Lonicera japonica Thunb. (L. japonica) has the functions of clearing away heat and detoxifying, broad-spectrum antibacterial and anti-virus, etc. More than 70% of anti-inflammatory and cold Chinese patent medicines contain L. japonica. Trichomes comprise specialized multicellular structures that have the capacity to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. The extraction of trichome secretions has great commercial value. However, little is known about the trichome formation mechanism in L. japonica. Therefore, the study of trichome development between different varieties provides a basis for selecting suitable planting resources.L. japonica cultivars, toward the identification of biological processes and functional gene activities that occur during flowering stage trichome development. In this study, the density and average lengths of flower trichomes were at their highest during three-green periods (S2). Using the Illumina RNA-Seq method, we obtained 134,304 unigenes, 33,733 of which were differentially expressed. In an analysis of 40 differentially expressed unigenes (DEGs) involved in trichome development, 29 of these were transcription factors. The DEGs analysis of plant hormone signal transduction indicated that plant growth and development may be independent of gibberellin (GA) and cytokinine (CTK) signaling pathways, and plant stress may be independent of jasmonic acid (JA) and ethylene (ET) signaling pathways. We screened several genes involved in the floral biosynthesis of odors, tastes, colors, and plant hormones, and proposed biosynthetic pathways for sesquiterpenoid, triterpenoid, monoterpenoid, flavonoid, and plant hormones. Furthermore, 82 DEGs were assigned to cell cycles and 2616 were predicted as plant resistance genes (PRGs).Here, we present a genome-wide comparative transcriptome analysis between two L. japonica, thereby offering valuable insights into the molecular networks that underly flower development in L. japonica.This study provides a comprehensive characterization of the expression profiles of flower development during the seven developmental stages of  Lonicera japonica Thunb. (L. japonica) is an extensively used traditional Chinese medicine that may be employed in both food and medicine [L. japonica is incorporated within approximately 1/3 of traditional Chinese medicine preparations, and is also widely used in health care products, cosmetics, foods, and more [L. japonica, which are used for the prevention and treatment of severe acute respiratory syndromes, H1N1 influenza, and hand-foot-and-mouth disease [L. japonica flowers possess antibacterial, anti-inflammatory, anticancer, anti-diabetic, anti-oxidative and antiviral properties, as well as various other pharmacological effects [L. japonica is referred to as a “plant antibiotic” [L. japonica are highest in Fengqiu of Henan and Shandong Provinces. A new variety of L. japonica (Yujin 1) was selectively bred by our research group from the main cultivar of Fengqiu ‘Damaohua’, which possesses the attributes of large buds, high yields, strong resistance, and a high content of active elements (chlorogenic acid (CGA) and luteoloside) [medicine . L. japo disease . Pharmac effects . Consequibiotic” ; its adaibiotic” . Howeveroloside) .Plant trichomes are epidermal outgrowths that protect plants from the attack of herbivorous insects, which develop even when plants are grown under optimal conditions . InteresR2R3 MYB protein GLABROUS1 (GL1), two redundant trichome formation bHLH proteins, GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), a WD40 repeat containing protein, TRANSPARENT TESTA GLABRA 1 (TTG1), and the enhanced expression of TRIPTYCHON AND CAPRICE 2 (TC2) and GLABRA2 (GL2), which promote trichome differentiation [R3 MYB transcription factor family, which can also form complexes with TRANSPARENT TESTA GLABRA 1 (TTG1) and GL3 to inhibit the differentiation of epidermal hair cells [The processes of initiation and development for trichomes involve a complex genetic network. Our knowledge about this developmental process is still limited, but genes controlling glandular trichome initiation and morphogenesis have recently been identified . These gntiation , 14. Supir cells , 16. In ir cells , 17–20, ir cells .SlCycB2 plays a critical role in reproductive organ development, multicellular trichome initiation [Arabidopsis nucleoporin CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) controls trichome cell death through the core cell cycle regulator CYCLIN-DEPENDENT KINASE INHIBITOR (CKI) [Cyclins are the best-known positive regulators of cell proliferation, and their molecular mechanisms in the cell-cycle transition are conserved in eukaryotes . It has itiation , secondaitiation . The stuOR (CKI) . PhytohoOR (CKI) . HoweverOR (CKI) .L. japonica flowers. The quality of L. japonica primarily depends on the developmental period of the flower [L. japonica through genetic engineering technologies, while cultivating new varieties with high yields, high quality, multi-resistance, and high efficiency.Trichome is closely related to plant resistance and the formation of volatile oils; thus, it is an important index for resource evaluation and species identification. However, there are no reports to date that elucidate the molecular mechanisms of trichome formation on e flower . In addie flower . TherefoL. japonica, designated as ‘Yujin 1’, was identified and developed by our research group, which has the characteristics of large flower buds, as well as dense and long trichomes  and de novo assembled into transcripts using the Trinity in paired-end method, which yielded a total of 134,304 unigenes with an average length of N50 of 1642 bp, and GC content of 45.75%  was conducted on the expression levels of DEGs by using the RPKM method for the seven developmental stages, respectively. All references to the direction of differential expression refer to expression in the ‘Yujin 1’ cultivar relative to the ‘Damoahua’ cultivar. There were 7725, 7132, 3290, 3826, 5245, 8145 and 8907 DEGs at seven stage, respectively  were significantly upregulated or downregulated in the seven stages , and BLISTER were involved in trichome branching (GO:0010091), eight unigenes, including FAS1, GL2, and GDPDL3 were involved in trichome differentiation (GO:0010026), 17 unigenes, including GTL1, SPIRRIG, and FPP4 were involved in trichome morphogenesis (GO:0010090). Further, ECR was involved in trichome papilla formation (GO:1905499); RAC1 was involved in seed trichome differentiation (GO:0090379); and RNF115_126 was involved in trichome elongation (GO:0090378). Of the 40 trichome development-related unigenes, 29 were transcription factors, which belonged to 12 transcription factor families, including bHLH, bZIP, C2H2, and so on  signaling pathway, phospholipase D signaling pathway, and transforming growth factor beta (TGFβ) signaling pathway were enriched Fig. S. Among tSecondary metabolites play major roles in the adaptation of plants to the environment and in overcoming stress conditions, while also contributing to the specific odors, tastes, and colors of plants. Glandular hairs can secrete chemicals to protect against biological and abiotic stresses and signal transduction, including organic acids, polysaccharides, proteins, polyphenols, alkaloids, and terpenoids. The analysis of pathways enriched by KEGG revealed that phenylpropanoid biosynthesis related to plant disease resistance was enriched at S1-S7 stages. The biosynthesis of sesquiterpenoid, triterpenoid, and monoterpenoid related to odors and tastes were enriched at S1, S2, S3, and S6. The biosynthesis of flavonoids related to colors was enriched at S1. The biosynthesis of tryptophan metabolism, glycerolipid metabolism, terpenoid backbone, and brassinosteroid, related to plant growth- and development-related hormones were enriched at different stages. The biosynthesis of carotenoid, cysteine and methionine metabolism, unsaturated fatty acids and phenylalanine, tyrosine and tryptophan were also enriched at different stages Table .Table 1APC11, CDC7, CDC53, CDC54, and CDC47 were up-regulated in S1, whereas CDK2 was down-regulated; CDC7, CDC45, GSK3B, YWHAE, and MCM2 were up-regulated in S2, and no gene was down-regulated; MCM6 and CHK2 were up-regulated in S3; CHK2, CDK2, BUB1, and ORC1 were up-regulated in S4; No gene was upregulated in S5; RBX1, CCNA, SKP1, and MCM2 were up-regulated in S6, with the important thing being that CDK2 expression was down-regulated; and 35 genes, including CCNB, were up-regulated in S7, with only BUB1, TFDP1, and GSK3B being down-regulated  at different flowering stages of ‘Yujin 1’ vs ‘Damaohua’, and found that a total of 2616 DEGs annotated to the PRG database and distributed across 13 classes, including CN, CNL, Mlo-like, N, NL, Pto-like, RLK, RLK-GNK2, RLP, RPW8-NL, T, TN, TNL, and unknown (Table S11). Among these, the distribution of PRGs in the RLP class was the highest, with the TNL class being second; However, the proportion of up-regulated PRGs was highest in the TNL class  covered the entire life cycle of the plant. Based on these transcriptomic data, we obtained 33,733 candidate genes that were differentially expressed at seven flowering stages.Although the DEGs analyses of nine tissues and transcriptional regulation during flower development in  RNA-Seq , 30, theArabidopsis thaliana. They found that the formation and development of trichomes are regulated by GA and CTK signaling, and transcription factors including MYB, bHLH, C2H2 [GIS, GIS2, and ZFP8 [glh6, which promoted trichome papillae development [GL2, GTL1, and TRY was downregulated at S1. However, ABIL3 and ECR were significantly increased at S1, which may have caused the trichome of ‘Yujin 1’ to be denser and longer.At present, extensive research on the formation and development of trichomes in LH, C2H2 . Sequencand ZFP8 . The triand ZFP8 , 31. GTLand ZFP8 . ECR, alelopment . It was elopment . In thisSKD1 were upregulated at S2 and S3, which may have been correlated with the secretion of metabolites in glandular trichomes.A key and unique feature of glandular trichomes, relative to their size, is the capacity to synthesize and secrete large quantities of a limited number of metabolites: primarily terpenoids, but also phenylpropanoids and flavonoids . In thisAPC11, CDC7, CDC47, CDC53, and CDC54 were up-regulated at S1, and CDK2 was down-regulated. It was reported that the ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) (APC11) is critical for cell cycle synchronization in the endosperm of Arabidopsis thaliana [APC11 may be closely related to the density of ‘Yujin 1’. The roles of CDC7, CDC47, CDC53, and CDC54 in plants are rarely reported, which warrant further study.Trichome development is intimately related to the cell cycle . It was thaliana . The uprPlant trichomes frequently function as the first line of defense against biotic and abiotic stresses via space hindrance . In thisL. japonica cultivars, toward the identification of biological processes and functional gene activities on trichome development that occur during flowering stage. This study provides a comprehensive characterization of the expression profiles of flower development during the seven developmental stages of L. japonica, and screened several genes may involve in the floral biosynthesis of odors, tastes, colors, and plant hormones, and proposed biosynthetic pathways for sesquiterpenoid, triterpenoid, monoterpenoid, flavonoid, and plant hormones. Furthermore, 82 DEGs were assigned to cell cycles and 2616 were predicted as plant resistance genes. In all, this study provides a theoretical basis for the identification of Lonicera japonica Thunb. varieties and the selection of new varieties, and lays a solid theoretical foundation for the further study of the molecular mechanism of its specific characteristics. It has important academic and application value. In the subsequent work, genetic transformation and phenotypic identification of the selected genes will be conducted to further study their effect on trichome development.This work presents a genome-wide comparative transcriptome analysis firstly between two L. japonica were five-year ‘Damaohua’ and ‘Yujin 1’ that grow in the resource garden of the College of Life Sciences, Henan Normal University . ‘Damaohua’ and ‘Yujin 1′ were identified as honeysuckle by Li at Henan Normal University, China. The fresh flower buds or flowers of ‘Damaohua’ and ‘Yujin 1′ were collected separately from five plants at seven stages: (S1) young bud stage; (S2) three-green stage; (S3) two-white stage; (S4) great-white stage; (S5) silver stage; (S6) golden stage; (S7) fade stage. During sample collection, the flowers of five plants were combined and regarded as one biological replicate that representing each stage, and three independent replicates were performed. Partial flower materials were flash frozen in liquid nitrogen following collection and stored at − 80 °C.The two breeds of L. japonica bud or flower samples. According to methods outlined by Ning et al. [The fresh buds or flowers of two varieties at S3 were sliced and added to distilled water on microscope slides for observation and photography. In addition, we used a scanning electron microscope (SEM)  to observe the arrangement of trichomes on g et al. , the budThe total RNA was extracted using a mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol . RNA intThe libraries above were sequenced using an Illumina HiSeq X Ten sequencer  and 150 bp paired-end reads were generated. The preparation and sequencing of the cDNA library were performed at Shanghai OE Biotech. Co., Ltd., Shanghai, China. Raw data (rawreads) were processed using Trimmomatic . Reads c− 5.The functions of the unigenes were annotated as NCBI non-redundant protein (NR), Clusters of orthologous groups for eukaryotic complete genomes (KOG), Gene Ontology (GO), Swiss-Prot, evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using diamond software, and mapped to Pfam databases by HMMER. The search was conducted using Blastx  with a tP value < 0.05 and foldchange > 2 or foldchange < 0.5 was set as the threshold for significant differential expression. Hierarchical DEGs cluster analysis was performed to explore transcript expression patterns. DEGs GO enrichment and KEGG pathway enrichment analyses, respectively, were performed using R, based on hypergeometric distribution.The FPKM  and readhttp://planttfdb.cbi.pku.edu.cn/index.php) is a plant transcription factor database that includes the sequences of 58 plant transcription factor families from 165 plant species [p value ≤0.05 and q value ≤0.05 [PlantTFDB ( species . The seque ≤0.05 .http://prgdb.crg.eu/wiki/Main_Page) contains more than 112 resistance genes and 104,335 candidate Resistance Genes [http://prgdb.crg.eu/wiki/Category:Classes. The sequence of unigenes were Blastx aligned to the PRG database, and the best of these with an E value of less than 10− 5 was screened as the annotation information of the unigene. The Expression analysis of the PRGs were the same as the TFs.The Plant Resistance genes database , and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide. Quantification was performed using a two-step reaction process: reverse transcription (RT) and PCR. Quantification was performed using the 2anscript .p ≤ 0.05 and p ≤ 0.01 using SPSS 19.0 software. All expression analyses were performed in three replicates. The reported values represented arithmetic averages of three replicates, and the data was expressed as a mean plus or minus standard deviation (mean ± SD).Significant differences were calculated using a one-way ANOVA analysis with a Turkey test and a significance level at Additional file 1: Figure S1. Summary of top 10 GO terms and top 20 KEGG pathway assignments for the L. japonica flower transcriptome. A-G: The top 10 GO terms in S1, S2, S3, S4, S5, S6, and S7. H-N: The top 20 KEGG pathways in S1, S2, S3, S4, S5, S6, and S7.Additional file 2: Figure S2. Transcript expression analysis for unigenes associated with signal transduction in different stages of L. japonica. A: Plant hormone signal transduction; B: phosphatidylinositol signaling system; C: Wnt signaling pathway; D: AMPK signaling pathway; E. phospholipase D signaling pathway; F: TGF-beta signaling pathway. Changes in expression levels are represented by color; blue indicates a lower expression level and red indicates a higher expression level.Additional file 3: Table S1. Comparison of the density of glandular and non-glandular hairs in ‘Damaohua’ and ‘Yujin 1’ (mm-2). Table S2. Comparison of the lengths of glandular and non-glandular hairs in ‘Damaohua’ and ‘Yujin 1’ (μm). Table S3. Assembly summary of transcriptomic data. Table S4. Preprocessing results of sequencing data quality. Table S5. Annotation results of the unigenes. Table S6. Summary of top 10 GO terms and top 20 KEGG pathway assignments for the L. japonica flower transcriptome. Table S7. DEGs involved in trichome development. Table S8. Primers used for analysis of gene expression by qRT-PCR. Table S9. DEGs involved in signal transduction. Table S10. DEGs involved in secondary metabolites. Table S11. DEGs involved in plant resistance.


Clinically, high cyclooxygenase-2 expression in malignant glioma correlates well with poor prognosis and the use of aspirin is associated with a reduced risk of glioma. To extend the current understanding of the apoptotic potential of aspirin in most cell types, this study provides evidence showing that aspirin induced glioma cell apoptosis and inhibited tumor growth, in vitro and in vivo. We found that the human H4 glioma cell-killing effects of aspirin involved mitochondria-mediated apoptosis accompanied by endoplasmic reticulum (ER) stress, Noxa upregulation, Mcl-1 downregulation, Bax mitochondrial distribution and oligomerization, and caspase 3/caspase 8/caspase 9 activation. Genetic silencing of Noxa or Bax attenuated aspirin-induced viability loss and apoptosis, while silencing Mcl-1 augmented the effects of aspirin. Data from genetic and pharmacological studies revealed that the axis of ER stress comprised an apoptotic cascade leading to Noxa upregulation and apoptosis. The apoptotic programs and mediators triggered by aspirin in H4 cells were duplicated in human U87 glioma cell line as well as in tumor-bearing BALB/c nude mice. The involvement of ER stress in indomethacin-induced Mcl-1 downregulation was reported in our previous study on glioma cells. Therefore, the aforementioned phenomena indicate that ER stress may be a valuable target for intervention in glioma apoptosis. Cyclooxygenase (COX) is a rate-limiting enzyme involved in the synthesis of eicosanoids from membrane-released arachidonic acid. The constitutive COX-1 isozyme is present in most tissues and serves to help maintain normal physiological functions. COX-2 expression is low under normal conditions, but is rapidly induced in stressed situations to mediate pathological responses . TheoretNonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed COX inhibitors, and include aspirin, indomethacin, and ibuprofen nonselective COX inhibitors, as well as celecoxib and NS398 selective COX-2 inhibitors. Monotherapy and combinatory therapy with COX inhibitors have been reported in the treatment of various types of cancers. Cell cycle progression, migration, invasion, angiogenesis, autophagy, apoptosis, and resistance can be classified as biochemical events that are vulnerable to either direct or indirect actions of COX inhibitors ,5,6,7,8.Glioma is an aggressive brain cancer, particularly the most malignant type known as glioblastoma multiforme, which originates from astrocytes. Despite advances in surgical techniques, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the responses and prognosis of patients with malignant glioma remain unsatisfactory ,10. FromIncreased expression of anti-apoptotic Bcl-2 family proteins and/or decreased expression of pro-apoptotic members have been demonstrated in patients with malignant glioma, revealing a crucial role of apoptosis resistance in drug resistance, poor prognosis, and recurrence ,17,18. IAspirin caused a reduction of cell viability, which was related to the concentration effect A and timTo further explore the apoptotic actions caused by aspirin, cell proliferation and apoptosis-related regulators were identified using Western blotting. An upregulation of p27 and a downregulation of cyclin D1 protein were found in aspirin-treated cells A. AspiriBH3-only Bcl-2 family proteins promote the transition to apoptosis, while Mcl-1 and Bcl-2 are the key regulators involved in antagonizing the apoptotic program . Thus, tSeveral upstream regulators participate in the expression of Noxa including ER stress, TP53, and the FoxO family members ,35,36. ITo demonstrate the apoptotic potential of aspirin directed against glioma cells, human U87 cell line was analyzed for comparison. As with H4 cells, in U87 cells aspirin still caused loss of cell viability A, increaTo determine whether aspirin could alter the ER stress/Noxa axis, induce apoptosis, and retard tumor growth in vivo, U87 cells were inoculated subcutaneously into the flanks of BALB/c nude mice. Aspirin treatment caused a reduction in tumor volume A and tumThe therapeutic use of aspirin in clinical practice yields diverse and conflicting results, but studies have revealed a promising association of aspirin use and a reduced risk of glioma ,31,37. IAspirin has a profound apoptotic effect on cancer cells via Akt, β-catenin, NF-κB or ER stress. The diverse signals converge at the mitochondria where they are integrated, resulting in anti-apoptotic protein downregulation and/or pro-apoptotic protein upregulation ,42,43,44TP53 [The levels of Mcl-1 and Noxa are regulated at several steps, particularly the transcriptional level ,45,46. ETP53 ,35,36,50It should be noted that growth arrest may represent an alternative mode of action in aspirin-induced viability loss due to results of flowcytometry and protein expression of p27 and cyclin D1. Activation of ER stress is able to induce glioma cell cycle arrest . The invThe current standard treatment for malignant glioma is limited to surgical resection, followed by radiotherapy and chemotherapy ,10. AmonDespite these interesting findings, our study has certain limitations. The concentrations of aspirin in current cell studies (3 mM) are higher than the therapeutic aspirin concentrations in humans (~0.5 mM). These relatively high concentrations of aspirin may cause adverse effects, particularly in the gastrointestinal tract. Thus, a careful risk-benefit evaluation is required.Human U87 MG glioblastoma (ATCC HTB-14) and H4 neuroglioma (ATCC HTB-148)  cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). To conduct experiments, cells were placed in DMEM containing 2% FBS.® AQueous Non-Radioactive Cell Proliferation Assay kit)  and LDH Assay Kit , respectively.Cells were seeded onto a 96-well plate and subjected to treatments. At the end of treatments, cell viability and damage were measured using a MTS reduction assay kit (CellTiter 96x 380 nm and Em 460 nm) and the fluorescence signals were normalized by protein contents.Cells were seeded onto a 6-well plate and subjected to treatments. At the end of the treatments, cells were homogenized and subjected to enzymatic reaction of caspase using the Caspase Fluorometric Assay kit  instructions. The liberated fluorescent AMC moiety was measured with a fluorometer (ECells (500 cells/well) were seeded onto 6-well plates in DMEM supplemented with 2% FBS. Two days after seeding, the cells were treated with various concentrations of aspirin (0–10 mM) for 6 days. At the end of treatments, cells were fixed and stained with crystal violet.Cells were seeded onto a 6-well plate and subjected to treatments. At the end of treatments, the detached cells were fixed, incubated with 100 µg/mL RNase, and stained with propidium iodide  to assess cell cycle distribution.Cells were seeded onto a 6-well plate and subjected to treatments. At the end of treatments, mitochondrial and cytosolic fractions were prepared using a Mitochondria/Cytosol Fractionation Kit  according to the manufacturer’s instructions.TM siRNA transfection reagent  according to the manufacturer’s instructions.The siRNAs against human Bax, Bcl-2, Mcl-1, Noxa, Bim, PERK, eIF2α, and ATF4 and control siRNA were purchased from Santa Cruz Biotechnology . Cells were transfected with siRNAs using INTERFERinProteins were extracted from treated cells and resected tumors using Tissue Protein Extraction Reagents . After routine SDS-PAGE separation, transfer onto PVDF membrane, and blocking, the membranes were sequentially incubated with primary antibodies, horseradish peroxidase-labeled IgG, followed by detection using enhanced chemiluminescence (ECL) Western blotting reagents. The intensity of each signal was measured by a densitometer. The used primary antibodies recognized PARP-1, p27, cyclin D1, Bad, Bid, Bim, Puma, Noxa, Bax, Bak, Bcl-2, Mcl-1, FLIP, PERK, phospho-PERK, eIF2α, phospho-eIF2α, IRE1, phospho-IRE1, ATF4, FoxO1, CHOP, cytochrome oxidase IV (COX IV), cleaved caspase 3 , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , andβ-tubulin .6 cells in 100 µL of serum-free DMEM) were inoculated subcutaneously into the right flank of 8-week-old BALB/c nude mice. One week later, mice were randomly divided into two groups (n = 6 per group): one group of mice received once daily treatment with aspirin  for three weeks and the other group, which served as the control, received saline vehicle. The dose of aspirin was considered according to our pilot studies (data not shown). After three weeks of treatments, tumor volume was measured using the Peira TM900 system  [The Animal Experimental Committee of Taichung Veterans General Hospital reviewed and approved the protocols of the animal study . U87 cells (1 × 10Belgium) . Then, tp value less than 0.05 was considered statistically significant.Data are expressed as means ± standard deviations. Statistical comparisons were analyzed using one-way analysis of variance followed by Tukey’s or Dunnett’s test. A


C9orf72 (chromosome 9 open reading frame 72), MAPT (microtubule-associated protein tau) and GRN (granulin), and their impact on neuronal and glial pathology. Further, evidence for white matter dysfunction in the aetiology of FTD and the clinical, neuroimaging and genetic overlap between FTD and leukodystrophy/leukoencephalopathy are discussed. The review highlights the role of common variants and mutations in genes such as CSF1R (colony-stimulating factor 1 receptor), CYP27A1 (cytochrome P450 family 27 subfamily A member 1), TREM2 (triggering receptor expressed on myeloid cells 2) and TMEM106B (transmembrane protein 106B) that play an integral role in microglia and oligodendrocyte function. Finally, pharmacological and non-pharmacological approaches for enhancing remyelination are discussed in terms of future treatments of FTD.Frontotemporal dementia (FTD) is a common cause of presenile dementia and is characterized by behavioural and/or language changes and progressive cognitive deficits. Genetics is an important component in the aetiology of FTD, with positive family history of dementia reported for 40% of cases. This review synthesizes current knowledge of the known major FTD genes, including  GRN), have a significant impact on glia function, and mutation carriers show a specific pattern of white matter abnormalities. Further, genome-wide association studies (GWAS) and next-generation sequencing (NGS) of clinical and neuropathological FTD cohorts have identified a number of novel FTD genes associated with myelin or white matter abnormalities. In this review, we will synthesize current knowledge of the known FTD genes in terms of their impact on neuronal and glia neuropathology and function. We will examine the role of white matter in the aetiology of FTD and the clinical, neuroimaging and genetic overlap between FTD and conditions with white matter dysfunction . These insights could lead to a better understanding of pathogenic mechanisms and identify potential therapeutics for this complex disease.Frontotemporal dementia (FTD) syndromes are a heterogeneous group of proteinopathies, characterized by progressive degeneration of the frontal and/or temporal lobes. Clinically, they are divided into three subtypes. The behavioural variant FTD (bvFTD) is characterized by progressive deterioration of personality, social comportment and cognition , while tMAPT) [GRN) [C9orf72) [TBK1) is found in 1–2% of total FTD cases [VCP), chromatin-modifying protein 2B (CHMP2B), TAR-DNA-binding protein 43 (TARDBP), fused in sarcoma (FUS), coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) and triggering receptor expressed on myeloid cells 2 (TREM2) are known to contribute to <5% of all FTD cases [CYLD as a causal gene for FTD and amyotrophic lateral sclerosis (ALS), but it appears to be a relatively rare cause of the disease [Genetics is an important component in the aetiology of FTD, with approximately 10% of cases being autosomal dominant and 40% of patients having a family history of dementia . BvFTD iT) [GRN) ,6 and chC9orf72)  are the TD cases ,12. LessTD cases ,14. Our  disease ,16.The three FTD clinical sub-types display significant regional difference in grey matter structural atrophy and their connecting white matter tracts, with bvFTD showing specific prefrontal cortex and striatum grey matter atrophy and greater cerebellar white matter loss compared to other FTD subtypes . CorrespMAPT variants account for 20% of heritable FTD cases and are responsible for most familial cases with FTLD-Tau pathology [MAPT mutations [athology , which iutations ,19. Baseutations .MAPT mutation carriers is the behavioral variant (bvFTD), which can be concomitant with a dominant Parkinsonism phenotype such as CBS or PSP [The main clinical subtype for S or PSP ,23,24. NS or PSP ,26 that GRN, C9orf72 and TBK1, whose features will be discussed below.Approximately 50% of all FTLD cases are FTLD-TDP . FTLD-TDC9orf72 expansions are associated with the bvFTD subtype [C9orf72 expansion carriers display additional degeneration of motor neurons or FTLD with amyotrophic lateral sclerosis (FTLD-ALS) [C9orf72 is the most common cause of FTD and ALS in Europe and North America [C9orf72 expansion in Australian and Spanish populations [As much as 85% of FTD cases caused by  subtype , while 2TLD-ALS) ,32. The TLD-ALS) ,33. Alth America , it is e America ,35,36,37ulations ,39.C9orf72 expansion is a combination of typical ALS and Type B or pure Type A [C9orf72 expansion was shown to impair the ability to degrade TDP-43, leading to TDP-43 accumulation in neurons and, occasionally, oligodendrocytes [The neuropathology associated with e Type A ,40,41,42drocytes . Both RNTBK1 are found in ALS, FTD and ALS-FTD cases [TBK1 variants are also associated with PNFA [TBK1 mutations are known to give rise to both FTLD-TDP subtype A [Loss-of-function mutations in TD cases , with bvTD cases ,32,45. Hith PNFA . Phenotyith PNFA . TBK1 muubtype A  and B [1ubtype A ,32,45,48ubtype A ,45,46, nubtype A ,47 and gGRN mutations are loss-of-function [GRN mutations to FTLD-TDP pathology have been proposed: progranulin-mediated caspase 3-dependent cleavage of TDP-43 [GRN is a growth factor and modulator of microglia, and mutation carriers have enhanced microglia activation [The majority of function  and accofunction , giving function . This FTfunction , as wellfunction . To datef TDP-43 , sortilif TDP-43  and impaf TDP-43  in the ntivation  and gliotivation ,56 upon GRN is highly heterogeneous in age of onset and clinical presentations, even for the same variant [GRN variants have been associated with parkinsonism, CBS and Alzheimer’s disease (AD) [GRN variant carriers present bvFTD followed by PNFA, and less than 10% are affected by CBS, AD and schizophrenia [GRN variant carriers than in carriers of other mutations [GRN-associated FTD [FTD associated with  variant . GRN varase (AD) ,59. Overophrenia ,60. Indeutations ,31,32, cutations . Neuroimutations ,26,58. Oated FTD ,64,65,66White matter involvement in FTD has been widely reported ,75,76,77White matter alteration is prevalent in early symptomatic phase ,75,81 anMAPT and C9orf72 mutations exhibited a relatively discrete and distinctive white matter profile that showed alterations in white matter within the left anterior temporal pole when compared to control and Alzheimer’s disease subjects [C9orf72, MAPT and GRN mutations have revealed early and widespread loss of white matter integrity in pre-symptomatic FTD, and there were clear genotypic “fingerprints” of white matter loss associated with each disease gene [Consistent and widespread white matter tract pathology has been reported in bvFTD patients, compared to clinically normal subjects and those with AD and other forms of dementia ,89. A susubjects . Furtherase gene ,83,84,85The human myelin lipidome consists of 700 different lipid moieties, 60% of which are species that are classified as phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, cerebrosides and sulfatides . The molLipid molecules in the brain are involved in energy storage, maintaining the structural integrity of the nervous system, signal transduction, modulation of membrane fluidity, trafficking of membrane proteins/receptors, cytoskeletal organization and neurotransmission ,93. The Changes in brain lipid metabolism are also evident in FTD. Lipidomic analysis performed on the plasma of bvFTD and AD patients has revealed a significant increase in triglycerides and decreases in phosphatidylserine and phosphatidylglycerol in bvFTD patients compared to AD patients ,108. FurGRN variants frequently lead to extensive gliosis and loss of myelin in the underlying white matter [GRN mutations [GRN mutation. Indeed, homozygous carriers of GRN mutations develop young adult-onset neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease characterized by lipofuscin deposition [e matter ,110,111.utations ,64,65,66utations . These oposition ,114.GRN mutations to dysregulation of lipid metabolism remains to be elucidated, the presence of the lipofuscinosis phenotype in GRN mutation carriers implicates lysosome regulation [GRN mutation carriers and Grn−/− mice [Grn−/− mice and FTD patients carrying GRN mutations [While the exact mechanism that links gulation ,116. Indgulation , has beegulation . In addi−/− mice . It was −/− mice . Of inteutations .Leukoencephalopathy is a collective term for a group of heterogeneous primary white matter disorders that can be either acquired or hereditary. Leukodystrophies—a term that derives from “leuko” = white, “dys” = lack of and “trophy” = growth—are heritable leukoencephalopathies. Leukodystrophy is defined as “heritable disorders affecting the white matter of the central nervous system, sharing glial cell or myelin sheath abnormalities, the neuropathology of which is primarily characterized by the involvement of oligodendrocytes, astrocytes and other non-neuronal cell types, although in many disorders the mechanism of disease remains unknown, and in other cases is suspected to include significant axonal pathology” .Leukodystrophies are highly variable in age of onset and clinical manifestations ,121, whiArylsulfatase A (ARSA) mutations [The diagnosis of leukodystrophies is slow and challenging, and the prognosis of patients is poor as a result of very limited therapies, which are only beneficial in the early onset of the diseases . Recent utations .MAPT [GRN [C9orf72 [It is of interest to note that while the major FTD causal genes such as MAPT , GRN [5,APT [GRN  and C9or[C9orf72  were ide[C9orf72 . These lTMEM106B causes childhood-onset leukodystrophy, a hereditary white matter disease [APOE, TOMM40 and LRRK2 are involved in lipid metabolism [GWAS is based on the rationale of the “common disease, common variant” hypothesis that attributes common diseases to allelic variants that are present in more than 1–5% of the population . The ass disease , and APOtabolism ,146,147.The “rare variants” hypothesis postulates that rare variants, primarily defined as having allele frequency <0.001 in the general population, could provide an explanation for a portion of missing heritability observed in twin studies  and impaARSA [CSF1R [TREM2 [TYROBP [NOTCH3 [CYP27A1 [To date, there are over 60 genes with diverse biological functions that have been associated with leukodystrophy/genetic leukoencephalopathies ,170, andARSA ,172, CSFA [CSF1R ,173,174,R [TREM2 ,176,177, [TYROBP , NOTCH3  [NOTCH3  and CYP2[CYP27A1  have bee[CYP27A1  showed t[CYP27A1 . RespectTMEM106B belongs to the TMEM106 family of Type II transmembrane proteins, mainly localized in lysosomes ,183,184.TREM2 and TYROBP variants are found in patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) or Nasu–Hakola disease (NHD), an autosomal recessive hereditary disease characterized by early-onset dementia and bone cysts [TREM2 variants [TYROBP variants [In the brain, TREM2 is exclusively expressed in microglial cells, where it interacts with TYROBP to initiate signaling cascades that promote microglial cell activation, phagocytosis and microglial survival . TREM2 ane cysts ,204, conne cysts , althougne cysts ,176,206.variants ,176,177 variants  are assoMicroglial cells are pivotal in remyelination by clearing myelin debris from sites of demyelination. Macrophages deficient in TREM2 were reported to have defects in the phagocytic pathways. A recent study of TREM2-deficient mice revealed cholesteryl ester accumulation in the brain and microglia, due to the transgenic mice’s inability to upregulate lipid metabolism genes. This led to an impaired ability of the microglia to phagocytose an influx of cholesterol brought about by chronic demyelination .CSF1R encodes the colony-stimulating factor 1 receptor, a transmembrane tyrosine kinase receptor found in mononuclear phagocytotic cells and microglia in the brain that is crucial for microglial function [CSF1R variants are associated with hereditary diffuse leukoencephalopathy (HDLS) with axonal spheroids and pigmented glia (ALSP) [CSF1R variants were identified in patients with presentations similar to that of parkinsonism [function ,209. CSFa (ALSP) , bvFTD [a (ALSP) , CBS, ADa (ALSP) . Heterozinsonism  and bvFTinsonism ,174. Indinsonism .Neuropathologically, CSF1R-related leukoencephalopathy resulted in patchy white matter degeneration, featuring atrophy and confluent bilateral white matter hyperintensities, accompanied by loss of myelin and axons, presence of neuroaxonal spheroids and lipid-laden and pigmented macrophages ,212,213 CYP27A1 encodes sterol 27-hydroxylase, a mitochondrial cytochrome p450 oxidase involved in cholesterol metabolism and the synthesis of bile acids. Homozygous or compound heterozygous mutations in CYP27A1 lead to cerebrotendinous xanthomatosis (CTX), a lipid storage disorder associated with a diverse range of neurological dysfunctions [CYP27A1 was reported in a patient with clinical FTD, but without the typical CTX neuroimaging changes when examined using MRI [unctions . Of notesing MRI . Criticasing MRI , making The consistent changes in white matter observed in FTD brains, as well as the genetic overlap between FTD and leukoencephalopathies/leukodystrophies, strongly support the hypothesis that therapies targeting white matter integrity, or remyelination, would be efficacious. Both processes of myelination and re-myelination after injury are mediated by oligodendrocytes . The matTMEM106B is one of the genes that straddle the genetic spectrum of FTD and leukodystrophy, and knock-down of its expression appears to have a protective role in myelination [TMEM106B expression was found to be effective in rescuing the FTD-like phenotypes in a transgenic mouse model carrying the GRN [C9orf72, transgene [C9orf72 mutation carriers compared with GRN mutation carriers [The genetic overlap between leukoencephalopathies/leukodystrophies and FTD has opened up exciting opportunities for FTD therapeutics. lination . Genetic the GRN , but notransgene . Howevercarriers . The concarriers , while Pcarriers . Recentlcarriers ; also, pcarriers .Transcranial magnetic stimulation (TMS) is a non-invasive form of neural stimulation that applies local magnetic fields to generate electric currents in the brain, leading to an increase in neuronal activity  that is GRN, MAPT and C9orf72 mutation carriers, demonstrated efficacy, with significant increase of intracortical connectivity and improvement in clinical scores and behavioral disturbances in both symptomatic patients and pre-symptomatic mutation carriers [Currently, clinical applications of TMS include diagnosis, monitoring and treatment of neurodegenerative diseases such as epilepsy , PD 230230, AD  and ALS carriers .Recent advances in sequencing technology have enabled the rapid discovery of novel FTD genes that enhance our understanding of this complex disease. While previous investigation into the pathogenesis of causal FTD genes has focused on their impact on neuronal functions, GWAS and NGS have revealed a number of novel FTD genes that affect both neurons and glia, whose mutations are associated with white matter abnormalities in carriers. In particular, our review highlights the extensive clinical and genetic overlap between FTD and leukoencephalopathy/leukodystrophy. This, in turn, has implications for pharmacological and non-pharmacological strategies to enhance oligodendrocyte numbers and the myelination process.


Lung cancer is the most common cancer and the most common cause of cancer-related death worldwide. However, the molecular mechanism of its development is unclear. It is imperative to identify more novel biomarkers.  Two datasets (GSE70880 and GSE113852) were downloaded from the Gene Expression Omnibus (GEO) database and used to identify the differentially expressed genes (DEGs) between lung cancer tissues and normal tissues. Then, we constructed a competing endogenous RNA (ceRNA) network and a protein-protein interaction (PPI) network and performed gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and survival analyses to identify potential biomarkers that are related to the diagnosis and prognosis of lung cancer.  A total of 41 lncRNAs and 805 mRNAs were differentially expressed in lung cancer. The ceRNA network contained four lncRNAs , 21 miRNAs, and 48 mRNAs. Functional analyses revealed that the genes in the ceRNA network were mainly enriched in cell migration, transmembrane receptor, and protein kinase activity. mRNAs DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 had the highest connectivity in the PPI network. Immunohistochemistry (IHC) demonstrated that mRNAs DLGAP5, MCM7, RACGAP1, and RRM2 were upregulated in lung adenocarcinoma (LUAD). Survival analyses showed that lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 were associated with the prognosis of LUAD.  lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 might be the biomarkers of LUAD. For the first time, we confirmed the important role of lncRNA CLDN10-AS1 in LUAD. Lung cancer has the highest incidence of all cancers  and the highest death rate  . The undProtein-coding genes are widely studied in lung cancer over the past decades, but the human genome transcribes less than 2% of the protein-coding genes. 85% are noncoding RNAs, including long noncoding RNAs (lncRNAs) . lncRNAshttps://www.ncbi.nlm.nih.gov/gds/), which provides us a large amount of genomic data about lung cancer. Two datasets (GSE70880 and GSE113852) were downloaded from GEO and used to identify the differentially expressed genes (DEGs) between lung cancer tissues and normal tissues. Then, we constructed a competing endogenous RNA (ceRNA) network and a protein-protein interaction (PPI) network and performed gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and survival analyses to identify potential biomarkers that are related to the diagnosis and prognosis of lung cancer. We verified the expression differences and measured the diagnostic roles of lncRNAs through The Cancer Genome Atlas (TCGA) database .This study aimed to explore more biomarkers of lung cancer via integrated bioinformatics analysis. Gene Expression Omnibus (GEO) is a NCBI's publicly available genomics database (We downloaded gene expression microarray datasets GSE70880 and GSE113852 from the GEO database. The criteria they met were as follows: (1) the studies were about lung cancer, (2) tissue samples included the tumor and corresponding adjacent tissues or normal tissues, (3) the number of total samples was not less than 40, and (4) the datasets must include lncRNAs and mRNAs.P value <0.05  were considered statistically significant for the DEGs.We merged the two datasets not only to increase the sample size but also to facilitate subsequent analyses. Based on different platforms, days, environments, and people, the samples had heterogeneity and potential variables, which might lead to a bias. Therefore, we batch-normalized the merged dataset by suing limma and sva packages in software R (v3.6.1). Next, we performed gene differential analysis between tumor and normal tissues by limma package. |LogFC| > 1 and adjusted http://www.mircode.org/) [http://mirtarbase.mbc.nctu.edu.tw/) [http://www.targetscan.org/) [http://www.mirdb.org/miRDB/) [In order to find out the competing endogenous regulating network mediated by lncRNAs and miRNAs, the ceRNA network was constructed. CeRNA networks link the functions of protein-coding mRNAs with that of noncoding RNAs such as microRNA, long noncoding RNA, pseudogenic RNA, and circular RNA . The conde.org/) . lncRNAsde.org/) . (2) miRedu.tw/) , TargetSan.org/) , and miR/miRDB/) . The inthttp://string‐db.org/cgi/input.pl).Functional enrichment analysis includes GO and KEGG analysis. Genes in the ceRNA network were analyzed by GO and KEGG analysis by package clusterprofiler in software R. In GO analysis, functions were divided into biological processes (BP), molecular functions (MF), and cellular component (CC). PPI network was constructed by using STRING , respectively [P < 0.05 was considered statistically significant. Genes related to the prognosis of LUAD or LUSC would be considered as the hub genes.To confirm whether lncRNA is associated with the survival of LUAD and LUSC was one of our research contents. Due to lack of clinical information, we performed Kaplan–Meier (KM) analyses of lncRNAs in the ceRNA network and genes in the PPI network through the Kaplan–Meier Plotter  curves by GraphPad Prism 7.0, and the values of cutoff, specificity, sensitivity, and area under the curve (AUC) were also calculated. The cutoff value is the best expression of a lncRNA for the diagnosis of lung cancer. P < 0.05 indicated significant difference.To confirm whether the expression levels of lncRNAs in the ceRNA network were different between normal and tumor tissues, we downloaded the gene expression profile from TCGA database and performed gene differential analysis of lncRNAs selected from the ceRNA network. There were two datasets and a total of 47 lung cancer tissue samples and 47 normal tissue samples in this study . We mergA total of 4 lncRNAs , 21 miRNAs, and 48 mRNAs were involved in the ceRNA network . In tumoGO terms and KEGG pathway analyses were performed by package clusterProfiler in software R. The results showed that biological processes were mainly associated with epithelial cell migration, regulation of epithelial cell migration, ameboidal-type cell migration, endothelial cell migration, endothelium development, and so on, while molecular functions were associated with carbohydrate binding, transmembrane receptor protein serine/threonine kinase activity, transmembrane receptor protein tyrosine phosphatase activity, transmembrane receptor protein phosphatase activity, carboxylic acid binding, and organic acid binding. Cellular component gathered in the basal part of cell, MCM complex, pore complex, cell-cell adherens junction, and basal plasma membrane . KEGG paP value was 1.07e − 07. We calculated the number of genes connected to each gene in the PPI network. Results revealed that five genes had the highest connectivity . All of them were upregulated. Immunohistochemistry (IHC) in The Human Protein Atlas (THPA) (https://www.proteinatlas.org/) database demonstrated that DLGAP5, MCM7, RACGAP1, and RRM2 were upregulated in LUAD  to perform survival analyses in LUAD and LUSC, respectively , SFTA1P (logrank P=0.00071), and ADAMTS9-AS2 (logrank P=0.0082) were associated with prognosis of LUAD. Upregulation of CLDN10-AS1 predicted poor prognosis, while upregulation of the SFTA1P and ADAMTS9-AS2 was related to good prognosis. mRNAs DLGAP5 (logrank P=4.7e − 05), E2F7 (logrank P=9.8e − 05), MCM7 (logrank P=0.031), RACGAP1 (logrank P=2.3e − 05), and RRM2 (logrank P=0.0062) were associated with prognosis of LUAD. Their high expression related to poor prognosis. No lncRNAs and mRNAs were associated with the prognosis of LUSC. DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 were considered as hub genes. The heatmap showed the expression changes of the three lncRNAs and five mRNAs in the merged dataset  and ADAMTS9-AS2 (logrank P=0.00048) and mRNAs RACGAP1 (logrank P=1.7e − 06) and RRM2 (logrank P=0.015) were associated with the prognosis of stage I LUAD patients (P=0.031) was associated with the prognosis of stage II LUAD patients (P=0.006), ADAMTS9-AS2 (logrank P=0.0012), and mRNA RACGAP1 (logrank P=0.00019) were associated with the prognosis of LUAD patients with smoking history (P=0.0057), ADAMTS9-AS2 (logrank P=0.027), and mRNA DLGAP5 (logrank P=0.0074) were associated with the prognosis of LUAD patients without smoking history  of LUAD were downloaded from the TCGA database. After gene differential analysis, we observed that, in tumor samples, CLDN10-AS1 was upregulated, while SFTA1P, SRGAP3-AS2, and ADAMTS9-AS2 were downregulated . The resIn the present study, we investigated the gene expression patterns of lncRNAs and mRNAs in lung cancer cells and corresponding adjacent tissues or normal tissues. We found that 525 genes were downregulated and 321 genes were upregulated in lung cancer cells. The ceRNA network revealed the correlation among lncRNAs, miRNAs, and mRNAs. Four lncRNAs  were involved in ceRNA network and survival analyses showed that CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 were associated with prognosis of LUAD. The PPI network showed the interaction among these mRNAs in ceRNA network and five mRNAs  had the highest connectivity. Survival analyses also revealed the relationship between the five mRNAs and the prognosis of LUAD, and they were confirmed as hub genes of LUAD finally. However, survival analyses indicated that these lncRNAs and mRNAs were not related to the prognosis of LUSC. To increase the credibility of our results, we verified the expression levels and measured the prognostic values of the four lncRNAs in LUAD through TCGA database. The results confirmed our conclusions and revealed that all the four lncRNAs were associated with the diagnosis of LUAD. lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2, and their related mRNAs DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 might be biomarkers of LUAD.Previous studies have explored the functions of lncRNAs as diagnostic and prognostic biomarkers . HOTAIR lncRNA SFTA1P had been reported previously. Huang et al. demonstrated that SFTA1P and CASC2 were associated with the regulation and development of LUSC and could be used as prognostic and predictive indicators of LUSC via integrated bioinformatics analysis . Zhang eThe number of miRNAs that connected with lncRNA ADAMTS9-AS2 was the largest among the four lncRNAs, suggesting it was a critical lncRNA. Studies about lncRNA ADAMTS9-AS2 in lung cancer were rare. Liu et al. demonstrated that lncRNA ADAMTS9‐AS2 was lowly expressed in lung cancer tissues by qRT-PCR. In their study, high expression of lncRNA ADAMTS9-AS2 reduced proliferation ability and inhibited migration and elevated their apoptosis rate. They also verified the relationship among lncRNA ADAMTS9-AS2, mRNA TGFBR3, and miRNA miR-223-3p. ADAMTS9-AS2 increased TGFBR3 expression, but miR-223-3p decreased both of them. miR-223-3p targeted TGFBR3 to enhance the ability of proliferation, migration, and invasion of lung cancer. They came to a conclusion that DAMTS9-AS2, TGFBR3, and miR-223-3p might provide potential therapeutic targets in lung cancer .P=0.026), indicating the potential functions in the prognosis of LUAD patients. It is noteworthy that CLDN10-AS1 has not been reported in lung cancer. For the first time, we confirmed the crucial role of lncRNA CLDN10-AS1 in LUAD. It might be related to the diagnosis, ability of migration, and prognosis of LUAD.miRNAs miR-125a-5p and miR-125b-5p only connected with lncRNA CLDN10-AS1, indicating lncRNA CLDN10-AS1 had a different binding trend among the four lncRNAs. CLDN10-AS1 might play a role in the development and progression of lung cancer, by regulating the G1/S transition of mitotic cell cycle through miR-125b-5p/PPAT and by regulating endothelium development, angiogenesis, and cell-cell adherens junction through miR-125b-5p/CDH5, respectively. Furthermore, CLDN10-AS1 was associated with the prognosis of LUAD . (4) We did not validate our results by experiment, giving us a direction for future research.Limitations existed in our study. (1) We only included two datasets. Although there were some other datasets, their sample size was too small. In order to increase the quality of our study, we excluded datasets with a sample size less than 40. (2) The two datasets are noncoding mRNA and lncRNA microarray, so we used the probe reannotation method to annotate gene symbol, which might drop some genes due to the failure of matching the probes. (3) Neither dataset contained clinical information. We could only perform survival analyses through the Kaplan–Meier Plotter (In our study, we identified the differentially expressed lncRNAs CLDN10-AS1, SFTA1P, SRGAP3-AS2, and ADAMTS9-AS2 by analyzing gene expression profiles from GEO. Among them, CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 and their related mRNAs DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 were associated with the prognosis of LUAD, suggesting they were more critical in LUAD. What is more, the four lncRNAs had diagnostic values in LUAD. lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 and their related mRNAs DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 might be biomarkers of LUAD. For the first time, we confirmed the important role of lncRNA CLDN10-AS1 in LUAD. It might be related to the diagnosis, ability of migration, and prognosis of LUAD.


The highest diagnostic accuracy  in identifying MT1 was provided by an irregular, yet intact endplate contour. This may be a useful MR feature for the differentiation between MT1 and spondylodiscitis, particularly in its early stage. On magnetic resonance (MR) imaging, Modic type 1 (MT1) endplate changes and infectious spondylodiscitis share similar findings. Therefore, this study investigated vertebral bone marrow and endplate changes to enable their differentiation. The lumbar spine MR examinations of 91 adult patients were retrospectively included: 39 with MT1; 19 with early spondylodiscitis without abscess; and 33 with advanced spondylodiscitis with abscess. The assessment included percentage of bone marrow edema on sagittal short tau inversion recovery images, and the signal ratio of edema to unaffected bone and endplate contour  on sagittal unenhanced T1-weighted images. Differences were tested for statistical significance by Chi-square test and mixed model analysis of variance. The MR diagnostic accuracy in differentiating MT1 and spondylodiscitis was assessed by cross-tabulation and receiver-operating characteristic analysis. The endplate contours, edema extents, and T1-signal ratios of MT1  were significantly different ( Propionibacterium acnes and Corynebacterium propinquum [Modic type 1 (MT1) vertebral endplate changes have been controversial findings since first defined in the late 1980s to describe endplate degeneration and subchondral bone marrow edema on MR imaging ,3. In thopinquum ,13,14,15opinquum ,18,19,20This is where the current MR imaging study becomes relevant. Vertebral bone marrow edema is a non-specific finding associated with low back pain, including a myriad of differential diagnoses: degeneration; Schmorl nodes; trauma; inflammation or infection; or sickle cell disease, as well as an iatrogenic, neurological, or neoplastic origin ,22,23. AConsequently, this study investigated the MR imaging features of the vertebral bone marrow and endplate in patients with MT1 and spondylodiscitis, particularly in its early stage, to obtain differentiating features that would aid in diagnostic accuracy.This retrospective study was approved by our institutional review board , and the study procedure was performed in accordance with the Declaration of Helsinki. Written, informed consent for clinical lumbar MR imaging was obtained from all patients, and informed consent for study participation was waived. Due to organizational reasons, the retrospective study evaluation was not started until June 2019. For this study, we reviewed the medical records from lumbar spine MR examinations, performed between January 2016 and April 2018, of adult patients (18 years of age or older), who had been referred to our tertiary referral center to undergo work-up because of low back pain.The following inclusion criteria were used to select patients:1. Patients with MT1 endplate changes :Available, standard, unenhanced MR examination of the lumbar spine that yielded a tentative diagnosis of MT1, as well as follow-up MR imaging (two to eight weeks afterward) confirming the diagnosis, with mono- or multisegmental involvement, without osseous destruction and vertebral height decrease, and without severe paravertebral phlegmonous changes involving the paravertebral musculature.Non-specific low back pain for at least three months, with or without signs of spinal segmental instability on clinical examination . 2. Patients with early stage spondylodiscitis :Available, standard MR examination, including contrast-enhanced images, of the lumbar spine that yielded a tentative diagnosis of early spondylodiscitis, defined as mono- or multisegmental involvement, without osseous destruction and vertebral height decrease, without severe paravertebral phlegmonous changes involving the paravertebral musculature, and without abscess formation. Clinical symptoms suggestive of spondylodiscitis: lumbar pain on heel strike, impaction, and percussion; pain on inclination and re-erection; constant lumbar pain worsening at night, with or without neurological deficits . Increase of inflammatory parameters , with or without fever.Positive proof of florid infection and/or pathogen, obtained by biopsy, needle aspiration, or blood culture.3. Patients with advanced stage spondylodiscitis :Available, standard MR examination, including contrast-enhanced images, of the lumbar spine that yielded a tentative diagnosis of spondylodiscitis, defined as mono- or multisegmental involvement, with or without destructive vertebral height decrease, with severe paravertebral phlegmonous changes involving the paravertebral fat tissue and musculature, and with abscess formation. Clinical symptoms suggestive of spondylodiscitis: lumbar pain on heel strike, impaction, and percussion; pain on inclination and re-erection; constant lumbar pain worsening at night with or without neurological deficits . Increase of inflammatory parameters , with or without fever.Positive proof of florid infection and/or pathogen, obtained by biopsy, needle aspiration, or blood culture.Exclusion criteria included: vertebral fractures; spondylolysis; severe scoliosis and congenital vertebral abnormalities; tuberculous spondylodiscitis; sero-positive rheumatoid arthritis and sero-negative forms of spondyloarthritis; myositis; previous spinal trauma; previous spinal surgery or interventional therapy; pregnancy; and metastatic tumor diseases.The MR examinations of the lumbar spine were performed on a 3.0 Tesla MR unit  using a 15-channel spine coil. All MR scans were performed in the supine position and head-first on the scanner table, with the patient’s arms positioned beside the body. All patients had a standard wedge-shaped foam lower leg support (15 cm maximum height), which resulted in slight bending of the hip and knee joints. Sagittal MR images were oriented along the posterior edge of the vertebral bodies, covering the lumbar spine in a craniocaudal direction from the upper endplate of vertebral body Th11 to at least vertebral body S2. In the dorsoventral direction, the lumbar spine was visualized, including at least 5 cm of antevertebral soft tissue and the skin of the dorsum. In the lateral direction, the lumbar spine was visualized, including the transverse process on both sides. The 3.0 Tesla MR protocol to image the lumbar spine included the following MR sequences relevant to this study:(a) A sagittal T1-weighted (w) fast-spin-echo (FSE) sequence (repetition time (TR), 500–650 msec; echo time (TE), 8 msec; flip angle, 90°; field of view (FOV), 300 × 300 mm; matrix, 800 × 800; in-plane resolution, 0.38 mm × 0.38 mm; slice thickness, 3 mm; interslice gap, 0.3 mm); (b) A sagittal T2-w FSE sequence ; (c) A sagittal short tau inversion recovery (STIR) sequence , 180 msec; flip angle, 90°; FOV, 300 × 300 mm; matrix 528 × 528; in-plane resolution, 0.57 mm × 0.57 mm; slice thickness, 3 mm; interslice gap, 0.3 mm).In addition, in all patients with spondylodiscitis, a contrast-enhanced sagittal T1-w FSE sequence and a transverse T1-w FSE sequence with selective fat supression were obtained. Please note that our retrospective evaluation was based on unenhanced MR images alone, because contrast-enhanced MR images were not available in all MT1 patients.All the MR examinations were anonymized and randomly presented on a picture archiving and communication system  to an MR imaging specialist , who was not aware of any patient data. For the assessment of the interreader agreement, a radiological fellow  evaluated the same images in an independent reading session, and was also unaware of any patient data. For the assessment of the intrareader agreement, the images were re-evaluated by the MR specialist five weeks after the first evaluation. First, the whole stack of the sagittal STIR images was used to confirm the presence of bone marrow edema of the vertebral body, defined as an area of high signal intensity within at least the subchondral vertebral bone marrow adjacent to the endplate . The edeSecond, a sagittal STIR image, showing the maximum hyperintense bone marrow edema changes, was manually selected, and the corresponding T1-w image was indicated by a reference tool. On this sagittal T1-w image, using a workstation-integrated software tool, a preformed circular region of interest (ROI) was manually adjusted to include an area of 200 pixels² and placed within the center of the visible signal changes, corresponding to the STIR-hyperintense edema area D. FurtheThird, on unenhanced sagittal T1-w images, the appearance and integrity of the vertebral endplate contour (upper and lower), representing a thin hypointense, linear layer, were evaluated as follows: (0) normal intact appearance: continuous sharp linear contour; (1) irregular, yet intact appearance: irregular but sharp continuous contour; (2) blurred appearance: fuzzy discontinuous contour, without linear appearance; and (3) destructive appearance: irregular defect(s) with discontinuous contour, with or without vertebral body height decrease .Fourth, on unenhanced sagittal T1-w and T2-w images, the intervertebral disc was evaluated for the presence of a vacuum phenomenon (gas content), seen as low signal within the disc ; and on Fifth, unenhanced sagittal T1-w and T2-w images were used to identify possible additional Modic type 2 changes of the vertebral endplates and subchondral bone, seen as T1-w and T2-w signal hyperintensity A, which p-value equal to or less than 0.05 was considered to indicate a significant difference.Statistical analysis was performed using software . A Descriptive statistics, including mean, median, minimum, maximum, standard deviation, and range, were calculated for the percentage extent of vertebral body edema and T1-w signal ratios of edema to unaffected vertebral bone, as observed in MT1, and early and advanced spondylodiscitis, including all measured values of affected vertebral bodies. Cross-tabulation was used to demonstrate the absolute frequencies and percentages of visually assessed T1-w signal changes, as well as of the upper and lower endplate contour assessment, as observed in MT1, and early and advanced spondylodiscitis. Taking into account that patients had multisegmental involvement, and thus, multiple measured values, for edema extent and T1-w signal ratios, differences were tested for statistical significance by using a mixed model analysis of variance; and for the visual T1-w signal changes and endplate contour by using a Chi-square test. Please note that, in this study, the assessment of the diagnostic accuracy  referred to the correct identification of MT1. A receiver-operating characteristic (ROC) analysis was performed, and the area under the time-intensity curve (AUC) was calculated to assess the optimal threshold, and thus, the diagnostic accuracy of the percentage of edema extent and the T1-w signal ratio of edema to unaffected bone to differentiate between MT1 and spondylodiscitis. In addition, 95% confidence intervals were calculated. Based upon cross-tabulation, the diagnostic accuracy, and the positive and negative predictive values of the contour evaluation, using the feature “irregular, yet intact” for the differentiation of MT1 and spondylodiscitis were assessed, considering the abnormal or most affected endplate in each individual. A logistic regression analysis was performed to assess the diagnostic accuracy of MR imaging in the differentiation of MT1 and spondylodiscitis, when combining the percentage of edema extent, T1-w signal ratio of edema to unaffected bone, and endplate contour. Furthermore, a multinominal logistic regression analysis was performed to demonstrate the percentages of correct classification of MT1, and early and advanced spondylodiscitis based upon the combined features of edema extent, T1-w signal ratio, and endplate contour. This regression analysis addressed MT1, and early and advanced spondylodiscitis separately without considering their differential diagnostic relationship.Cross-tabulation was also used to demonstrate the absolute frequencies and percentages of vertebral body involvement at Th12 to L1, the vacuum phenomenon, the discal STIR hyperintensity, and any additional Modic type 2 and 3 changes in MT1, and early and advanced spondylodiscitis. Differences were tested for statistical significance using a generalized estimating equation (GEE).Using the intraclass correlation coefficient (ICC), the intrareader and interreader agreement were assessed for the quantitative evaluation of the percentage of edema extent and the T1-w signal intensity measurements (continuous variables). Using weighted κ statistics, the intrareader and interreader agreement were assessed for the visual, qualitative evaluation of the vertebral endplate contour and the T1-w signal intensity within the edema area . Based upon the aforementioned criteria, we included a total number of 91 patients. There were 39 patients with MT1 changes , and 52 patients with infectious spondylodiscitis , including 19 patients with early spondylodiscitis, and 33 with advanced spondylodiscitis. Of 39 patients with MT1, 28 had monosegmental and 11 multisegmental involvement. Of 52 patients with spondylodiscitis, 46 had monosegmental and six multisegmental involvement . Of 33 patients with advanced spondylodiscitis, seven had paravetrebral, 21 paravetrebral and epidural, and five epidural abscess formations.Staphylococcus aureus in 16; Staphylococcus epidermidis in two; Streptococcus species in four; Escherichia coli in four; Pseudomonas aeruginosa in four; Enterococcus species in two; and Klebsiella species in two patients.In 18/52 patients with spondylodiscitis, there was florid infection shown by biopsy, needle aspiration, or blood culture, but without proof of a specific pathogen. In 34/52 patients, a specific pathogen could be identified: p < 0.001) among MT1 , early spondylodiscitis , and advanced spondylodiscitis . The T1-w signal ratios of edema to unaffected bone of MT1  were significantly different (p < 0.001) from early spondylodiscitis , and advanced spondylodiscitis ; there was no significant difference (p = 1.000) between early and advanced spondylodiscitis. The visually assessed T1-w signal changes within the edema area of MT1 were significantly different (p < 0.001) from early spondylodiscitis, and advanced spondylodiscitis; there was no significant difference (p = 0.837) between early and advanced spondylodiscitis. A mixed hyper-/hypointense signal was seen in 72.4% of MT1, a hypointense signal in 47.5% of early spondylodiscitis, and a hypointense signal in 39.7% of advanced spondylodiscitis  from early spondylodiscitis, and advanced spondylodiscitis; and between early and advanced spondylodiscitis as well (p = 0.015). The upper endplate was irregular, yet intact in 88.1% of MT1, blurred in 68.2% of early spondylodiscitis, and blurred in 68.6% of advanced spondylodiscitis; the lower endplate was irregular, yet intact in 94.9% of MT1, blurred in 68.2% of early spondylodiscitis, and blurred in 65.7% of advanced spondylodiscitis ; specificity, 86.50% (45/52); accuracy, 84.62% (77/91)), and a T1-w signal ratio of edema to unaffected bone of ≥0.80 ; specificity, 86.50% (45/52); accuracy, 84.62% (77/91)) as the optimal thresholds for identifying MT1, and thus, differentiating spondylodiscitis . The prep = 0.999) between MT1 and early and advanced spondylodiscitis , L4 (23.3%), and L5 (24.8%) were most commonly involved, without statistically significant differences (discitis .p = 0.001) and abnormal STIR signal hyperintensity (p < 0.000) between MT1 and early and advanced spondylodiscitis  and type 3 (p < 0.001) changes between Modic type 1 and early and advanced spondylodiscitis  and type 3 (30.5%) changes were most commonly found in MT1 patients. There were statistically significant differences for additional Modic type 2  and the T1-w signal intensity measurements . There was also high reader agreement for the visual, qualitative assessment of the T1-w signal intensity within the edema area  and the vertebral endplate contour .At 3.0 Tesla state-of-the-art spinal MR imaging, this study systematically investigated imaging features for the differentiation between MT1 and early and advanced spondylodiscitis, all presenting with bone marrow edema. The endplate assessment on unenhanced T1-w images provided high diagnostic accuracy for identifying MT1, and thus, differentiating spondylodiscitis. This may be particulary useful in early-stage infection in the absence of distinctive features, such as vertebral destruction and abscess formation.In our study, the vertebral bone marrow edema extent was evaluated by using fat-suppressed, fluid-sensitive MR images, which have been shown to reveal more MT1 changes, compared to the standard T2-w images used for their original description . DetaileThis study included previous insights derived from MR imaging of pedal osteomyelitis that considered T1-w signal assessment of the edema area . Fat-supTechnically, unenhanced T1-w MR images, as currently and previously used, may be best suited for evaluating the vertebral endplate contours ,29,30,36Vertebral endplate destruction is typical for spondylodiscitis ,28,29,30In this study, among edema and T1-w signal assessment, an irregular, yet intact endplate contour showed the highest diagnostic accuracy  in the identification of MT1, thus differentiating spondylodiscitis. In this specific research setting, the combination of endplate contour, edema extent, and signal ratio improved the diagnostic accuracy again, with a sensitivity of 100%. Practically, the endplate evaluation seems most simple and effective. These endplate irregularities in the context of osteochondrosis, characterized by tiny endplate defects, associated with microfractures and fragmentations, have been termed erosive osteochondrosis, and underscore its active degnerative state ,8,30,32.Both MT1 and spondylodiscitis most commonly affected L3 to L5, possibly explained by the lower lumbar biomechanical alignment and vascular anatomy, respectively . TherefoIn this study, discal STIR-hyperintense signal changes occurred in both MT1 and spondylodiscitis, with significant differences. In infection, this typically involves the entire disc space, with a loss of the intranuclear cleft, known as the hot disc sign, with disc hyperintensity and enhancement reported in > 90% of cases ,44. In cAs a result of it being largely available and a rapid alternative when MR imaging is unavailable or contraindicated, multidetector CT has been recently investigated for the assessment of suspected spondylodiscitis ,48. CT wn = 19). Second, biopsy or needle aspiration results were not available in MT1 patients, which were diagnosed based only upon clinical features, negative blood culture results, and imaging findings, including follow-up. Thus, this study did not provide insights into the debated infectious pathogensis of MT1 [Our study has several limitations, mostly due to its retrospective nature. First, this study included a total of 91 patients, but a limited number with early spondylodiscitis (s of MT1 ,11,12,13s of MT1 . Our stus of MT1 . DespiteIn conclusion, in patients with vertebral bone marrow edema, the presence of an irregular, yet intact T1-w endplate contour, provides high diagnostic accuracy for identifying MT1, with a sensitivity of 94.9% and a specificity of 94.2%. In contrast, the absence of an irregular, yet intact endplate contour is highly suggestive of infection, and particularly of early stage spondylodiscitis in patients without abscess formation. This simple and robust assessment of the endplate contour, on unenhanced standard MR images, may be applied by general and musculoskeletal radiologists, as well as by orthopedic specialists on a routine clinical basis to aid in the patient´s diagnostic and therapeutic management.


When facing an acute viral infection, our immune systems need to function with finite precision to enable the elimination of the pathogen, whilst protecting our bodies from immune-related damage. In many instances however this “perfect balance” is not achieved, factors such as ageing, cancer, autoimmunity and cardiovascular disease all skew the immune response which is then further distorted by viral infection. In SARS-CoV-2, although the vast majority of COVID-19 cases are mild, as of 24 August 2020, over 800,000 people have died, many from the severe inflammatory cytokine release resulting in extreme clinical manifestations such as acute respiratory distress syndrome (ARDS) and hemophagocytic lymphohistiocytosis (HLH). Severe complications are more common in elderly patients and patients with cardiovascular diseases. Natural killer (NK) cells play a critical role in modulating the immune response and in both of these patient groups, NK cell effector functions are blunted. Preliminary studies in COVID-19 patients with severe disease suggests a reduction in NK cell number and function, resulting in decreased clearance of infected and activated cells, and unchecked elevation of tissue-damaging inflammation markers. SARS-CoV-2 infection skews the immune response towards an overwhelmingly inflammatory phenotype. Restoration of NK cell effector functions has the potential to correct the delicate immune balance required to effectively overcome SARS-CoV-2 infection. Natural killer (NK) cells form part of the innate immune system, where they serve as a first-line defense against acute infection and cancer, whilst also regulating the adaptive immune response . NK cellCellular stress, impaired KIR engagement and MHC 1 downregulation, associated with infection or cancer growth, lower the inhibitory signalling threshold, resulting in NK cell activity receptor upregulation ,4,5. NK DIM and CD56BRIGHT subsets ) and directly through interactions with the T cells themselves . During acute infection, NK cells can promote the differentiation of naïve CD4+ T cells into Th1 T cells through the secretion of IFNγ, thereby leading to increased pathogen control, they can also decrease T cell priming through IL-10 . NK cell promotion of mature DCs leads to increased antigen presentation and thus an increased CD8+ T cell response . Otherwise, NK cells also regulate the adaptive immune response through cytotoxic killing of activated T cells. Activated T cells often express higher levels of activating ligands (NKG2D) and decreased inhibitory ligands (MHC I), resulting in their recognition and elimination . Resultant reduction in T follicular helper (Tfh) responses hampers the development of the B cell response, as Tfh are responsible for driving B cell differentiation into memory B cells and plasma cells that produce antibodies .There are many parallels between infection, genetics and physiology and NK cell dysregulation. In this section we share aspects of NK cell dysfunction that are not related to infections agents.Ageing is associated with the development of chronic inflammation and a general reduction in immune diversity . The ageNK cell production and proliferation are reduced in ageing, though the absolute number of NK cells is higher, likely due to the accumulation of long-lived NK cells . Express+), and an increased CD56DIM:CD56BRIGHT ratio. The increase in CD56DIM cells may be compensatory to the loss of natural killer cell cytotoxicity observed in older individuals . Pa. Pa36]. BRIGHT NK cell subset has been shown to be increased in AP, particularly in symptomatic patients, suggesting their preferential accumulation in unstable plaques ..39].Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by the presence of autoantibodies, immune dysregulation and damage to kidney, skin, heart and lung tissues. In SLE apoptosis is either increased or clearance suboptimal, leading to an increase in autoantigen-antibody complexes stimulating interferon alpha (IFN- α). Increased expression of IFN- α activates lymphocyte, DCs and natural killer (NK) cells, and leads to the upregulation of several inflammatory proteins . PatientBRIGHT NK cells [Juvenile idiopathic arthritis (JIA) presents as chronic synovitis and is the most common cause of chronic arthritis in children . SystemiNK cells . This paNK cells .BRIGHT/CD56DIM NK cells has been found in cerebrospinal fluid (CSF) of MS patients with respect to controls [+ T cell evasion due to impaired DNAM-1 (DNAX accessory molecule-1) interactions [Multiple sclerosis (MS) is characterised by demyelination involving the central nervous system, which leads to a progressive cognitive decline and physical disability . MS clincontrols . This reractions .Hematologic malignancies include leukemia, chronic myeloid neoplasms, B-, T- and NK cell lymphomas, as well as multiple myeloma. In general, cancers evade the host immune system through mechanisms of apoptotic resistance and immune cell deactivation. Specifically, however, hematological malignancies result in metabolic changes that directly suppress effector immune responses ,45,46,47Moreover, hematological malignancies may release other immune suppressive signals such as adenosine and IL-10. Extracellular adenosine (ADO), indirectly inhibits the maturation, cytotoxicity and effector function of NK cells, through modification of specific cellular pathways . Increas+, CD16+ and CD56+ cells positive for cytotoxic molecules. High serum levels of CXCR1 indicated increased CD56DIM NK cell activity resulting in necrosis, apoptosis and organ failure [BRIGHT NK cell subset, the CD56DIM cells, if present are characterised by functional impairment [Aggressive natural killer leukemia (ANKL) is a rare systemic proliferation of mature NK cells largely associated with Epstein Barr virus (EBV) . ANKL is failure . In starpairment , togethepairment .Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease associated with an overwhelming systemic immune activation. HLH can be either genetic  or acquired, the latter being associated with viral infection, malignancies and autoimmune disease . The mos+, result in an increase in NK cell cytokine secretion [Acquired HLH is most commonly associated with haematological malignancies, such as T cell and NK cell leukaemia  and has ecretion .The 2009 influenza pandemic occurred between early 2009 and middle 2010, and it is estimated that between 700 million to 1.4 billion people were infected and that between 150,000 and 410,000 deaths may have occurred . PandemiPandemic A(H1N1) 2009 infects DCs, macrophages, and NK cells through cell surface sialic acids, where it fails to effectively activate IFNγ and TNFα gene expression, and induces only minimal antiviral and pro-inflammatory cytokines, allowing for potential immune escape. Disease severity has been correlated to an increase in the inflammatory markers IL-6, IL-10, IL-15 and MCP-1 . InfluenSevere acute respiratory syndrome first emerged in China in November 2002, where after it rapidly spread across the world, 8096 infections were reported with a mortality rate of 9.6% . SARS is+, CD8+, CD20+, DC, macrophage and NK cell populations are all decreased in SARS patients. In addition to reduced numbers of NK cells, a reduction in immunoglobulin-like receptor CD158b+ NK cells has also been observed [CD4observed , suggestobserved ,74. Elevobserved ,75,76.Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease, and is the leading cause for mortality in infants and the elderly ,77. InfeSevere RSV disease is characterised by a skewing of the immune response towards a dysregulated Th2-type response, where the production of IFN-I is inhibited, decreasing the Th1 antiviral response ,81. RSV SARS-CoV-2 is the causal agent of coronavirus disease 2019 (COVID-19). This ongoing pandemic has infected over 1.7 million people and resulted in over 800,000 deaths ,83. SARSDIM cells [The immunopathology of COVID-19, as was the case for SARS, is based on the dysregulation of the innate and cell-mediated immune responses. SARS-CoV-2 has also been shown to utilize ACE2 as a cell entry receptor ,90, intrIM cells . All theBRIGHT subpopulation, indicating that the balance of NK cell subsets may be skewed towards inflammation, rather than cytotoxicity. Indeed, Mazzoni et al., found an inverse correlation between serum levels of IL-6 and the frequency of NK cells expressing Granzyme A, particularly in intensive care unit (ICU) patients [COVID-19 patients display high levels of inflammatory cytokines and chemokines , with severe cases showing elevation in TNFα, IL-1, IL-6, IL-18, IL-8, IL-10, MCP-1 and MIP-1A, leading to severe pulmonary tissue damage ,86,95,99patients .Little is known about the role of NK cell activating receptors in SARS-CoV-2 infection. In other respiratory viruses such as RSV, Influenza and adenovirus, various mechanisms of inhibition have been described ,115,116.BRIGHT NK cells, dampening overactive inflammatory responses. Several trials are also investigation the use of oxygen therapy in the early treatment of SARS-CoV-2 infection, as previous studies have shown that this treatment effectively decreases inflammation, reducing IL-12 and TNF-α, and increasing IL10 [Mycobacterium tuberculosis), may offer heterologous protection against SARS-CoV-2 through trained immunity [Numerous clinical trials are investigating treatment strategies that directly affect NK effector cell function ,120. Theing IL10 ,129. Numimmunity . BCG vacimmunity .The flavonoids Quercetin and Luteolin, have been shown to reduce IL-6 expression in Mast cells, and may be a safer alternative to corticosteroid treatment ,133. TheNatural killer cells play a central role in maintaining immune homeostasis, a critical requirement when facing the challenge of a novel pathogen. SARS-CoV-2 infection has been shown to impede NK cell function, thus disrupting this vital balance. With factors such as ageing and other comorbidities, leading to further skewing, the current trials investigating drugs and biologicals, which improve NK cell function, may prove to be monumental in our fight against COVID-19.


Consequently, CD30 has been advocated as a screening test to differentiate benign seromas. Here, we report a case of BIA-PTCL, not otherwise specified (NOS) presenting as a periprosthetic mass with no CD30 expression that was fatal. While resembling the infiltrative variant of BIA-ALCL, cell surface markers were most similar to that of monomorphic epitheliotropic T-cell lymphoma (MEITL). While this case is atypical, it suggests that even with CD30 negativity, clinicians should maintain a high index of suspicion in patients with concerning clinical or pathologic features, and BIA-PTCL, NOS with a silent phenotype should be considered. Further research into implant-associated PTCL is needed.Breast implant–associated peripheral T-cell lymphoma (BIA-PTCL) was first reported in 1997.A 67-year-old Hispanic-American female presented to the emergency room after being found wandering in the street, disoriented. She described gradual loss of vision and could only appreciate light perception and finger movements. When her vision worsened, she saw an optometrist and started prednisone, now at 20 mg oral daily, without relief. History was remarkable for bilateral breast implant placement 42 years prior for augmentation. She denied smoking or alcohol and was a retired clerical worker. Review of systems noted significant weight loss and left breast swelling. On examination, there was a large breast mass lateral to the prosthesis with induration extending medially.Peripheral blood testing showed normal cell counts and normal immunophenotyping by flow cytometry. Positron emission tomography-computed tomograph imaging confirmed a markedly fluorodeoxyglucose avid, dominant 10.6 × 6.7 × 12.3 cm mass within the left breast adjacent to the left breast implant . No definite hypermetabolic foci were seen outside of the dominant breast mass. However, fundoscopy showed dense vitritis and deposits in the right eye. B-scan ultrasonography confirmed posterior hyaloid separation and vitreous opacities, without retinal detachment.The patient underwent core biopsy of the left breast mass with extensive testing done both in house as well as at a commercial and reference facility. A summary of findings is reported in the Table. Histology showed a diffuse lymphoid infiltrate composed of large atypical cells with irregular nuclear contours, fine chromatin, conspicuous mitotic activity, and abundant eosinophilic cytoplasm. IHC was done for CD20, CD3, CD5, CD2, CD7, CD4, CD8, CD10, CD56, CD138, CD117, CD123, CD30, CD68, CD45, CD33, CD34, kappa, lambda, IgD, IgG, IgM, cyclin D1, BCL-6, BCL-2, MUM1, S-100, granzyme B, perforin, ALK1, c-MYC, PAX5, HHV8, TdT, Ki-67, pan-cytokeratin, EBER ISH, TIA-1, TCR delta, Beta F1, and ALK. The neoplastic cells were positive for CD2, CD3, CD56, Granzyme B, Perforin, c-MYC, BCL-2, MUM1, and CD20 (weak), while the rest were noncontributory. Ki-67 proliferation index was high (~90%).Fluorescence in situ hybridization studies were negative for BCL-6 rearrangement, MYC rearrangement, MYC amplification, or IgH/BCL-2 translocation. The interphase study revealed a deletion signal pattern in the 14;18 (IgH/BCL2) probe set suggestive of a BCL2 gene deletion or chromosome 18 monosomy. Polymerase chain reaction studies were positive for T-cell receptor gamma gene rearrangement and negative for B-cell gene rearrangement, confirming a clonal T-cell population. Sampling of both vitreous and cerebrospinal fluids was performed and flow cytometry confirmed central nervous system (CNS) infiltration, with distinct populations ranging from 18% to 37% cellularity exhibiting a cytotoxic phenotype and falling within the high forward scatter region indicating large size.The patient was diagnosed with BIA-PTCL, NOS with CNS involvement. After an extensive discussion on treatment options, she decided on comfort-directed measures and was discharged in the care of her family with hospice care; she died 4 weeks later.7 Breast PTCL is even more infrequent, representing 8%-10% of all breast lymphomas.3 With the first BIA-PTCL case reported in 1997, the next 2 decades saw increasing reports, and in 2016, provisional classification for BIA-ALCL was published.5 The exact incidence of BIA-ALCL is unknown but it is estimated that the lifetime prevalence is 1 per 30,000 patients with textured breast implants, although a prospective study suggested that the incidence may be closer to 1 case per 4000.8 Most cases present after 7-10 years and at a median age of 50-60 years,9 although there are reports >30 years from surgery, as in the present case.10Primary non-Hodgkin lymphomas of the breast constitute 0.01%-0.5% of all malignant breast tumors and 1% of all non-Hodgkin lymphoma.4 These 2 variants have different prognoses, with the infiltrative variant having more aggressive biology and worse outcomes. However, no specific pathologic features differentiating these 2 groups have been elucidated.11 Indeed, it is unclear if these are distinct subtypes, or simply represent a spectrum of disease.Our current understanding is that there are 2 clinical variants of BIA-ALCL, presenting as either the more common periprosthetic seroma or an infiltrative mass.12 Added to this is a recent hypothesis on prosthesis-associated tissue hypoxia.11 Aberrant gene expression is of course most likely involved in the oncogenic progression from a chronic inflammatory state to a full-blown malignancy. Recurrent JAK1 and STAT3 gene mutations have been identified and, like many other ALCLs, also identified have been mutations in DNMT3A, TP53, and SOCS.15 However, even in the analyses of these small series, there have been cases evaluated with no somatic mutations. Recently, RNA sequencing and gene set enrichment analysis comparing eleven BIA-ALCLs to 24 non–BIA-ALCLs identified upregulation of hypoxia signaling genes including VEGFA, VEGFB, SLC2A3 (encoding GLUT3), and carbonic anyhydrase-9.11BIA-ALCL is thought to develop after years of implant-induced inflammation inducing dysplastic changes from chronic antigen stimulation. To date, the 2 most frequently discussed etiological candidates are inflammation from textured implants and chronic bacterial stimulation.16 Expression of CD5, CD7, CD8, CD15, or CD56 is less common. MUM1, Granzyme B, TIA-1, and BCL2 have a high prevalence.16 Our case expressed CD2, CD3, CD56, Granzyme B, Perforin, C-MYC, BCL-2, MUM1, and CD20 (weak). Weak expression of CD20 has been reported in PTCL, NOS.Currently, diagnosis of BIA-ALCL is based on cytology, evidence of T-cell clonality, and IHC. Morphologically, the tumor is comprised of large, pleomorphic cells with abundant cytoplasm, and horseshoe-shaped or “embryoid” nuclei with prominent nucleoli. With the exception of prominent nucleoli, the case presented had this morphology, and as noted, also T-cell clonality. On IHC, BIA-ALCL typically demonstrates strong and uniform membranous expression of CD30 and lacks ALK expression. Other T-cell antigens have been reported expressed variably, with the most common being CD4 (79%-84%), CD43 (80%-95%), CD3 (28%-46%), CD45 (36%), and CD2 (30%-41%).2 Nevertheless, IHC has not been reported in all cases, and for example, in this series, 57% of cases did not have CD30 results available.2 National Comprehensive Cancer Network guidelines now recommend that scant or rare CD30-positive lymphocytes with normal morphology can be considered a normal finding with no further investigation.17 One group has recently examined a CD30 enzyme-linked immunosorbent assay with the aim of a lower-cost screening test more widely available than flow cytometry and IHC, and use to screen suspicious peri-implant fluid collections.18 Other groups are investigating unique cytokine profiles including IL-13, and molecular signatures to differentiate BIA-ALCL.19 Apart from the significance of CD30 in diagnosis, it is also important for management. Brentuximab vedotin has been used in cases of BIA-ALCL.While T-cell phenotype has been variably reported, cases to date have been consistently CD30 positive. For instance, in a 2019 expanded safety analysis, 246 cases reported positive.20This case of BIA-PTCL, NOS presented a large periprosthetic mass with the dilemma of no CD30 expression. The disease was extremely aggressive with a high proliferation index and CNS involvement, and ultimately resulted in death. Clinically, the disease course resembled the infiltrative type of BIA-ALCL. Cytology, ALK testing, and T-cell clonality were similar to reported cases of BIA-ALCL. However, the cell surface markers were most similar to that of MEITL. Both MEITL and BIA-ALCL are novel diseases classified in 2016 and driven by T-cell activation. MEITL was previously known as type II enteropathy–associated T-cell lymphoma but it was given a new name due to its distinctive nature and lack of association with celiac disease. It should be noted that MEITL is an intestinal lymphoma and does not occur in the setting of implants. In this case, it could easily be excluded based on the clinical setting, tumor site, as well as histology—MEITL tumor cells are monomorphic and small to medium in size, infiltrating the intestinal epithelium. The adjacent “normal” mucosa demonstrates villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis. There is no inflammatory background in MEITL. Most cases of MEITL are derived from γδ T cells and, similar to BIA-ALCL, the JAK-STAT pathway has been implicated in MEITL.While this case is rare, it suggests that even with CD30 negativity, clinicians should maintain a high index of suspicion in patients with concerning clinical or pathologic features, and consider BIA-PTCL, NOS with silent phenotype. As more cases of BIA-ALCL are reported, we anticipate more data on atypical cases. Further research into BIA-PTCL, NOS is needed.We have presented the first case of BIA-PTCL, NOS occurring as CD30 negative disease with a surface phenotype similar to MEITL. The disease was clinically similar to the infiltrative mass variant of BIA-ALCL, being aggressive and resulting in death. This case suggests that CD30 by itself is not 100% sensitive to exclude BIA-PTCL, NOS. Similar cases need to be reported to help define this disease better and further research into implant-associated PTCL in general is urgently needed.