. In patients who displayed an intracerebral hemorrhagic injury, FPR1 mRNA and protein levels were both significantly upregulated compared to healthy controls, with the receptor being the most abundantly expressed PRR amongst those reported, more so than more classically studied Toll-like receptors 2 and 4, and the P2 × 4 purinoceptor . These ereceptor . The impd oedema . HoweverFpr1 knockout reduces tissue damage and acute neuroinflammation 24 h post-injury, but was associated with reduced neurogenesis 4-weeks post-injury [The role of FPR1 within the CNS does appear to vary depending on the original inflammatory insult, though, its importance in initiating the response to injury appears to be conserved across different models of disease. In a mouse model of traumatic brain injury, t-injury . The impt-injury . BecauseThe importance of FPR2 for inflammatory resolution has become more evident in recent years , with maThe importance of FPR2 in resolving sterile peripheral inflammatory responses has been reported for many disease models, including promoting muscle fiber regeneration , alongsi4, has become more visible in recent years. Administration of ANXA1 following intracerebral hemorrhage injury in mice reduced microglial activation, brain oedema and acute neurological deficiencies, as determined with the sensorimotor Garcia testing paradigm [2 and platelet function in both mice and humans, alongside promoting neutrophil elicited platelet phagocytosis [N-terminal peptide Ac2-26 was also reported to shift microglia towards pro-resolving phenotypes at 3 days post-transient middle cerebral artery occlusion/reperfusion injury, highlighted by the reduction in pro-inflammatory  and the increase in resolving markers , respectively [The role of FPR2 within the CNS, primarily through agonism by ANXA1 or lipoxin Aparadigm . Howeverocytosis . Intraveectively . These oectively . In humaectively ,112, whiFurther supporting protective roles of FPR2 in the CNS, ANXA1sp—a bioactive ANXA1 peptide—improved inflammatory profiles and neurological scores in a rat model of exsanguinating cardiac arrest . InteresAβ peptides [Aβ1-42 mediated proinflammatory cytokine release and increased an anti-inflammatory cytokine profile in organotypic hippocampal slice cultures (OHCs). Repeated intraperitoneal injections of MR-39 resulted in similar findings in 29-week old APP/PS1 transgenic mice, wherein neuronal apoptosis within the cortex and Aβ plaques in the hippocampus were both significantly reduced [4 was sufficient to prevent an increase in fibrillar Aβ1-42-induced IL-6 and TNFα in OHCs, although the statistical significance of the latter was not determined. Because Aβ is a known FPR2 ligand [Aβ1-42 aggregates can partially trigger inflammatory responses via FPR2 modulation. Additional studies have reported that pan-antagonism of FPR1/FPR2 with Boc-2 can also reduce neuronal Aβ pathology, increase the mRNA expression of several Aβ-degrading enzymes, decrease microglial ameboid morphology, and improve spatial memory in APP/PS1 transgenic mice [Aβ interaction, may prove to be tactical research approaches in deciphering novel neuroprotective pathways in models of AD.There is also direct evidence to support a neuroprotective function of FPR2 in neurodegenerative disease, particularly in AD. Firstly, in 5XFAD AD transgenic mice, ANXA1 protein expression is reduced in both the brain and capillaries of the BBB ,82. Simipeptides . The FPR reduced . Interes2 ligand ,124, thinic mice . Thus, mN-formyl peptides) are universally expressed in mitochondria and bacterial pathogens, several FPR2 ligands display specific endogenous expression patterns. Arguably one of the most important is the previously described ANXA1, a highly potent FPR2 agonist widely acknowledged to be involved in inflammatory resolution [As described above, FPRs are promiscuous receptors which interact with a wide range of ligands. Although many of these ligands have been chemically synthesized, such as Compound 43 and Quin-C1 , there asolution ,127,128.solution . In partsolution ,131,132.solution . While rsolution ,134,135.solution ,136,137.solution ,139, altsolution . Interessolution , hippocasolution ,142, embsolution , and retsolution . While Asolution ,142,144,The formyl peptide receptor family are crucial pattern recognition receptors that respond to both infection and sterile inflammation. As such, these receptors are an attractive drug target for therapeutic interventions. The utilization of high throughput drug candidate screens , in tand1-25, which can activate FPR1 at high concentrations, triggering pro-inflammatory signaling responses similar to traditional FPR1 agonists [We have previously described the therapeutic promise of targeting the FPR system for neurodegenerative diseases, with a particular focus on FPR2 and AD . Howeveragonists . As suchAβ pathology in AD models [Aβ displays agonism for both of these receptors [The ability for ligands to stimulate conformational changes in FPRs, facilitating both homo- and heterodimerization , may conD models , a pathoeceptors ,124, decWhile our knowledge of FPR1 and FPR2 signaling has improved in recent years, the physiological role of FPR3 remains relatively elusive. It was previously reported in HEK293T cells that the receptor displays a marked phosphorylation state under resting conditions compared to both FPR1 and FPR2 . The subFormyl peptide receptors are complex, multifunctional, and promiscuous receptors which display central roles in initiating, propagating, and resolving the inflammatory response. While most research has focused on their roles in infectious and sterile inflammation within the periphery, newer insights indicate their importance within the central nervous system. Thus, their responses to neuroinflammatory insults in neurodegenerative conditions cannot be overlooked. As such, future FPR research may be of critical importance in the development of neuroinflammatory-associated therapeutics.


This study aims to investigate the prognostic value of composition and spatial architecture of tumor-infiltrating lymphocytes (TILs) as well as PDL1 expression on TILs subpopulations in nasopharyngeal carcinoma (NPC).A total of 121 patients with NPC were included and divided into two groups: favorable (n = 68) and unfavorable (n = 53). The archived tumor tissues of the included patients were retrieved, and a tissue microarray was constructed. The density and spatial distribution of TILs infiltration were analyzed using the multiplex fluorescent immunohistochemistry staining for CD3, CD4, CD8, Foxp3, cytokeratin (CK), PDL1, and 4′,6-diamidino-2-phenylindole (DAPI). The infiltration density of TILs subpopulations and PDL1 expression were compared between the two groups. The Gcross function was calculated to quantify the relative proximity of any two types of cells. The Cox proportional hazards regression model was used to identify factors associated with overall survival (OS) and disease-free survival (DFS).TC : Treg (Tregs engagement surrounding TCs) and GCTL : PDL1+ Treg were identified as independent factors correlated with poor outcomes.The densities of regulatory T-cells (Tregs), effector T-cells (Teffs), PDL1+ Tregs, and PDL1+ Teffs were significantly higher in patients with unfavorable outcomes. PDL1 expression on tumor cells (TCs) or overall TILs was not associated with survival. Multivariate analysis revealed that higher PDL1+ Tregs infiltration density was independently associated with inferior OS and DFS, whereas Tregs infiltration density was only a prognostic marker for DFS. Spatial analysis revealed that unfavorable group had significantly stronger Tregs and PDL1+ Tregs engagement in the proximity of TCs and cytotoxic T lymphocyte (CTLs). Gcross analysis further revealed that Tregs and PDL1+ Tregs were more likely to colocalize with CTLs. Moreover, increased GTILs have a diverse infiltrating pattern and spatial distribution in NPC. Increased infiltration of Tregs, particularly PDL1+ Tregs, as well as their proximity to TCs and CTLs, correlates with unfavorable outcomes, implying the significance of intercellular immune regulation in mediating disease progression. Nasopharyngeal carcinoma (NPC) is characterized by its close association with Epstein–Barr virus (EBV) infection, poor differentiation, and sensitivity to radiotherapy and chemotherapy , 2. InteThe tumor microenvironment (TME) is an intricately organized landscape occupied by infiltrating immune cells, epithelial cells, vascular and lymphatic vessels, cytokines, and chemokines . The tumA few studies have been conducted over the last several decades to investigate the immunological landscape of NPC using hematoxylin and eosin (H&E) staining, immunohistochemical (IHC) staining, and flow cytometry. Recent studies have demonstrated that the immunological components such as CD8+ T-cell infiltration and PD1/PDL1 expression may have prognostic value, but the results are still controversial –24. AsidThe present study provides a comprehensive analysis of the composition and abundance of TILs, as well as PDL1 expression in the TME using the multiplex fluorescent immunohistochemistry (mfIHC) approach, aiming to evaluate the prognostic role of TILs in NPC. Furthermore, the spatial architecture of TCs and TILs is studied using multispectral imaging analysis. This allows researchers to assess the role of TILs’ intercellular proximity and distribution pattern in mediating disease progression, revealing a potential treatment-responsive biomarker for immune-modulatory therapy.th American Joint Committee on Cancer (AJCC) TNM staging system, had no concomitant immune system disease, received IMRT at our institution between March 2010 and July 2014, and had sufficient tumor sample collection prior to any anticancer treatment were included. Eligible patients were then divided into two groups with comparable clinicopathological characteristics but distinct posttreatment outcomes. For attaining a balance between the two groups, clinicopathological data such as age, sex, smoking history, histological classification, AJCC 8th TNM stage, Karnofsky performance status, lactate dehydrogenase level, hemoglobin level, platelet count, and treatment modality were considered. The 5-year disease progression rate was the main prognostic index in this study. Finally, 121 patients were included, with 68 in the favorable group surviving at least 5 years without disease progression (Group 1) and 53 in the unfavorable group having disease progression within 5 years (Group 2).In this study, patients with NPC who were staged as I–IVA according to the 8All fresh tumor samples were preserved at our institution at −80°C liquid nitrogen with signed written informed consent. Formalin-fixed, paraffin-embedded (FFPE) blocks were prepared using a standard method. All H&E-stained slides were reassessed independently by two pathologists, and they were blinded to clinical data. After reviewing H&E-stained slides, one 1.5 mm diameter tumor tissue core from representative sections of FFPE blocks was used to construct the tissue microarray (TMA) .The Opal 7-color manual IHC kit 50 slides  was used according to the manufacturer’s protocol. Briefly, TMA block sections were deparaffinized in an automatic dyeing machine  and subjected to antigen retrieval by microwave treatment in Citrate buffer (pH=6.0). Sections were then incubated in 3% hydrogen peroxide in methanol for 30 min at room temperature and subsequently with a blocking solution containing 0.3% bovine serum albumin in 0.05% Tween solution for 30 min. Then, the sections were incubated with primary antibody for 60 min at room temperature and its corresponding HRP-conjugated secondary antibody for 10 min, followed by opal fluorophores for 10 min. The staining sequence of primary antibodies and corresponding fluorescence channels was anti-CD4, CD3, PDL1, CD8, Foxp3 and CK, with the corresponding opal fluorophores 520, 690, 570, 620, 540 and 650, respectively. After staining the above markers in turn, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Tech) and mounted with VECTASHIELD fluorescence mounting medium .To study the infiltration composition of T-cell subpopulations in NPC and their potential correlation with posttreatment progression, the following markers were used: cytokeratin (CK), CD3, CD4, CD8, Foxp3, and 4′,6-diamidino-2-phenylindole . CK was used to identify epithelial cancer cells in NPC tumor samples. The detailed information on biomarkers and antibodies used is presented in 2). The spectral signature for each fluorophore was determined using single-antigen staining and captured using a multispectral fluorescent microscope, which records an image every 10 nm over the full-emission spectrum. This enabled the simultaneous capture of seven different fluorophores into a single composite image, which could then be unmixed and separated into six unique images representing each fluorophore and the nuclear stain DAPI, as well as the precise x and y spatial coordinates of each identified cell.Multiplex fluorescent-stained TMA slides were scanned using the image analysis software StrataQuest (TissueGnostics-StrataQuest 7.7.1.165 version) for multicellular contextual tissue analysis in both bright field and fluorescence images. The total counts of various cell phenotypes derived from all available cores were analyzed. Additionally, the core density was calculated by dividing the total number of cells by the area of each core . At the patient level, specific phenotypes of cells with a PDL1 staining score of at least 5% were considered positive PDL1 expression . Since tij(r)) was calculated to estimate the distribution probability of finding at least one specified point “j” within a given radius “r” (µm) of any specified point “i,” allowing quantification of the relative proximity of any two cell types  of the Gcross curve was calculated to represent the accumulated infiltration level of cell type “j” within a given distance from cell type “i.” Accordingly, larger AUCs indicate higher immune cell interaction around TCs. Typical Gcross function curves indicating high (left), intermediate (middle), and low (right) levels of infiltration are illustrated in The spatial distribution of TILs surrounding TCs was first analyzed in various tissue compartments (inner tumor vs. stroma). Next, the stroma area within 200 µm of the tumor edges was divided into 10 intervals, and the infiltration density of each type of TILs within each interval was quantitatively estimated. Furthermore, the Gcross function , the Statistical Package for the Social Sciences (SPSS) 22.0 software (IBM Inc.), and the R 3.6.3 software .The continuous variables, such as percentage, density, and Gcross value, were presented as median and interquartile range (IQR) and compared between two groups using the Mann–Whitney U-test. The categorical data were compared using the Chi-squared test. Disease-free survival (DFS) was defined as the time between the first date of diagnosis and disease progression or death, whereas overall survival (OS) was defined as the time between the first day of treatment and death from any cause or the last follow-up. The survival index was estimated using the Kaplan–Meier method, and the significance of the difference was assessed using the log-rank test. The Cox proportional hazards regression model was used to identify factors related to survival variables and to calculate the hazard ratio and corresponding confidence interval. To further evaluate the prognostic significance of TME, the densities and spatial architectures of TIL phenotypes were respectively assessed in multivariable Cox regression models that initially included age, sex, smoking history, histological type, N stage, T stage, and TNM stage. All statistical tests were two-sided, and a p < 0.001) and 100% vs. 0% (p < 0.001), respectively. In the final analysis, 121 patients with NPC were included and classified into two groups: favorable  and unfavorable . The overall population had a median follow-up time of 78.0 months (IQR: 57.5–93.2 months). The 5-year OS and DFS rates in the favorable and unfavorable groups were 100% vs. 45.3% (p = 0.023), whereas there was no significant difference in the proportions of TCs, total TILs, Teffs, and CTLs  and median Teffs density  were significantly higher in patients with an unfavorable outcome than in those with a favorable outcome, respectively  can reflect cell distribution to some extent. The median densities of the total TILs, CTLs, Teffs, and Tregs were 2260.1 (IQR: 1721.1–3213.1), 582.1 (IQR: 242.7–1225.1), 140.1 (IQR: 69.8–318.6), and 58.2 (IQR: 19.0–142.0) cells/mmp = 0.775) and 71.1% vs. 60.0% (p = 0.166), respectively. In the overall population, the median densities of PDL1+ TCs and PDL1+ TILs were 3113.8 (IQR: 1043.4–5461.6) and 1037.4 (IQR: 711.5–1709.1) cells/mm2, respectively. In terms of PDL1 expression on TILs subpopulations, densities of PDL1+ Tregs  and PDL1+ Teffs  were significantly higher in patients with unfavorable outcomes than in those with favorable ones, respectively, with no significant differences in densities of PDL1+ TCs, PDL1+ TILs, or PDL1+ CTLs between the two groups  and PDL1 positive TILs (PDL1+ TILs) in the favorable and unfavorable groups were 20.3% vs. 18.6% , Teffs (p < 0.001), Tregs (p = 0.04), and PDL1+ Tregs (p < 0.001) (p = 0.008) and PDL1+ Tregs (p = 0.015) within the stroma area , tumor-associated macrophages(TAMs), CD8+ T cells and regulatory T cells (Tregs) in TME also may impact the prognosis of sloid tumors . TherefoTregs play crucial roles in suppressing antitumor immunity in TME by expressing ligands for inhibitory checkpoint receptors and secreting suppressive cytokines, promoting the occurrence and development of tumors , 36. TheThe PD1/PDL1 axis is a well-known immune checkpoint that attenuates T-cells’ antitumor immune response and mediates immunological escape . Despitehi Tregs was positively correlated with PD-1-positive CD8 in the tumor stroma of non-small cell lung cancer  of Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College . All patients provided informed consent for the collection of tissue samples.FZ and GS: data curation, formal analysis, methodology, visualization, writing-original draft preparation. XC, YZ, RW, JZ, XH, KW, and YQ: investigation, data curation. SS, QL, YL, and XS: data curation, methodology. JL and Y-XL: investigation, supervision. BL and JW: software, visualization. JY and JW: conceptualization, project administration, writing - review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.This work was supported by the CAMS Innovation Fund for Medical Sciences (2021-I2M-C&T-B-070) to JW, the Beijing Hope Run Special Fund of Cancer Foundation of China (LC2021L06) and the National Natural Science Foundation of China grant (81172125) to JY.We would like to thank Dr. Chuqing Pan for her guidance with the spatial analysis.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Over the past decade, transcriptome profiling has elucidated many pivotal pathways involved in oncogenesis. However, a detailed comprehensive map of tumorigenesis remains an enigma to solve. Propelled research has been devoted to investigating the molecular drivers of clear cell renal cell carcinoma (ccRCC). To add another piece to the puzzle, we evaluated the role of anoctamin 4 (ANO4) expression as a potential prognostic biomarker in non-metastasized ccRCC. Methods: A total of 422 ccRCC patients with the corresponding ANO4 expression and clinicopathological data were obtained from The Cancer Genome Atlas Program (TCGA). Differential expression across several clinicopathological variables was performed. The Kaplan–Meier method was used to assess the impact of ANO4 expression on the overall survival (OS), progression-free interval (PFI), disease-free interval (DFI), and disease-specific survival (DSS). Univariate and multivariate Cox logistic regression analyses were conducted to identify independent factors modulating the aforementioned outcomes. Gene set enrichment analysis (GSEA) was used to discern a set of molecular mechanisms involved in the prognostic signature. Tumor immune microenvironment was estimated using xCell. Results: ANO4 expression was upregulated in tumor samples compared to normal kidney tissue. Albeit the latter finding, low ANO4 expression is associated with advanced clinicopathological variables such as tumor grade, stage, and pT. In addition, low ANO4 expression is linked to shorter OS, PFI, and DSS. Multivariate Cox logistic regression analysis identified ANO4 expression as an independent prognostic variable in OS , PFI , and DSS . GSEA identified the following pathways to be enriched within the low ANO4 expression group: epithelial–mesenchymal transition, G2-M checkpoint, E2F targets, estrogen response, apical junction, glycolysis, hypoxia, coagulation, KRAS, complement, p53, myogenesis, and TNF-α signaling via NF-κB pathways. ANO4 expression correlates significantly with monocyte  and mast cell  infiltration. Conclusions: In the presented work, low ANO4 expression is portrayed as a potential poor prognostic factor in non-metastasized ccRCC. Further experimental studies should be directed to shed new light on the exact molecular mechanisms involved. Renal cell carcinoma (RCC) is the most common genitourinary tumor in the United States, with a projected 79,000 new cases and 13,920 deaths in the year 2022 ,5. Each VHL (von Hippel–Lindau tumor suppressor) and PBRM1 (protein polybromo1) [SETD2 , KDM5C (lysine demethylase 5C), or BAP1 (BRCA1 associated protein 1) [ccRCC is one of the most inscrutable types of cancer . An arraybromo1) ,12. Otheotein 1) ,15. The otein 1) ,17.ANO4) expression as a potential prognostic biomarker in non-metastasized ccRCC. ANO4 belongs to the anoctamin (anion channels with 8 transmembrane domains) family [TMEM16 (transmembrane proteins with 16 domains) [ANO1 and ANO2 act as Ca2+-dependent ion channels, whereas others including ANO6 function as Ca2+-dependent scramblases [ANO4 is primarily expressed in the central nervous system and certain endocrine glands, and it has been linked to a variety of neurological disorders [ANO4 expression as a potential prognostic biomarker in ccRCC.Despite the development of numerous drugs targeting genomically prioritized pathways, patients with ccRCC have had limited responses to these treatments ,18. Thes) family . It was domains) . In vertdomains) . Despitedomains) . For exaamblases . The rolamblases . Howeveramblases ,24. ANO4isorders . To dateANO4 mRNA expression in non-metastasized ccRCC (KIRC). KIRC clinical and transcriptomic data from The Cancer Genome Atlas Program (TCGA) were accessed using the California Santa Cruz Cancer Genomics Browser ; a web-based platform for visualizing and analyzing public genomic data resources [ANO4 expression from tumor samples with normal adjacent tissues was downloaded in an RNA-Seq by expectation maximization (RSEM) normalized count transformed as ANO4 expression data. KIRC cohort was also divided into two subsets based on ANO4 expression (high vs low expression), with a cut-point determined by X-tile software [This work aims to investigate the prognostic utility of esources . Experimsoftware .ANO4 expression status against clinicopathological variables was performed as follows: Chi-square test or Fisher’s exact test for categorical variables, paired t-test for normally distributed paired samples, unpaired t-test and Welch’s corrected unpaired t-test for normally distributed non-paired samples according to variance equality, and finally, Wilcoxon matched pairs test and Mann–Whitney U-test for non-normally distributed data.IBM SPSS statistical package for Windows v.26  and GraphPad prism v.9.3.1  were utilized for statistical analysis and graph generation. Nominal data were presented as frequency (percentage). Mean ± standard deviation of the mean (SD) or standard error of the mean (SEM) were used to present normally distributed continuous variables, while non-normally distributed data were presented as median (interquartile range (IQR)). Normality was evaluated using the Kolmogorov–Smirnov test and the Shapiro–Wilk test aided with quantile–quantile (Q–Q) plots. Comparison between ANO4 expression status in relation to OS, PFI, DFI, and DSS. Statistical difference across survival curves was detected using a log-rank test reporting the p-value, 95% confidence intervals (95% CI), and hazard ratios (HR). Univariate and multivariate Cox logistic regression models were applied to test clinicopathological variables and ANO4 expression as independent prognostic indicators. Variables were dichotomized for Cox logistic regression analysis as follows: age (ref. ≤ 53 years), gender , pT stage (ref. T1 + T2), pN stage (ref. N0), AJCC stage (ref. Stage 1 + 2), ISUP grade (ref. Grade 1 + 2), and ANO4 expression (ref. Low). The age cut-off value (53 years) was defined previously based on age-related differentially expressed genes optimized by the TCGA and Surveillance Epidemiology and End Results (SEER) database [p ≤ 0.05 was considered statistically significant.Kaplan–Meier survival methods were utilized to evaluate the impact of database . All staANO4 signature. GSEA performs genome-wide transcriptional profiling across two expressional groups (high vs low ANO4 expression) against a set of genes representing pivotal processes involved in oncogenesis/tumorigenesis [p value ≤ 0.05 and a false discovery rate (FDR) < 0.25.GSEA was performed to discern a set of molecular mechanisms involved in the prognostic igenesis . Three mANO4-related interacting genes were browsed using STRING v.11 , an online PPI networking resource based on functional interactions annotated using gene-enrichment analysis, GO/KEGG classification systems, and high-throughput text-mining [Identification and retrieval of t-mining . Signifiρ) and the correlation significance.Immune cell infiltration was enumerated from transcriptomes using xCell, an online platform with pre-calculated TCGA immune infiltration estimates that deploys a curve-fitting approach with a novel spillover compensation method . SpearmaANO4 expression as a potential prognostic biomarker in non-metastasized ccRCC using a modified TCGA-KIRC cohort. It consisted of 422 patients with a median age of 61 (IQR: 51–71) and a male predominance . The majority of patients with non-metastasized ccRCC were found to have tumor grade 2 , followed by grade 3  and grade 4 . In regard to tumor staging, the cohort was mainly distributed across two stages including Stage 1  and stage 3 , which was definitely in concordance with the pathological T and N scoring distribution. ANO4 expression harboring two groups .The presented work studies the usefulness of ANO4 was significantly upregulated (p < 0.0001) in the non-metastasized KIRC (ANO4 using age and gender-matched participants (p < 0.0001), as illustrated in ANO4 expression compared to normal tissue, intriguingly, low ANO4 expression within tumor samples is associated with advanced demographics and clinicopathological features. ANO4 expression did not exhibit a tractable significant difference across age subgroups (p = 0.1989), as seen in ANO4 expression (p < 0.0001) in contrast to female samples (ANO4 expression (p = 0.0497) in comparison to grades 1 and 2  was observed  on the OS, PFI, DFI, and DSS. The low ANO4 expression group has a poor OS  compared to the high expression group  as seen in ANO4 expression on the OS in specific sub-groups. In patients below the age of 53, ANO4 expression did not impact the OS  vs  as presented in ANO4 expression still exhibits the same survival effect  vs  as presented in ANO4 expression effect , grade , stage , pT , and ANO4 expression , while the gender (p = 0.596) and pN (p = 0.349) were not prognostic. However, multivariate Cox logistic regression highlighted the age  and ANO4 expression  as significant predictors, while the grade (p = 0.082), stage (p = 0.617), and pT (p = 0.912) were not significant. In regard to PFI, univariate Cox logistic regression analysis illustrated a prognostic utility of all indicators except for the pN (p = 0.617) which moves in concordance with the OS results. The significant PFI predictors are age , gender , grade , stage , pT , and ANO4 expression . Upon pooling proposed predictors in PFI multivariate Cox logistic regression, stage  and ANO4 expression  were the only significant predictors. A total of six predictors were identified in the univariate Cox logistic regression analysis of the DSS. They include age , grade , stage , pT , and ANO4 expression . On the other hand, the DSS multivariate Cox logistic regression portrayed ANO4 expression as the only predictor . Tumor stage was the only predictor in the DFI-related univariate  and multivariate  Cox logistic regression analysis. Univariate Cox logistic regression analysis of the OS identified five independent prognostic indicators, including age . The enrichment results include the following sets: epithelial–mesenchymal transition , G2-M checkpoint , estrogen response-late , E2F targets , apical junction , glycolysis , hypoxia , coagulation , KRAS signaling pathway , complement , p53 pathway , estrogen response-early , myogenesis , and TNF-α signaling pathway via NF-κB . The high ANO4 expression group enrichment analysis revealed a total of 16 enriched gene sets out of 50, with only three genes with FDR < 25% and a p ≤ 0.05. The enriched sets include fatty acid metabolism , pancreatic beta cells , and bile acid metabolism . ANO4 expression groups.The first GSEA involved the hallmark gene sets in which a total of 34 out of 50 gene sets were enriched in the low ANO4 expression groups. The poor prognostic effect of ANO4 downregulation is hypothesized to be mediated through the following pathways: ribosome , α-linolenic acid metabolism , glycosaminoglycan biosynthesis–keratan sulfate , glycosaminoglycan biosynthesis–chondroitin sulfate , complement and coagulation cascades , arachidonic acid metabolism , NOD-like receptor signaling pathway , proteasome , basal cell carcinoma , base excision repair , cell cycle , homologous recombination , p53 pathway , taste transduction , and Wnt signaling pathway . On the other hand, the high ANO4 expression group was associated with significant enrichment of 11 pathways out of the top 15 pathways, with an FDR < 0.25. The pathways are propanoate metabolism , valine, leucine, and isoleucine degradation , PPAR signaling pathway , fatty acid metabolism , peroxisome , renin–angiotensin system pathway , pyruvate metabolism , proximal tubule bicarbonate reclamation , TCA cycle , nitrogen metabolism , and histidine metabolism . ANO4-related PPI. The generated PPI contains 11 nodes and 12 edges with an average node degree of 2.18, an average local clustering coefficient of 0.883, and PPI enrichment p-value of 0.329. The nodes include the following PLSCR5, CCDC181, ACOT7, SLC17A8, ASCL1, ASH2L, TSPAN16, DYSF, SPIC, and GAS2L3.ANO4 expression correlates significantly with monocyte  and mast cell  infiltration. On the other hand, non-significant correlations were observed regarding neutrophils , macrophages , NK cells , basophils , B-cells , plasma cells , CD4+ T-cells , and CD8+ T-cells . Pre-calculated TCGA immune infiltrate estimates were obtained from xCell. +2 activated C-l channel (CaCC) functionality, other members did not pursue such function, since they could not produce a C-l current through Ca+2 activation [Anoctamins (ANO) are a group of anion channels with eight transmembrane domains, and numerous cellular functions . The codtivation . Furthertivation . Despitetivation .+2 [ANO1 has been the most extensively studied ANO protein and has been shown to mediate Cl−1 secretion in secretory epithelia of the respiratory, gastrointestinal and renal systems, and sweat glands [ANO1 regulates vascular and bronchial smooth muscle tone. Because of this, ANO1 may play a role in the pathophysiological mechanisms underlying hypertension and asthma, respectively [et al., the interaction between RANK and ANO1 in osteoclasts causes increased bone resorption and decreased bone mass. In individuals with osteoporosis, this makes ANO1 a viable therapeutic target [ANO1 antagonist can have a synergistic effect with commercially used tocolytics on human uterine smooth muscles [ANO2, a member of the ANO family that is expressed in olfactory sensory cells of the olfactory epithelium may have a function in the olfaction process [ANO3 is expressed in the dorsal root ganglia, which controls nociception [ANO5 is most abundantly expressed in the musculoskeletal system including bones, chondrocytes, cardiac, and skeletal muscles [ANO6 as a procoagulant has been confirmed through human and animal studies resulting in Cl−1 influx and swelling of platelets [ANO6 has a well-known scramblase activity, which is also shared with ANO3, ANO4, and ANO7 [ANO1, which has been thoroughly investigated, other members of the ANO family need even more in-depth research, and we are only at the beginning.Anoctamins have been involved in a wide variety of cellular functions, including epithelial cell secretion, neuronal activation, smooth muscle contractions, skeletal muscle membrane repair, sensory transduction, and carcinogenesis ,31,33. O+2 ,31,33. At glands . Under tt glands . Furtherectively ,36. Accoc target . ANO1 an muscles . Finally muscles . ANO2, a process . It has iception . ANO5 is muscles . The rollatelets . ANO6 haand ANO7 . OverallANO1, which is found on locus 11q13, is amplified in multiple tumors, particularly in gastrointestinal stromal tumors (GIST) and head and neck squamous cell carcinoma (HNSCC), in addition to numerous others, such as lung adenocarcinoma, chondroblastoma, esophageal squamous cell carcinoma, salivary gland tumors, oral squamous cell carcinoma, leiomyosarcoma of the uterus, glioma, breast, colorectal and prostate cancer [ANO1 was a cancer biomarker prior to it is identification as a chloride channel in GIST known as DOG1, and it has received several other names including ORAOV2, and TAOS-2 among oncologists [ANO1 has implicated numerous cell signaling pathways. ANO1-mediated proliferation was shown by Duvvuri et al. to be accompanied by extracellular signal-regulated kinase (ERK)1/2 activation, cyclin D1 evocation, and mitogen-activated protein kinase (MAPK) activation [ANO1 upregulates calcium/calmodulin-dependent protein kinase II (CaMKII) and epidermal growth factor receptor (EGFR) expression, the latter of which regulates the MAPK or PI3K-AKT pathway. Therefore, ANO1 plays a role in governing cell variability through EGFR-AKT/SRC/MAPK and CaMKII signaling pathways [ANO1, this might provide a clue to the role of ANO1 in EGF-driven migratory and invasive properties [ANO1 which is known to coordinate cellular growth and differentiation [e cancer ,59,60,61ologists ,39. Howeologists ,47,50,61tivation . ANO1 uppathways . ezrin-roperties . In addioperties . Anotherntiation .ANO1 overexpression; ANO1 knockdown greatly reduces the tumor’s aggressive behavior [ANO5 has been linked to a negative prognostic role; when it is knocked down, it causes apoptosis, reduces cell proliferation, and arrests the cell cycle at the G1/S transition [et al., osteosarcomas express ANO5, a pro-tumorigenic factor that increases tumor size, grade, and metastasis. The instability and destruction of nel-like proteins 1 and 2 enable such activity [ANO5 expression is scarce in healthy pancreatic tissue, but it is elevated in pancreatic cancer, where it contributes to the disease’s proliferation and migration [ANO7 is only expressed in prostate cells, has an unknown function, and is negatively correlated with the prognosis of prostate cancer. Its low expression has been linked to positive surgical margin, lymph node metastasis, high classical and quantitative Gleason grades, advanced tumor stage, high Ki67 labelling index, and early biochemical recurrence [Anaplastic thyroid carcinoma appears to acquire its undesirable traits and aggressiveness through behavior . In gastansition . Accordiactivity . ANO5 exigration . ANO7 iscurrence .ANO4 because it is one of the ANO family members that has not received enough attention. It is mostly expressed in the cervix, ovaries, prostate, adrenal glands, and central nervous system [ANO4 acts as a Ca2+-dependent phospholipid scramblase and monovalent nonselective ion channel [ANO4 to modulate disintegrin-like metalloproteases ADAM 10 and 17 sheddase activity, evident through the increasing ADAM10 and 17 substrates: transforming growth factor alpha (TGF-α), amphiregulin (AREG), and betacellulin. They also demonstrated that the effects brought upon by the overexpression of ANO4 are due to it is scramblase activity, consequently resulting in diminishing AREG and increased cellular proliferation [ANO4 as a significant gene expressed in the zona glomerulosa cells despite its contradictory effects on aldosterone secretion [ANO4 with tumor involvement.We focused our bioinformatic research on s system . Given ts system ,72,73,74 channel . Leitzkeferation . Manieroecretion ,77. To tANO4 expression was upregulated in tumor samples compared to normal kidney tissue. Albeit the latter finding, low ANO4 expression is associated with advanced clinicopathological variables such as tumor grade, stage, and pT. In addition, low ANO4 expression is linked to shorter OS, PFI, and DSS. Multivariate Cox logistic regression analysis identified ANO4 expression as an independent prognostic variable in OS , PFI , and DSS . GSEA identified the following pathways to be enriched within the low ANO4 expression group: epithelial–mesenchymal transition, G2-M checkpoint, E2F targets, estrogen response, apical junction, glycolysis, hypoxia, coagulation, KRAS, complement, p53, myogenesis, and TNF-α signaling via NF-κB pathways. ANO4 expression correlates significantly with monocyte  and mast cell  infiltration.In conclusion,