Haploinsufficiency of A20 (HA20) is a novel genetic disease presented by Zhou et al in 2016. A20 is a protein encoded by TNFAIP3. Loss-of-function mutation in TNFAIP3 will trigger a new autoinflammatory disease: HA20. HA20-affected patients may develop a wide range of clinical manifestations, such as Behcet disease, rheumatoid arthritis, rheumatic fever, juvenile idiopathic arthritis, and systemic lupus erythematosus. HA20 is rarely reported, thus remaining far from thoroughly understood. Sixty-one cases of HA20 have been reported worldwide, among which 29 cases were diagnosed with Behcet disease ultimately. Moreover, 3 cases have been reported in China, which was the first report of HA20 characterized by Behcet disease. A comprehensive understanding of the pathogenic genes of HA20 could help us apply targeted therapy as soon as possible to improve patients’ survival rates.A 2-year-old 3-month-old child was presented to our hospital with recurrent infectious enteritis and stomatitis.∗7) accounted for familial Behcet-like autoinflammatory syndrome in the child suffering from HA20, while no variation in this locus was found in her parents.Genetic mutations were detected immediately, and a novel pathogenic mutation was found in TNFAIP3. A heterozygous mutation (c.436-437deTC) located at TNFAIP3 was confirmed. The present research indicated that the TNFAIP3 mutation of c.436-437deTC  and iron supplement were performed, and repeated infection was no longer observed in the child. Pain and activity limitation was found in the knee joints. The treatment regimen was adjusted to oral prednisone  and subcutaneous injection of rhTNFR:Fc (12.5 mg/week).Outcomes: At the last follow-up, the limbs’ activities were normal, the inflammatory indicators were reduced or within the normal range. The prednisone dose was reduced to 7.5 mg/d, while the dose of rhTNFR:Fc was not changed.We have identified a novel pathogenic HA20 mutation. In this article, 1 case was analyzed in-depth in terms of clinical manifestations of the patient and new sources of such a novel disease, which might improve our understanding of this disease. Loss-of-function mutation in TNFAIP3 may weaken the negative regulation of NF-κB signaling pathway while enhancing the inflammasome activation of the nucleotide-binding domain-like receptor protein, both of which may lead to an overproduction of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18, and TNF-α. As a result, patients with congenital immune dysfunction may develop various immune disorders.,4,5 Currently, relevant cases reported were diagnosed with multiple immune diseases, including Behcet's disease, rheumatic fever, systemic lupus erythematosus, juvenile idiopathic arthritis, synovitis, arthritis, and lymphoma.–8 Moreover, for some cases, there remained no definite or multiple clinical diagnoses. These patients may present with various symptoms, including recurrent fever, oral ulcers, genital ulcers, joint pain, polyarthritis, bilateral anterior uveitis, or even no clinical specificity.–8 So far, a total of 61 cases from 26 families have been reported, including sporadic cases. Clinical manifestations vary considerably, even within 1 family. In addition, there were 29 cases with Behcet disease from 13 families. In this report, we summarized the gene locus and clinical manifestation of a total of 30 cases, including this case, hoping to provide some information about the diagnosis and treatment of this disease.A20 haploinsufficiency (HA20) is a novel genetic disease presented by Zhou et al in 2016.2The patient was a 2-year-old and 3-month-old girl. She was admitted to our hospital complaining of “recurrent fever, diarrhea, and vomiting for more than 2 years.” Her mother had no abnormality during pregnancy and delivery. Three months after birth, the child had a recurrent fever, diarrhea, and vomiting. Each attack lasted for about 1 to 2 weeks, with an interval of 1 to 5 months. So far, 11 attacks have occurred. During the onset of the disease, procalcitonin (PCT), C-reactive (CRP), and white blood cell (WBC) count were increased, and many WBC could be seen in the stool. Therefore, the patient was treated with cephalosporins for about 1 week based on the diagnosis of infectious diarrhea, and her condition had improved. The child was prone to recurrent oral ulcers, and she was diagnosed with iron deficiency anemia previously. Still, her symptoms had not been alleviated after the oral administration of iron dextran, with hemoglobin fluctuation between 80 to 90 g/L. In addition, she was allergic to milk, eggs, mango, seafood, and other foods, which may lead to severe vomiting and diarrhea.9/L, NE 8.52 × 109/L, HB 87 g/L; PCT 0.24 ng/mL, CRP 65.5 mg/L, IL-6 65.5 mg/L, VitB12 1118 pmol/L, APTT 69 s, DRJT 942 ng/mL, ESR 58 mm/h, VitB12 118.2 pmol/L, IgE 96.81 IU/ml, natural killer cells 25.2%, total B cells 27.8%, CD4/CD81.0, ANA 1:100, and anticardiolipin antibody 18 RU/mL. Gastrointestinal ultrasound showed an uneven thickening of the ascending colon and thickening of the surrounding mesentery, about 0.4 cm, suggesting inflammatory changes , and it was a zero effect mutation (frameshift mutation) that may lead to loss of gene function (PVS1). The family verification analysis showed no mutation in the parents; thus, it was a spontaneous mutation (PS2). Based on the analyses above, this variation in the normal population database was a low-frequency variation (PM2), so it was preliminarily determined as pathogenic variation according to the ACMG guidelines.Since the allergen test indicated multiple allergens and that autoimmune diseases could not be ruled out, whole-exome gene sequencing was carried out from the child's peripheral blood . The Sanger sequencing of their parents for pedigree verification is shown in Figure After the genetic test results were obtained on December 19, 2019, symptomatic treatments including oral administration of prednisone (12.5 mg/d) and iron supplement were performed, and repeated infection was no longer observed in the child. The dose of prednisone was reduced gradually to 10 mg/d. Pain and activity limitation was found in the knee joints in May 2020, and MRI of joints showed synovitis and joint effusion signs. The inflammatory indicators also increased: PCT 16.2 ng/mL, CRP >90 mg/L, and IL-6 218 pg/mL. Therefore, the treatment regimen was adjusted to oral prednisone  and subcutaneous injection of recombinant human type II tumor necrosis factor receptor-antibody Fc fusion protein . Afterward, the disease was stable, and only gastrointestinal manifestations, including fever and vomiting, were found after intake of cold drink in October 2020, which improved after treatments, including anti-inflammation therapy. Reexamination by colonoscopy showed that the intestinal wall was smooth and with no evident abnormality. Pulmonary CT showed no signs of granuloma. Ultrasound examination of the knee joints showed normal results.Further reexamination on January 25, 2021, showed that the anemia was corrected, and the treatment with iron supplement was stopped. The limbs’ activities were normal, the inflammatory indicators were ESR 11 mm/h, PCT 0.02 ng/mL, CRP 12 mg/L, and IL-6 8 pg/mL, and TNF was in the normal range. The prednisone dose was reduced to 7.5 mg/d, while the dose of rhTNFR:Fc was not changed.3 The mutations in the first 2 families were detected using whole-exome sequencing and confirmed by Sanger sequencing. Three subsequent mutations were observed in 3 of 150 probands with a similar disorder who were directly screened for TNFAIP3 mutations. The sixth mutation was found in one of 768 individuals diagnosed with Behcet disease via targeted sequencing. In vitro studies on functional cellular expression demonstrated that all mutations failed to suppress TNF-induced NF-κB activity, although not in a dominant-negative fashion, which suggested haploinsufficiency as the underlying pathogenic mechanism. Compared with control cells, patient-derived cells exhibited decreased recruitment of TNFAIP3 to the TNFR complex, and increased phosphorylation of IKKA and IKKB and subsequent degradation of IκBα, with nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines, consistent with activation of the NF-κB pathway. Cells expressing the mutant proteins showed defective removal of lys63-linked ubiquitin from TRAF6, NEMO, and RIP1 after stimulation with TNF, indicating inefficient deubiquitinating. The proinflammatory cytokines’ levels were substantially higher in patients’ serum compared to healthy controls, as robust evidence for enhanced IL1B signaling.,11In affected cases from 6 independent families with familial Behcet-like autoinflammatory syndrome, Zhou et al identified 6 different heterozygous truncating mutations in the TNFAIP3 gene.Cytokines such as TNF exert a profound impact on the function of endothelial cells. For instance, TNF can promote the interaction with leukocytes and induce a procoagulant phenotype. Changes of this nature might play a pivotal role in the proinflammatory effects of TNF. TNF-induced primary response genes were analyzed in human umbilical vein endothelial cells. Of the 6 induced cDNAs identified, 2 encoded paracrine factors (neutrophil chemotactic factor and monocyte chemotactic factor), 1 encoded a membrane receptor for neutrophils , and 3 encoded undescribed TNF primary response genes hitherto. On exposure of endothelial cells to TNF, there was a rapid and substantial increase in mRNA levels encoding the 6 genes, which were further superinduced by cycloheximide. Thus, these represent primary response genes, as their induction does not depend on protein synthesis. One of the 3 new proteins, designated A20, was found on sequence analysis to code for a novel zinc finger protein.Lu et al generated 2 lines of gene-targeted mice via abrogating either the deubiquitinating activity of A20 (Tnfaip3-OTU mice) or zinc finger-4 of A20 (Tnfaip3-ZF4 mice). Both strains exhibited increased responses to TNF and sensitivity to colitis. The deubiquitinating motif restricted both lys48- and lys63-linked ubiquitination of Rip1. ZF4 was required for the recruitment of A20 to ubiquitinated Rip1. Two mutant A20 proteins complemented each other through dimerization to regulate Rip1 ubiquitination and NF-κB signaling in compound mutant Tnfaip3-OTU/Tnfaip3-ZF4 cells. generated A20-deficient mice by targeted disruption. A20 +/− mice appeared normal without evidence of pathology. A20 −/− mice, born from interbred A20 +/− mice in Mendelian ratios, developed runting as early as 1 week old. Mice deficient for A20 developed severe inflammation and cachexia, were hypersensitive to both lipopolysaccharide and TNF, and died prematurely.Lee et al,5,14–16 For example, in this case, the patient had synovitis and pulmonary granuloma in addition to Behcet. In this report, the clinical symptoms, supplementary examination, gene locus, and other characteristics of this case and the 48 cases reported so far are summarized in Table At present, there were 29 cases of Behcet disease with various clinical symptoms.HA20 is a genetic disease about which we are still ill-informed, whose definite treatment and prognosis remained poorly explored. Using second-generation sequencing and according to the ACMG guidelines, the gene locus of this case was newly discovered to account for HA20. In this article, we reported and analyzed 1 case in detail and expected to offer clues for the future diagnosis and treatment of this disease.Conceptualization: Danlu Li.Data curation: Danlu Li.Formal analysis: Hongtao Zhu.Funding acquisition: Shakan. Aknai.Investigation: Shakan. Aknai.Methodology: Mei Yan.Project administration: Mei Yan.Resources: Danlu Li.Software: Mayila. Abudureyim.Supervision: Mei Yan.Validation: Hongtao Zhu.Visualization: Danlu Li.Writing – original draft: Danlu Li.Writing – review & editing: Danlu Li.


Background: Non-small cell lung cancer (NSCLC) accounts for the largest pathological type of lung cancers, and it is characterized by high incidence and poor prognosis. However, the molecular mechanisms involved in development and progression of NSCLC are not well elucidated. In this study, we aimed to explore the role and regulatory mechanism of RPS9 in NSCLC.Materials and methods: The RPS9 expression in NSCLC tissues and cell lines was assessed by qRT-PCR and western blot. Knockdown of RPS9 induced by RNA interference (RNAi) method in PC9, A549 and H1299 cells. Overexpression of RPS9 induced by transient transfection in H292 cells. Cell proliferation, colony formation, metastasis and apoptosis abilities were determined by CCK-8 assay, colony formation assay, transwell assay and flow cytometry, respectively. The host signaling pathways affected by RPS9 were screened by antibody library and proved by western blot.Results: RPS9 was significantly upregulated in NSCLC tissues and cell lines. Overexpression of RPS9 predicted poor prognosis of NSCLC patients. Knockdown of RPS9 obviously repressed cell proliferation, metastasis, and induced apoptosis. Mechanistically, suppression of RPS9 inhibited the expression level of phosphorylation of Stat3 and Erk.Conclusion: Our study clarified that knockdown of RPS9 inhibits the progression of NSCLC via inactivation Stat3 and Erk signaling pathways. Lung cancer is one of the most frequently diagnosed malignant cancer and the first cause of cancer-related death in the world RPS9 is ribosomal protein gene and located at chromosome 19ql3.4 Previous studies have suggested that RPS9 dysregulation was involved in tumorigenesis, such as pancreatic cancer, head, and neck cancer (such as head and neck squamous cell carcinoma) and so on In this research, we provide evidence for the first time that RPS9 is upregulated in NSCLC patients and cell lines and overexpression of RPS9 indicates poor prognosis. Strikingly, RPS9 knockdown inhibits NSCLC cell proliferation, colony formation and metastasis. In addition, RPS9 suppressed apoptosis in NSCLC cells. More interestingly, we found that knockdown of RPS9 could inhibit the progression of NSCLC via regulating Stat3 and Erk signaling pathways. This study highlights the function of RPS9 in NSCLC growth and metastasis and may facilitate development of new diagnostic or therapeutic biomarker to improve the outcome of NSCLC patients.A total of 143 pairs of primary NSCLC and matched adjacent normal lung tissues were collected from patients who underwent surgery between 2017 and 2019 in Huashan Hospital, Fudan University. Written informed consents were signed before the research started. And this research was authorized by the Ethics Committee of Huashan Hospital, Fudan University. All procedures were carried out in accordance with the ethical guidelines of the Declaration of Helsinki. Three NSCLC cell lines  and normal human bronchial epithelial cells (16HBE) were obtained from the Cell Bank of the Chinese Academy of Sciences . All the cells were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 ºC. Cells were used when they were in the logarithmic growth phase.The total RNA from NSCLC tissues and cell lines was purified using RNAiso Plus reagent . Then, RNA was converted to complementary DNA (cDNA) by the PrimeScript RT Reagent Kits  according to the manufacture's protocol. The qRT-PCR assays were carried out using SYBR Premix Ex Taq II Kit . The following primer sequences were used: RPS9: Forward: 5'-GAAGCGGAGCCAACATGC-3', Reverse: 5'-ATACTCGCCGATCAGCTTCAG-3'. GAPDH served as an internal control.The siRNAs targeting RPS9, and corresponding negative control siRNAs (si-NC) were designed and synthesized by Genechem . Plasmid used for transient transfection were designed and synthesized by Ribobio . Cells were plated at 60-70% confluence in a 6-well plate. Cell transfection was performed using Lipofectamine 2000 reagents . After transfection for 48 hours, the knockdown and overexpression efficiency were validated by both qRT-PCR and western blot.For transwell migration assays, cells were resuspended in DMEM without FBS and 4 × 10^5 cells were seeded into the upper chamber of a 24-well insert  precoated with Matrigel. 800-µL DMEM supplemented with 10% FBS was placed in the lower chamber. After incubation for 24 hours, the invaded cells on the lower surface were fixed with methanol, stained with crystal violet and the number of invaded cells from five random fields was counted under a light microscope. For the invasion assay, 1 × 10^6 cells were seeded into the upper Matrigel invasion chambers , and the following procedures were carried out similarly as what was described in the transwell migration assays.Cells were seeded into 96-well plates in triplicate and cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay  according to the manufacturer's instructions. For colony formation assay, cells were plated in 6-well plates (1000 cells/well) and cultured for 14 days. After washed with phosphate-buffered saline (PBS) for three times, the colonies were fixed in methanol and subsequently stained with crystal violet for 30 min.For cell cycle analysis, after washed with PBS for three times, cells were collected and fixed with 70% ethanol. Following PBS washing, the cells were stained with 500μl propidium iodide (PI)  contained RNaseI  according to the manufacturer's instructions. Subsequently, the cell cycle was analyzed by a FACSCalibur flow cytometer . For apoptotic assay, after washed with PBS for three times, cells were stained with Annexin V-FITC and PI  at room temperature for 30 min. Then, the apoptotic cells were assessed using flow cytometer.Protein lysate were extracted using RIPA lysis buffer  contained protease and phosphatase inhibitors . Subsequently, proteins were electrophoresed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes . After blocked with 5% bovine serum albumin for 1 hour, the membranes were incubated with following primary antibodies at 4℃ overnight: RPS9 ; P- mTOR, mTOR , P-FAK, FAK, P-FoxO3a, FoxO3a, P-FoxO1, FoxO1, P-Stat3, Stat3, NF-κB, P-AMPK, AMPK, P-Smad2, Smad2, P-AKT, AKT, TGF-β, P-Erk and Erk ; β-actin . Next day, after washed in PBST for three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit antibodies  at room temperature for 1 hour. Finally, the bands were visualized and analyzed by SuperSignal West Femto Maximun Sensitivity Substrate .The results were presented as mean ± SEM (Standard error of mean). The unpaired two-tailed Student's t-test was used to compare the differences between two groups and the data were visualized by Graphpad prism version 8.0 software. All statistical analysis was conducted with SPSS version 22.0 software. A p-value less than 0.05 was considered statistically significant.To examine the potential role of RPS9 in NSCLC tissue, we firstly analyzed RPS9 mRNA levels in NSCLC data from TCGA database via UALCAN, the results confirmed that RPS9 was highly expressed in primary adenocarcinoma samples compared with normal samples Figure A. KaplanTo identify the biological function of RPS9 in NSCLC, we assessed the RPS9 expression levels in three NSCLC cell lines  and the normal human bronchial epithelial cells (16HBE) by qRT-PCR and immunoblotting analysis. As shown in Figure in vitro.To investigate the impact of RPS9 in NSCLC, we next silenced RPS9 expression in PC9, A549 and H1299 cell lines using si-RNA. The effective knockdown efficiency was validated by both qRT-PCR and immunoblotting analysis, demonstrating that RPS9 was significantly inhibited in PC9, A549 and H1299 cell lines after transfection with si-RPS9 Figure A-C. The To further examine whether RPS9 affects cell apoptosis capacities of NSCLC, flow cytometry was also utilized to determine the percentage of cell apoptosis. As shown in Figure Seeking to define the potential mechanism of RPS9 promoting NSCLC progression, the common phosphorylated protein related antibodies and their total protein antibodies including P- mTOR, mTOR , P-FAK, FAK, P-FoxO3a, FoxO3a, P-FoxO1, FoxO1, P-Stat3, Stat3, NF-κB, P-AMPK, AMPK, P-Smad2, Smad2, P-AKT, AKT, TGF-β, P-Erk and Erk from antibody library were utilized. Subsequently, the above phosphorylated protein and total protein levels were examined by western blot. As shown in Figure Ribosomal proteins, as indispensable components of living cells, are long considered as highly conserved and exert diverse roles in different cells Previously, RPS9 could act as a tumor suppressor in breast cancer, pancreatic cancer, and glioma Reversely, we revealed that RPS9, as a member of ribosomal proteins, was significantly up regulated in NSCLC tissue samples compared to the corresponding non-cancerous tissues. In addition, high levels of RPS9 in NSCLC tissues are related to poor prognosis. In the present study, RPS9 knockdown suppressed tumor growth, metastasis and promoted cell apoptosis, which is consistent with the previous researches in osteosarcoma, glioma and cervical carcinoma In NSCLC, it has been reported that RPS9 was involved in the activation of JAK-STAT and calcium signaling pathways. In the previous study, knockdown of RPS9 offset the promoting effects of si-BRCAT54 on cell proliferation and silencing of RPS9 activated JAK-STAT pathway and suppressed calcium signaling pathway gene expressions In conclusion, our findings exposed that RPS9 acted an oncogene in NSCLC via regulating the phosphorylation expression of Stat3 and Erk, promoting cell proliferation, migration, invasion, and inhibiting apoptosis in NSCLC. The role of RPS9 on the occurrence and metastasis of lung cancer needs more research. With the emergence of more studies, RPS9 may serve as an important marker for clinical prediction and diagnosis.Yiru Kong involved in data curation, methodology, writing—review and editing. Shuangshuang Deng involved in formal analysis, investigation, writing—review and editing. Xiaohua Liang involved in resources, writing—review and editing. Xinli Zhou involved in conceptualization, resources, writing—review and editing.


APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF), a plant-specific transcription factor (TF) family, plays an essential role in the growth and development of plants, and in their response to biotic and abiotic stresses. However, information on AP2/ERF in Cucurbita moschata (pumpkin), an edible and medicinal vegetable used worldwide, is scarce. A total of 212 AP2/ERF genes were identified in the C. moschata genome (CmoAP2/ERFs). Based on phylogenetic analysis, they were divided into four groups—28 AP2s, 92 ERFs, 86 dehydration-responsive element-binding (DREB) factors, and 6 ABI3/VPs (RAV). The 212 AP2/ERF genes were unevenly distributed on the 20 chromosomes of C. moschata. The results of structural analysis showed the absence of introns on 132 CmoAP2/ERFs. Four pairs of tandem duplication and 155 pairs of segmental duplication events were identified, which indicated that segmental duplications might be the main reason for the expansion of the CmoAP2/ERF family. The analysis of cis-regulatory elements (CREs) showed that most of the CmoAP2/ERFs contained hormone response elements  in their promoters, suggesting that AP2/ERFs could contribute to the processes regulated by ethylene and abscisic acid. By comparing the transcriptome of ethephon-treated and control plants, we found that 16 CmoAP2/ERFs were significantly upregulated after ethephon treatment. Furthermore, we determined the expression patterns of these genes at different developmental stages of female and male flowers. This study provides insights into the identification, classification, physicochemical property, phylogenetic analysis, chromosomal location, gene structure, motif identification, and CRE prediction of the AP2/ERF superfamily in C. moschata. Sixteen CmoAP2/ERF genes were identified as ethylene-inducible genes. The results of this study will be valuable for understanding the roles of CmoAP2/ERFs in ethylene response and should provide a foundation for elucidating the function of AP2/ERF TFs in C. moschata. All the searched sequences were then submitted to the SMART tool3 and NCBI CDD4 to detect the AP2/ERF domain and to remove the sequences without the AP2/ERF domain.The 5The protein features, including amino acid number, isoelectric point (pI), molecular weight (MW), aliphatic index, grand average of hydropathicity (GRAVY), and instability index, were predicted using ExPASy.AP2/ERF genes was plotted with TBtools6 based on the C. moschata genome database. Collinearity analysis within C. moschata was performed using the TBtools and MCScanX , were used to explore the expression patterns of CmoAP2/ERFs in response to ethylene. The transcript levels of CmoAP2/ERFs were calculated with the fragments per kilobase of exon per million fragments mapped (FPKM) method. The obtained FPKM values of CmoAP2/ERFs were Log2 normalized. Heatmap was drawn using the TBtools software for further visualization.The RNA-Seq data of shoot apices subjected to ethephon (100 mg/L) treatment for 4 h, which were previously obtained by our group  and then reverse transcribed into cDNA using the PrimeScript™ RT Master Mix  Reagent Kit . Real-time PCR was performed on a Bio-Rad IQ5 instrument . Based on the results of ethylene response analysis of t method . The expC. moschata genome database, members of the AP2/ERF family were searched using HMMsearch with a HMM profile of PF00847. After SMART tool and NCBI CDD verification, a total of 212 sequences were identified as candidate CmoAP2/ERF genes across the whole genome. The basic features, including amino acid number, aliphatic index, GRAVY, pI, MW, and instability index of the CmoAP2/ERF family genes, are listed in Additional File 2. The identified CmoAP2/ERF genes ranged in length from 416 to 22,786 bp. These genes were predicted to encode proteins with 132–1183 amino acids. The MW of CmoAP2/ERF proteins ranged from 14.79 (CmoERF42) to 130.61 (CmoERF36) kDa and the pI varied from 4.36 (CmoERF19) to 11.54 (CmoERF25). There are 73 CmoAP2/ERF proteins with pI > 7 and 139 with pI < 7, which indicates that most of these proteins have a higher proportion of acidic amino acids. The GRAVY score of CmoAP2/ERF proteins ranged from −1.104 (CmoERF68) to −0.018 (CmoDREB12). Most of the CmoAP2/ERF genes were predicted to encode unstable proteins with the instability index >40, and only 11 family members, namely CmoRAV2, CmoDREB38, CmoERF34, CmoERF39, CmoERF40, CmoRAV3, CmoDREB65, CmoERF54, CmoRAV5, CmoERF78, and CmoRAV6, were predicted to be stable proteins based on the instability index.With reference to the CmoAP2/ERF genes, we constructed a phylogenetic tree using the amino acid sequences of proteins encoded by these genes  and DREB  subfamilies.To evaluate the evolutionary relationship of the se genes . The CmoCmoAP2/ERF members, the introns and exons of the CmoAP2/ERF genes were examined. As shown in DREB genes have no introns, except for 26 genes, in which the number of introns range from one to nine ; 9 genes lack 5′-UTR, and 10 genes lack 3′-UTR.To characterize the gene structure of  to nine . All the introns . Only onron each . OverallTwenty important conserved motifs in the CmoAP2/ERF proteins were analyzed . The lowest number of CmoAP2/ERF genes was found on chromosome 13 (3 genes), which were all identified as ERF genes. Six RAV genes were found to be distributed on four chromosomes, chromosomes 04, 11, 15, and 18. Most of the chromosomes contained a mixture of different family genes, except chromosomes 09, 13, 19, and three chromosomes only contained the EREBP family (ERF and DREB subfamily) genes. The 212 CmoAP2/ERF genes were renamed from CmoERF1 through CmoERF92, CmoDREB1 through CmoDREB86, CmoAP2-1 through CmoAP2-28, and CmoRAV1 through CmoRAV6, respectively, based on their order on the chromosome. The precise location of each CmoAP2/ERF gene on the chromosomes of C. moschata is detailed in Additional File 3.To further investigate the gene duplication events in omosomes . ChromosCmoAP2/ERF genes, we analyzed tandem and segmental duplication events of these genes. Four pairs of tandem duplication genes were detected, of which two pairs were of ERF genes, and two were of DREB genes , RAV (2 pairs), DREB (53 pairs), and ERF (79 pairs) genes  elements were involved in the response of plants to hormones, such as ethylene (ERE), abscisic acid (ABRE), auxin , methyl jasmonate , gibberellin , and salicylic acid (TCA-element). Among 212 CmoAP2/ERF genes, 183 contained abscisic acid responsive elements, ABREs, which accounted for 9.04% of the total number of CREs in the CmoAP2/ERF family. A total of 166 CmoAP2/ERF genes contained ethylene responsive elements, EREs, which accounted for 13.34% of the total number of CREs in the CmoAP2/ERF family. The results indicated that most of the CmoAP2/ERFs might play a key role in phytohormone regulation, especially for abscisic acid and ethylene. The promoter regions of many CmoAP2/ERF genes contain different binding sites involved in stress response. A total of 1121 (19.87%) elements, such as ARE, W box, TC-rich repeats, LTR, MBS, WUN-motif, GC-motif, were involved in stress response. Among 212 CmoAP2/ERF genes, 174 contained ARE element, which accounted for 7.02% of the total number of CREs in the CmoAP2/ERF family. A total of 121 CmoAP2/ERF genes contained the W box element, which accounted for 3.49% of the total number of CREs in the CmoAP2/ERF family. Overall, 2064 (36.58%) elements, such as G-box, Box 4, O2-site, CAT-box, CCGTCC-box, MRE, circadian, were involved in plant growth and development. G-box and Box 4 were commonly found in the promoter regions of CmoAP2/ERF genes. The number of CmoAP2/ERF genes that possess G-box and Box 4 elements in the promoter regions was 189 and 188, respectively. This suggests that some members of the CmoAP2/ERF family might play important regulatory roles in the growth and development of plants.To better understand the regulatory mechanisms of CmoAP2/ERF genes respond to ethylene. To explore the roles of those genes in the response to ethylene, the RNA-Seq data of shoot apical meristem of pumpkin at the seedling stage after ethephon treatment were used to research their expression patterns. The results showed that 16 CmoAP2/ERF genes, including CmoERF24, CmoERF29, CmoERF53, CmoERF69, CmoERF76, CmoERF81, CmoDREB20, CmoDREB38, CmoDREB39, CmoDREB50, CmoDREB59, CmoDREB83, CmoRAV1, CmoRAV5, CmoRAV6, and CmoAP2-22, were significantly induced . Notably, all these 16 CmoAP2/ERF genes were upregulated after ethephon treatment  showed similar expression that they were up-regulated genes in the treatment of ethephon , jujube (18), and pineapple (27). This may be one of the reasons for the large number of AP2/ERF genes in C. moschata. In addition to tandem duplications and segmental duplications, whole-genome duplication events also affect the number of family members. Studies have found that C. moschata may have experienced whole-genome duplication events, core-eudicot common hexaploidization (ECH), cucurbit-common tetraploidization (CCT), cucurbita-specific tetraploidization (CST)  . TherefoCmoAP2/ERF genes, we found that 132 members of the CmoAP2/ERF family, including 67 ERF genes, 60 DREB genes, and five of the six RAV genes, had on introns . This indicates that these genes may play important roles in flower development. The expression levels of CmoAP2-22, CmoRAV1, CmoRAV5, CmoRAV6, CmoERF29, CmoDREB20, and CmoDREB50 in female flowers were significantly higher than that in male flowers. Furthermore, these 7 genes were up-regulated during ethylene induction of female flowers. Thus, they may post-ively regulate the formation of female flowers during floral sex differentiation of C. moschata. Further researches are required to understand the role of the ethylene-induced genes playing in the process of ethylene induction of female flowers. The results of this study confirm that some of the AP2/ERF genes play important roles in flower development.Several studies have shown that elopment . CsERF11cucumber . An applmoschata . The resCmoAP2/ERF genes were identified from C. moschata and their basic biochemical information, phylogenetic relationships, chromosome locations, gene structure, conserved motifs, expression profiles under ethylene treatment were determined. The expression levels of 16 ethylene-induced CmoAP2/ERF genes at different developmental stages of female and male flowers were further analyzed. These results should provide an opportunity to understand the roles of CmoAP2/ERFs in ethylene responses and in flower development.In this study, a total of 212 The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/QL and LZ conceived the study. QL and PC conducted the molecular experiments. CW, HZ, and JY provided software support. XL and JZ provided plant materials. QL wrote the manuscript. XL revised the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Small-cell lung cancer (SCLC) is an aggressive malignancy that exhibits a rapid doubling time, a high growth fraction, and the early development of widespread metastases. The addition of immune checkpoint inhibitors to first-line chemotherapy represents the first significant improvement of systemic therapy in several decades. However, in contrast to its effects on non-SCLC, the advantageous effects of immunotherapy addition are modest in SCLC. In particular, only a small number of SCLC patients benefit from immune checkpoint inhibitors. Additionally, biomarkers selection is lacking for SCLC, with clinical trials largely focusing on unselected populations. Here, we review the data concerning the major biomarkers for immunotherapy, namely, programmed death ligand 1 expression and tumour mutational burden. Furthermore, we explore other potential biomarkers, including the role of the immune microenvironment in SCLC, the role of genetic alterations, and the potential links between neurological paraneoplastic syndromes, serum anti-neuronal nuclear antibodies, and outcomes in SCLC patients treated with immunotherapy. Small-cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy that exhibits a rapid doubling time, a high growth fraction, and the early development of widespread metastases . DespiteThe expression of PD-L1 in cases of SCLC is reported to be less frequent than in cases of NSCLC. The scarce cellularity of SCLC specimens limits the ability to detect PD-L1 . In spitIn accordance with the analysis of the IMPower133 trial, the phase III CASPIAN trial, evaluating the use of durvalumab in combination with etoposide plus either cisplatin or carboplatin, showed no significant impact of PD-L1 expression on the effect of treatment. About half of the tumour specimens (277/531) were evaluable, showing low levels of PD-L1 expression. In particular, 22% and 5% of patients had tumours with expression levels ≥1% in immune and tumour cells, respectively. Neither the PD-L1 expression in tumour cells nor in immune cells correlated with better outcomes in patients treated with chemotherapy plus durvalumab  SimilarlIn a single-arm, phase II study investigating first-line maintenance pembrolizumab in patients with SCLC, 66% of the tumour specimens (30/50) were evaluable. Only three patients showed PD-L1 expression levels ≥1%. Among these, one had no measurable disease at study entry and two responded to therapy with a median PFS of 11 months. PD-L1 expression was also detected at the tumour–stromal interface in 8 out of 20 patients who responded or who had stable disease after induction chemotherapy. PD-L1 expression at the tumour–stromal interface resulted in a better outcome, specifically, an improvement of ORRs (37.5% versus 8.3%), PFS (6.5 versus 1.3 months), and median OS (12.8 versus 7.6 months) [n = 42) with patients characterized by a CPS <1% (n = 50). A CPS ≥1% was associated with an improvement of ORRs (35.7% versus 6%) and median OS (14.6 months versus 7.7 months), and an OS rate of 1 year for 53.1% patients with positive CPSs. The ORR was 27% (4 out of 15) among patients with unknown PD-L1 status (The combined positive score (CPS), consisting in the number of PD-L1-positive cells (tumour and immune cells) divided by the total number of viable tumour cells and multiplied by 100, was evaluated in the phase 1b multicohort trial KEYNOTE-028, involving patients with relapsed SCLC who were treated with pembrolizumab. In this trial, a CPS of ≥1% was an inclusion criterion for treatment with pembrolizumab, showing an ORR of 33% (8 of 24) . More re1 status , whereasn = 19) of the patients had a CPS ≥1%, showing a better outcome than patients with CPS <1% or those who were not assessable (n = 6). In particular, the post hoc analyses demonstrated a higher ORR (58% versus 33%), and mPFS  = 0.73, 95% CI, 0.25 to 1.91). Similarly, patients with higher levels of tumour-infiltrating lymphocytes (TILs) (n = 13) had a better ORR and mPFS [In a recent single-arm phase II Study, patients with relapsed SCLC were treated with pembrolizumab plus amrubicin until they showed progression. In total, 76% , intermediate (n = 44), and high (n = 47) TMB tertiles, with a median OS of 3.1 months, 3.9 months, and 5.4 months, respectively. At the same time, the analysis of data from the nivolumab plus ipilimumab group revealed similar results: the ORRs were 22%, 16%, and 46% in the low (n = 27), intermediate (n = 25), and high (n = 26) TMB tertiles, with a median OS of 3.4 months, 3.6 months, and 22 months, respectively  TMB was evaluated in SCLC patients treated with first-line atezolizumab plus chemotherapy. The bTMB assay is the same assay used to demonstrate the predictive value of bTMB in patients receiving atezolizumab for the treatment of relapsed NSCLC, obtained by collecting samples from two randomized trials, namely, the POPLARtrial and OAK study . Similarp = 0.027). In agreement with this, in a more recent study including 102 patients with histologically confirmed SCLC at stages I–III, high FOXP3 expression showed longer relapse-free survival times than the low-level group  [Due to the small number of biopsies with necrotic tissue and the uncommonness of the surgery treatment, studies concerning the immune microenvironment in cases of SCLC are few and retrospective. T cell infiltration in pretreatment biopsies correlates with an improved response to chemotherapy and a better prognosis. However, data concerning the characterization of T cell infiltration are contradictory. The higher expression of CD3, CD20, and CD45, the classic surface biomarkers of TILs, correlates with a better survival rate  Table 3Table 3. = 0.008) . On the = 0.008) ,29. In c= 0.008) .2 versus a median of 651 cells/mm2). In particular, the best discrimination was observed in the tumour stroma zone (p = 0.00003); this area revealed the highest number of TILs [Concerning macrophages (MPs), Eerola et al. evaluated 56 surgical samples from patients with SCLC, showing a link between a high number of intratumoural MPs and a favourable outcome. Moreover, intratumoural MPs directly correlated with microvessel density (MD). Nevertheless, the authors did not report the MP/T cell ratio, nor did they report the MP phenotype . In a re of TILs .Unfortunately, there is a lack of studies examining the tumour microenvironment as a predictive factor for immunotherapy in SCLC patients. A better knowledge of the relationship between tumour microenvironments and immune responses in SCLC patients could promote research into predictive factors for immunotherapy.Neurologic paraneoplastic syndromes (PNSs) associated with SCLC correlate with a favourable outcome ,39. The + CD4 T cells and memory CD45RO+CD4+ helper T cells. Nevertheless, the mechanisms that explain the better outcomes associated with neurologic PNSs remain unclear. Although neurologic PNSs occur in 3% to 5% of patients with SCLC, about half of all SCLC patients have neuronal antibodies (neur-Abs). Interestingly, SCLC patients with neur-Abs and without neurologic PNSs seem to have favourable outcomes as well. Gozzard et al. [p = 0.037) and in 28 patients (12%) with uncharacterised anti-neuronal nuclear antibodies (ANNA-U) . No differences have been found with any of the other neur-Abs, including anti-SOX2. Regarding anti-SOX2, two other studies showed that this antibody has diagnostic value in discriminating SCLC-LEMS from non-tumour LEMS but has no relation to survival in patients with SCLC [p = 0.032)  , whereasMore studies are needed to demonstrate whether neur-Abs are able to be used as biomarkers for immunotherapy efficacy and toxicity.Unlike the increasingly personalized approach to NSCLC, SCLC is still regarded as a single cancer type. SCLC is characterized by clinical and molecular heterogeneity. Firstly, SCLC is classified into neuroendocrine (NE)-high and NE-low subtypes, primarily based on the expression of different neuroendocrine markers, namely chromogranin, synaptophysin, neural cell adhesion molecule 1, and gastrin-releasing peptide ,50,51,52More recently, Gay et al.  identifiA pan-cancer, T cell-inflamed gene-expression profile (GEP) composed of 18 genes indicative of a T cell-activated TME resulted in a correlation with a better outcome in multiple tumour types treated with pembrolizumab . These 1The predictive value of inflammatory cytokines for immunotherapy has been evaluated in SCLC, comparing patients treated with chemotherapy and those treated with chemotherapy plus ipilimumab. The addition of ipilimumab resulted in an increase in various cytokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, Mip-1 alpha, GM-CSF, INF-γ, and TNF-alpha. In particular, higher baseline IL-2 levels and an increase in IL-4 levels during immunotherapy correlated with a better OS rate. On the other hand, high TNF-alpha and IL-6 levels predicted resistance to ipilimumab  Table 3Table 3.The lung immune prognostic index (LIPI), calculated from the ratio of derived neutrophils to lymphocytes (dNLR) and lactate dehydrogenase (LDH), was correlated with ICIs outcomes in patients with melanoma as well as NSCLC ,57,58. IGalectin-9 , a soluble lectin playing a major role in innate and adaptive immunity, was found to be correlated with the PD-L1, PD-1, FOXP3, CD3, CD4, and CD8 levels in SCLC patients. Moreover, higher Gal-9 expression in TILs correlated with better outcomes, suggesting a potential role of Gla-9 as a predictive biomarker in SCLC patients treated with immunotherapy .p = 0.014) [Human leukocyte antigen (HLA) plays a crucial role in the interaction between the immune system and cancer cells. Recently, a study concerning 102 patients with stage I–III SCLC who underwent radical surgery showed a correlation between HLA class II positivity on TILs and longer recurrence-free survival  . At the = 0.014) , GarassiFurther prospective studies concerning these factors linked to inflammation and host immune systems are needed in order to evaluate their predictive properties. On the other hand, due to the high plasticity of SCLC and the low quality of tissue samples obtained in SCLC patients, the study of host-related features could prove useful in the search for biomarkers that predict immunotherapy efficacy.Despite recent advances in SCLC research, there are still many crucial gaps in our understanding of this disease. The recent addition of ICIs to first-line chemotherapy treatment of ES-SCLC was the first significant improvement of systemic therapy in several decades. However, the magnitude of the treatment effect is very modest . TherefoAs is the case with NSCLC, several studies concerning strategies to improve the efficacy of ICIs are ongoing ,62. In p


Giant pituitary adenomas are highly invasive tumors whose treatment is challenging. Surgery is their management mainstay. However, there is no consensus about the type of approach. Open transcranial, microscopic, and endoscopic trans-sphenoidal approaches have all been employed, alone or in combination. Extended endoscopic endonasal techniques may represent a versatile and safe one-stage approach. Our research aimed at evaluating prospectively their applicability, effectiveness, and safety in a multicenter series, to acquire further evidence toward its use in the treatment of those challenging lesions. Ninety-six patients were recruited and followed-up for 52.4 months on average. Most of them (81.2%) presented with visual deficits and >50% had various degrees of adenohypophysis insufficiency. Resection of at least 75% of initial volume was achieved in all cases, with 98.7% visual improvement, >50% endocrine deficit recovery and a permanent complication rate of 4.2%, indicating extended endoscopic endonasal approaches as a valuable treatment option.Purpose: To evaluate factors influencing clinical and radiological outcome of extended endoscopic endonasal transtuberculum/transplanum approach (EEA-TTP) for giant pituitary adenomas (GPAs). Methods: We recruited prospectively all consecutive GPAs patients undergoing EEA-TTP between 2015 and 2019 in 5 neurosurgical centers. Preoperative clinical and radiologic features, visual and hormonal outcomes, extent of resection (EoR), complications and recurrence rates were recorded and analyzed. Results: Of 1169 patients treated for pituitary adenoma, 96 (8.2%) had GPAs. Seventy-eight (81.2%) patients had visual impairment, 12 (12.5%) had headaches, 3 (3.1%) had drowsiness due to hydrocephalus, and 53 (55.2%) had anterior pituitary insufficiency. EoR was gross or near-total in 46 (47.9%) and subtotal in 50 (52.1%) patients. Incomplete resection was associated with lateral suprasellar, intraventricular and/or cavernous sinus extension and with firm/fibrous consistence. At the last follow-up, all but one patient  with visual deficits improved. Headache improved in 8 (88.9%) and anterior pituitary function recovered in 27 (50.9%) patients. Recurrence rate was 16.7%, with 32 months mean recurrence-free survival. Conclusions: EEA-TTP is a valid option for GPAs and seems to provide better outcomes, lower rate of complications and higher EoR compared to one- or multi-stage microscopic, non-extended endoscopic transsphenoidal, and transcranial resections. Pituitary adenomas (PAs) are benign, slow growing tumors that may cause compression and/or encasement of surrounding neural structures, such as optic nerve, chiasm, pituitary stalk, and hypothalamus, and/or vascular structures, such as carotid artery, anterior cerebral artery, anterior communicating artery complex, and cavernous sinus (CS). Clinically, they may present with either signs and symptoms secondary to mass effect , or with clinical syndromes related to abnormal hormonal secretion ,2. TreatClinical data were prospectively collected and analyzed from a collaborative multicenter database including all cases of non-secreting GPAs undergoing EEA-TTP resection in 5 different European centers during a 5-year period (from January 2015 to December 2019).All patients had pre- and postoperative laboratory assessment  and imaging (CT and MRI scans). Invasion/extension of GPAs within CS was classified according to Knosp grade  by a neuBaseline tests included insulin-like growth factor 1 (IGF-1), adrenocorticotropic hormone (ACTH), 8 a.m. cortisol, prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), eostradiol, testosterone, sex hormone binding globulin (SHBG), thyrotropin (TSH), and total thyroxine (TT4).The aim of the evaluation was to assess visual acuity, color vision, peripheral vision, eye movements and appearance of retina and optic nerve. It included best-corrected visual acuity (BCVA), slit-lamp biomicroscopy, intraocular pressure, dilated fundus examination, visual field (VF), and optical coherence tomography (OCT) before and 6 weeks, 3, 6, 12, and 24 months after surgery.This study was conducted in accordance with the Principles of Ethics for Medical Research Involving Human Subjects set in the Declaration of Helsinki and its subsequent amendments. To report our results, we followed the recommendations of the STROBE  statement for observational studies .p-value < 0.05 was considered statistically significant. Graphpad online calculator was used for all statistical analysis of the study .Variables were classified as continuous or categorical. Mean, ranges, and medians were used for continuous data collected in the study. Categorical data were presented as total count and proportions. Extent of resection was analyzed regarding demographic, clinical, radiological, and intraoperative tumor characteristics using the chi-square and Fisher exact tests. A Description of EEA-TTP is based on previous technical reports ,16,17,18A total of 96 (8.2%) GPAs were treated by EEA-TTP and included in this prospective collaborative multicenter study among 1169 patients harboring PAs and endoscopically managed during a 5-year period. All centers contributed equally to patients’ recruitment. Demographics, patients’ clinical and radiological features, and outcome are summarized in p = 1.763) or hospital length of stay. Mean cranio-caudal diameter of lesion was 46.5 mm (range 41–61 mm). According to Knosp [Among the 96 patients included, 55 were men (57.3%) and 41 were women (42.7%), and the mean age was 52.2 years range 26–81 years). Gender and age were not significantly associated with EoR rate , while significant vascularization did not influence it. In multivariate analysis maximum diameter of the GPAs (p 0.021) and soft consistency of the lesion showed to be statistically significant factor influencing the EoR and favoring GTR (p = 0.021 and p = 0.043 respectively). All patients with preoperative visual impairment, except one developing a monocular blindness postoperatively, showed an improvement in both visual field and visual acuity deficits . Among 53 patients presenting preoperatively various degree of pituitary insufficiency, 27 (50.9%) showed a recovery of their pituitary function within 6 months from surgery. Regarding surgical complications, although a CSF leak was observed intraoperatively in 16 (16.7%) patients, it persisted postoperatively in 7 patients (7.3%), that underwent serial lumbar punctures (up to three deliquorations) or lumbar drain for 3 to 5 days. In 5 cases rhinorrhea dried up, while in 2 cases it was necessary a re-do surgical exploration, which revealed that the CSF fistula was caused by a necrosis of the pedicled flap used for primary repair, hence requiring rescue with a contralateral pedicled nasoseptal flap. Seven patients (7.3%) presented with postoperative bacterial meningitis, which was associated to postoperative CSF leakage in 3 cases only. All cases were successfully treated by targeted antibiotic therapy. Two patients (2.1%) had apoplexy of the residual tumor on 3rd and 5th postoperative day, respectively. Clinical presentation included abrupt headache, meningeal signs, severe hyperthermia, tachypnea with systemic alcalosis, arterial hypertension, 3rd nerve palsy, and DI. Both patients underwent prompt management in our endocrinology units with high dose steroids and close monitoring of vital parameters. In both cases, clinical improvement occurred within 7 days from symptoms onset. One case of postoperative monocular blindness (1%) and one case of postoperative hydrocephalus requiring ventriculoperitoneal shunt (1%) were recorded. Thirty-six patients (37.5%) developed transient diabetes insipidus, which resolved in 4 to 12 days after surgery, while delayed postoperative hyponatremia was observed in 20 patients (20.8%) and was resolved in all cases within 1 week from diagnosis. At a mean follow-up of 52.4 months (range: 24–88 months), 16 patients (16.7%) with a known residual tumor showed a progression of the disease after a mean recurrence-free survival of 32 months. Re-do EEA was carried on in 3 cases (3.1%), whereas 13 (13.5%) were referred for adjuvant radiotherapy .On univariate analysis, CS extension, large (>2.5 cm from diafragma sellae) suprasellar and/or intraventrincular extension, and firm/fibrous consistence showed to be significant factors in determining the EoR rate (GPAs are relatively uncommon, representing 5–14% of all adenomas ,18,19, aPostoperative visual field deficit improvement rate varies in the literature, and it is more likely to be obtained with transsphenoidal than transcranial surgery ,41. In aKoutourousiou et al.  found thManagement of GPAs is challenging and can be hardly standardized, as many factors affect surgical outcome. Therefore, a multimodal strategy should be tailored on a case-by-case basis. Although long-term disease control often needs adjuvant treatment, the initial course of action consists in an aggressive maximal resection, which should be pursued, with the aim of relieving mass effect in order to improve visual and endocrinologic function. EEA-TTP allows satisfactory resection rates, even for lesions extending into the CS, in the suprasellar compartment, in the ventricular and clival regions. However, tumor extension beyond the lateral walls of CS together with a firm/fibrous consistency continues to have negative predictive factors for total/near-total removal. Except for postoperative CSF leak, whose rate may be contained by careful application of pedicled flap reconstruction techniques, incidence of EEA’s complications appears to be lower than those reported from microscopic transnasal and open surgery. Thus, surgery through EEA-TTP approach should be promoted as the initial treatment for GPAs.


AtumOBP3, 26 and 28) and 3 CSPs  were highly expressed in antennae. Overall, this study could provide a basis for elucidating functions of these chemosensory genes at the molecular level.The small hive beetle is a destructive pest of honeybees, causing severe economic damage to the apiculture industry. Chemosensory genes play key roles in insect behavior, such as foraging and mating partners. However, the chemosensory genes are lacking in the small hive beetle. In order to better understand its chemosensory process at the molecular level, a total of 130 chemosensory genes, including 38 odorant receptors, 24 ionotropic receptors, 14 gustatory receptors, 3 sensory neuron membrane proteins, 29 odorant binding proteins, and 22 chemosensory proteins were identified from the transcriptomic data of antennae and forelegs. Reverse-transcription PCR showed that 3 OBPs , 24 ionotropic receptors (IRs), 14 gustatory receptors (GRs), 3 sensory neuron membrane proteins (SNMPs), 29 odorant binding proteins (OBPs), and 22 chemosensory proteins (CSPs) were identified. The analyses of tissue expression profiles revealed that genes encoding 38 ORs, 13 antennal IRs, 11 GRs, 1 SNMP, 24 OBPs, and 12 CSPs were predominately expressed in antennae. No significant differences in expression levels of these genes were found between males and females. Genes encoding 5 non-NMDA iGluRs, 3 GRs, 2 SNMPs, 5 OBPs, and 12 CSPs were predominately expressed in forelegs. RT-PCR assays for SNMPs, OBPs and CSPs further revealed that 3 OBPs  and 3 CSPs  were highly expressed in antennae. Our results enrich the gene inventory of A. tumida and facilitate the discovery of potential novel targets for developing new pest control measures. Insects rely on olfaction to recognize and discriminate chemical cues during foraging, mating, and oviposition ,2. The pInsect chemoreceptors consist of ORs, IRs and GRs are located in the dendritic membrane of olfactory receptor neurons (ORNs). ORs are receptors with two heteromeric subunits and each subunit with seven-transmembrane domains. ORs are composed of a highly conserved, universal co-receptor  and a variable partner (named ORX) that interacts with specific ligands . IRs areAethina tumida Murray (Coleoptera: Nitidulidae) is a destructive pest of honeybees. The beetle feeds on bee nest products and has serious negative impact on honeybees and other pollinators. The small hive beetle has spread to Sub-Saharan Africa and is now found in all continents except Antarctica [A. tumida pheromone [A. tumida. Also, A. tumida is highly attracted to volatiles emitted by adult honeybees (Apis mellifera), bumble bees (Bombus impatiens), stored pollen, wax, brood, and honey [A. tumida to perceive these volatiles needs to be revealed.The small hive beetle tarctica ,22,23. Iheromone . Recentlnd honey ,26,27. Tnd honey ,29,30. TThe objective of this study was to identify the repertoire of chemosensory genes  of the small hive beetle via a transcriptome analysis. We performed transcriptomes from dissected antennae and forelegs from both male and female adults. Insect forelegs are involved in sensing non-volatile chemicals after insects landing on the host ,32. BaseApis cerana cerana in the Shanwei city, Guangdong province, China . The colony was maintained at 20 °C, 65% RH, under darkness.A small hive beetle colony was maintained in Institute of Zoology, Guangdong Academy of Sciences in Guangzhou city and was originally established from a sample collected from naturally infested colonies of n = 80 each). Tissue samples were homogenized to powder immediately in liquid nitrogen and stored at −80 °C until RNA extraction.For transcriptome sequencing, 300 pairs of antennae and forelegs were dissected separately from females and males of the small hive beetle. Three replicates were included for each transcriptomic analysis. For RT-PCR analyses, we dissected the different tissues including the antennae, forelegs, wings, and genitals from adults  and potential genomic DNA contamination was removed by RNase-free DNaseI . RNA integrity was examined via agarose gel electrophoresis, and RNA purity and concentration were assessed using a NanoDrop spectrophotometer .One microgram purified RNA per sample was used as input material for library construction. Libraries were constructed using an Illumina’s TruSeq RNAseq Sample Prep kit  following the manufacturer’s instruction. cDNA libraries were evaluated using an Agilent 2100 Bioanalyzer, and sequenced on the Illumina HiSeq 4000 platform  with 150 bp paired-end module . Raw reads were firstly processed by removing adaptor sequences, unknown (poly-N) and low-quality reads and subsequently assembled into unigenes using Trinity (version: 2.0.6) Software  with default parameters.http://www.ebi.ac.uk/uniprot, accessed on 10 December 2019), Cluster of Orthologous Groups of proteins (COG) and Gene Ontology (GO) databases using BlAST program . To identify unigenes coding for ORs, IRs, GRs, SNMPs, OBPs, and CSPs, known protein sequences from other Coleopteran species . Open reading frame (ORF) of candidate chemosensory genes were predicted using the ORF Finder in NCBI . The conserved domain, signal peptide and cysteine location in candidate genes were analyzed by using the InterProScan tool plug-in in Geneious  [Gene annotation was achieved by searching these unigenes against the NCBI non-redundant protein database (NR), Swiss-Prot  . Candidahttp://tree.bio.ed.ac.uk/software/figtree, accessed on 11 December 2019). The data set contained genes identified in other Coleopterans as follows: 257 OR sequences from the OR data set ; 87 IR sequences from the IR data set ; 278 GR sequences from the GR data set ; 74 CSP sequences from the CSP data set . The data sets of chemosensory genes chosen from other insect species are listed in The amino acid sequences of candidate chemosensory genes were aligned using Clustal Omega . PhylogeTo estimate the expression levels of the candidate chemosensory genes in female antennae (FA), male antennae (MA), female forelegs (FL), and male forelegs (ML), the average FPKM (fragments per kilobase of exon per million fragments mapped) values were used . The estGAPDH and E-cadherin were used as internal reference genes. The PCR products were subjected to electrophoresis and the results were analyzed by gel imaging . At least three independent biological replicates were performed in this analysis. The gene-specific primers were designed using Primer5 software [Reverse-transcription PCR (RT-PCR) was employed to examine the expression pattern of 29 OBPs, 22 CSPs, and 3 SNMPs in different tissues including antennae, foreleg, wings, and genitals. Total RNA was extracted from these tissues, and the first-strand cDNA was synthesized by using a HiScrip III RT SuperMix Kit . PCR was performed under the following conditions: 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. software , and theWe sequenced the transcriptomes of female antennae (FA), male antennae (MA), female forelegs (FL), and male forelegs (ML) of A. tumida with three independent biological replicates. We obtained approximately 50.06 (FA1), 56.84 (FA2), 63.25 (FA3), 57.71 (MA1), 56.14 (MA2), 52.61 (MA3), 54.15 (FL1), 48.96 (FL2), 47.51 (FL3), 53.76 (ML1), 44.18 (ML2), and 48.87 (ML3) million clean reads from these samples. Clean reads were assembled into 34,531 unigenes with an average length of 1430 bp, and an N50 of 2403 bp . The datAfter annotation, there were 21,775 (63.05%), 15,912 (46.08%), 19,365 (56.08%), 7705 (22.31%), 19,561 (56.64%), 19,561 (56.64%), and 10,247 (29.67%) unigenes that homologous sequences were found in NCBI-nr, NCBI-nt, Swiss-Prot, KO, PFAM, GO and KOG databases, respectively. The overall 26,877 (77.83%) unigenes were annotated according to the homologous sequences .AtumOR1-38) were identified based on the combined transcriptome data from A. tumida (AtumOR1) had a high sequence homology with the conserved insect Orco gene family and named it AtumOrco. In previous studies, phylogenetic analysis has separated ORs in Coleopteran species apart from the Orco gene subgroup , into multiple subgroups numbered 1–7 [AtumOR8 and 26), 36 ORs were divided into six subgroups , with six ORs assigned to group 1, eight ORs assigned to group 2, six ORs assigned to group 3, two ORs assigned to group 4, two ORs assigned to group 5, and 11 sequences assigned to group 7, respectively. Group 7 was further assorted into two subsets: group 7a and group 7b. The rest one subgroup 6 contained only T. castaneum ORs. No AtumORs were clustered with high homology to known functional ORs, such as pheromone receptors from M. caryae [In total, 38 putative OR-encoding unigenes  had the highest level of expression in antennae from both males and females , followed by AtumOR24 . Genes encoding all 38 ORs exhibited similar expression patterns in the FA and MA.The expression levels of genes encoding all 38 ORs were assessed using FPKM-values b. Our trAtumIR8a, 21a, 25a, 41a, 60a, 68a, 75c, 75q1, 75q2, 75q3, 75s, 76b, and 93a, were clustered with their orthologs. Genes encoding all antennal IRs from A. tumida were expressed at relatively high levels in both FA and MA. In comparison, genes encoding a number of non-NMDA iGluRs  were expressed at higher levels in FL and ML  among all antennal IRs of the small hive beetle, followed by AtumIR8a  and AtumIR25a  (Twenty-four putative iGluR/IR-encoding genes were identified : Sheet 2L and ML b. The gean FPKM) .AtumGR1-14) were identified  were more highly expressed in the FL and ML (AtumGR14) was the highest in antennae. The expression of a gene encoding an unknown GR (AtumGR10) was the highest in forelegs  were identified. All these candidate genes had a full-length protein ranging from 121 to 303 amino acid, with a secretion signal peptide except AtumOBP2, 19, and 25 . Seven AtumOBPs clustered with other Coleopteran OBPs, while the remaining OBPs formed the sister pairs likely due to species-specific expansion (AtumOBP15 was the most abundantly expressed gene (FPKM > 1000), while AtumOBP10, 11, 24, 25 and 27 were moderately expressed (FPKM > 100). Among the OBP genes expressed highly in forelegs, AtumOBP21 was the most abundantly expressed OBP (FPKM > 1000).Twenty-nine putative OBP-encoding genes  were identified, with a full-length ORF ranging from 98 to 139 amino acid residues  were located in the same branch along with the orthologous sequences from other Coleopterans, while the remaining CBPs formed the sister pairs  and species-specific expansion   a. Based 9/14/16) b. AtumCSAtumOBP3, 26, and 28, and three CSP-encoding genes including AtumCSP7, 8, and 21, were abundantly expressed in antennae, whereas other AtumOBPs and AtumCSPs were expressed in multiple body parts. AtumSNMP1 was also expressed in antennae, forelegs, wings and genitals.Since SNMPs, OBPs and CSPs in insects were widely expressed in many tissues and might have different roles, we examined their expression levels in four tissues including antennae, foreleg tarsus, wings and genitals using RT-PCR . The resA. tumida. From these transcriptomes, we identified 130 putative chemosensory genes, including 38 genes coding for ORs, 24 for IRs, 14 for GRs, 3 for SNMPs, 29 for OBPs, and 22 for CSPs. Genetic and phylogenetic analyses were carried out on these genes to examine similarities and differences on related genes.Chemosensory proteins play an important role in insect behavior, including foraging, mating, and oviposition. In this study, we generated transcriptomes from antennae and forelegs of both males and females from AtumOR1 is highly conserved in comparison with Orcos from other Coleopterans. Other genes encoding ORs from A. tumida can be scattered into previously defined Coleopteran OR subgroups including subgroups 1–5, 7a and 7b [A. tumida have been found. Expanded OR subgroups in A. tumida include subgroup 3 with five members (AtumOR16/18/19/28/29), subgroup 7a with eight members (AtumOR20/32/33/34), and subgroup 7a with five members (AtumOR11/12/15/24/31). Expansion of OR gene subgroups specific to an insect species has also been found in other insect species, and it is generally acknowledged a strategy for a species to adapt to a distinct ecological niche [A. tumida [A. tumida uses vibrations to mediate sexual communication among adults rather than chemical signals among Coleoptera families including Anobiidae [a and 7b ,40. Seveal niche ,40. Stud. tumida . This minobiidae , Tenebrinobiidae , Cerambynobiidae , and Curnobiidae .AtumIR76b, AtumIR25a and IR8a, and 13 antennal IR-encoding genes including AtumIR21a, IR41a, IR60a, IR68a, IR75c, IR75q1, IR75q2, IR75q3, IR75s, and IR93a identified here have orthologs in other Coleopterans. Based on data from D. melanogaster, IR64a is sensitive to acetate, propionate and butyrate [IR41a, together with IR76b, mediate long-range attraction to odor [IR21a, together with IR25a, mediate behavioral responses to cool conditions [IR93a and IR68a mediate behavioral responses to both temperature and moisture [IR75q1 and IR75q2 are necessary for moths to conduct acetic acid preference, with IR75q1 recognizing acetic acid and IR75q2 amplifying sensitivity [A. tumida might play similar roles in sensory perception due to their high similarity. At present, the function of the IR60a remains to be determined and further investigation is needed to reveal its function. As expected, transcriptome analysis also showed all the antennal IRs had higher expression in the antennae as compared to the forelegs, and no sex-biased expression was found. This suggests that IR as an ancient chemosensory receptor family [IRs are the most ancient chemoreceptors for odor sensation and taste sensation ,49,50,51butyrate . IR41a, nditions . IR93a amoisture ,54. IR75sitivity . The IR r family  functionAtumOBP3, 26 and 28) were highly expressed in antennae, suggesting that they may play roles in olfactory perception. The two Classic OBPs, AtumOBP7 and 9, formed a cluster with other Coleopteran PBPs [AtumOBP7 and 9 are putative PBPs in A. tumida. Similar to OBPs, CSPs are also postulated to function as carriers of odorant molecules [AtumCSP7, 8, and 21, were highly expressed in antennae and their roles are likely involved in olfaction. Similar to the OR family, sex-biased expression was not detected in the antenna-predominant OBPs analyzed by RNA-seq. The expression profile of OBPs might function in the detection and discrimination of host volatiles or pheromone that elicit aggregation behaviors in both sexes [A. tumida are much smaller than most other Coleopterans [A. tumida may be due to host adaptation since A. tumida have relatively narrow host ranges. A. tumida is primarily a parasite of bee colonies. Further study on the function of the antenna specific OBPs and CSPs in A. tumida is needed to explore their functions.OBPs are commonly regarded as solubilizers and carriers of odorants  and can ran PBPs . We hypoolecules . Among tth sexes . RT-PCR opterans ,65,66. TA. tumida. From these transcriptomes, we identified 132 putative chemosensory genes, including 38 ORs, 24 IRs, 14 GRs, 3 SNMPs, 29 OBPs, and 22 CSPs. Furthermore, 3 OBPs  and 3 CSPs  were identified to be highly expressed in antennae. Our results might provide a foundation for the further study of olfactory function and the biological control of the small hive beetle.In the present study, we generated transcriptomes from antennae and forelegs of both males and females from


EFEMP1. Hence, we identified EFEMP1 as a novel disease-causing gene in the CL spectrum, differentiating it from other HDCT.Hereditary disorders of connective tissue (HDCT) compromise a heterogeneous group of diseases caused by pathogenic variants in genes encoding different components of the extracellular matrix and characterized by pleiotropic manifestations, mainly affecting the cutaneous, cardiovascular, and musculoskeletal systems. We report the case of a 9-year-old boy with a discernible connective tissue disorder characterized by cutis laxa (CL) and multiple herniations and caused by biallelic loss-of-function variants in  Hereditary disorders of connective tissue (HDCT) are a heterogeneous group of diseases, mainly affecting the cutaneous, ocular, cardiovascular, pulmonary, and musculoskeletal systems. HDCT are caused by pathogenic variants in genes encoding structural and regulatory components of the extracellular matrix (ECM) .FBLN4 and FBLN5 both cause severe recessive cutis laxa (CL), characterized by loose redundant skin folds and variable systemic involvement, including prominent emphysema [EFEMP1, is a secreted extracellular matrix glycoprotein, which is abundantly expressed in skin fibroblasts, retina, fascia, and vasculature [EFEMP1 has previously been associated with Doyne honeycomb retinal dystrophy [EFEMP1 were recently reported in two probands with a pronounced connective tissue phenotype, characterized by multiple herniations and joint hypermobility [Fibulins belong to a protein family implicated in both elastic fiber (EF) assembly and function. Loss-of-function variants in mphysema ,3. Fibulystrophy . Howevermobility ,8.EFEMP1.In this study, we present a 9-year-old Turkish boy with a novel, discernible connective tissue disorder characterized by cutis laxa and multiple herniations, associated with biallelic loss-of-function variants in The legal guardian of the patient in this manuscript gave written informed consent to publication of the case details. Specific informed consent was obtained for the publication of clinical pictures. This study was conducted in accordance with the 1984 Declaration of Helsinki and its subsequent revisions.EFEMP1 reference sequence (Refseq NM_001039348.3).Exome sequencing (ES) of the proband was performed on genomic DNA extracted from peripheral leukocytes. Target enrichment of the gDNA was obtained by the SureSelectXT Low Input Human All Exon V7 . Sequencing was performed on a NovaSeq 6000 platform  with a minimal expected coverage depth of 20×. All obtained variants were analyzed using the Seqplorer software, an in-house developed pipeline, which integrates population frequencies (GnomAD)  and in shttp://www.hgvs.org/VARONEM, accessed on 28 February 2021) [All variants were reported according to the nomenclature of the Human Genome Variation Society  .EFEMP1 ; weight: 15.9 kg (−2.5 SD)), despite reportedly normal intake. His head circumference was normal (50 cm (−1.28 SD)). He presented with discernable craniofacial characteristics, including a long face, a high anterior hairline, telecanthus, blepharochalasis, sagging lower eyelids and cheeks, a long philtrum, full lips, downturned mouth corners, retrognathia, dental caries, malocclusion, and cutis laxa A. He furEFEMP1 C. This vEFEMP1 (OMIM#601548), which encodes fibulin-3. Several lines of evidence support the causality of the identified loss-of-function variant for the observed phenotype in the reported individual. We present a 9-year-old Turkish boy with a discernible connective tissue disorder consisting of cutis laxa and multiple herniations with homozygous loss-of-function mutations in FBLN4 (EFEMP2) and FBLN5 cause autosomal recessive cutis laxa (ARCL) type 1b and 1a, which are associated with severe vasculopathy and developmental pulmonary emphysema, respectively. Of note, the latter condition is also associated with diverticula of the gastrointestinal and genitourinary system, as well as congenital diaphragmatic hernia. ARCL1c, a third disorder within the ARCL type 1 spectrum, shows considerable overlap with ARCL1a and is caused by pathogenic variants in LTBP4, which is an interaction partner of both FBLN4 and FBLN5 [Firstly, fibulin-3 belongs to a family of 8 ECM glycoproteins associated with basement membranes and elastic fibers and is abundantly expressed in skin fibroblasts and fascia . FibulinLN4 EFEMP and FBLNnd FBLN5 . Efemp1-knockout mice demonstrate herniations, loose skin, small body size or mass, atrophy of muscle and fat tissue, and dermal EF fragmentation. Interestingly, the resulting phenotype is highly dependent on the genetic background of the mice, as herniations are predominantly manifested in Efemp1−/− mice on the C57BL/6 background, but not the BALB/c background [Secondly, ckground . EFEMP1 have previously been reported in 3 individuals of 2 distinctive families with multiple herniations and joint hypermobility, supporting loss-of-function as the molecular mechanism of a novel heritable connective tissue disease (EFEMP1 [EFEMP1 are associated with a specific connective tissue disorders characterized by multiple hernias and skeletal and pulmonary manifestations. Although clinically variable, this disorder should be classified within the group of ARCL type 1 [Thirdly, biallelic loss-of-function variants in  disease . Our pat (EFEMP1 . Althoug (EFEMP1 ,8,21. OfL type 1 ,8,19,21.EFEMP1 cause a novel HDCT characterized by cutis laxa and recurrent herniations. As these patients additionally present with mild cutaneous and pulmonary manifestations, we propose this HDCT to be a classified as a subtype of ARCL type 1. This observation confirms that EFEMP1 has a pivotal role in abdominal wall integrity.In conclusion, biallelic loss-of-function variants in


Asthma is a mysterious disease with heterogeneity in etiology, pathogenesis, and clinical phenotypes. Although ongoing studies have provided a better understanding of asthma, its natural history, progression, pathogenesis, diversified phenotypes, and even the exact epigenetic linkage between childhood asthma and adult-onset/old age asthma remain elusive in many aspects. Asthma heritability has been established through genetic studies, but genetics is not the only influencing factor in asthma. The increasing incidence and some unsolved queries suggest that there may be other elements related to asthma heredity. Epigenetic mechanisms link genetic and environmental factors with developmental trajectories in asthma. This review provides an overview of asthma epigenetics and its components, including several epigenetic studies on asthma, and discusses the epigenetic linkage between childhood asthma and adult-onset/old age asthma. Studies involving asthma epigenetics present valuable novel approaches to solve issues related to asthma. Asthma epigenetic research guides us towards gene therapy and personalized T cell therapy, directs the discovery of new therapeutic agents, predicts long-term outcomes in severe cases, and is also involved in the cellular transformation of childhood asthma to adult-onset/old age asthma. Asthma is well known as a noncommunicable, chronic, and heterogeneous inflammatory condition of the lower airway tract characterized by various clinical conditions that vary in severity and frequency , 2. AsthThe common asthma phenotypes are type 2 asthma (T2 high) and non-type 2 asthma (T2 low). T2 asthma includes early-onset allergic and late-onset nonallergic eosinophilic asthma. The common Th2 biomarkers used in clinical practice are mainly blood eosinophils, fractional exhaled nitric oxide (FeNO), and IgE levels. Interestingly, the majority of people with T2 asthma respond well to standard therapy with inhaled corticosteroids . HoweverAlthough we have ongoing research and an improved understanding, prevalence of asthma has been increasing in recent years  and affeWaddington described the term epigenetics more than half a century ago . Later, Despite extensive knowledge, there is still much unknown about asthma. There are many new etiologic factors, variations in presentation, and new genetic linkage with asthma. Therefore, we must ask if there are any new factors driving asthma development and progression besides the known factors and etiology. This review is aimed at providing an overview on asthma epigenetics and its components, including asthma epigenetic studies, and discuss potential epigenetic links between childhood asthma and adult-onset/old age asthma.The answer is yes. Asthma epigenetics have received immense interest because genetic and environmental factors cannot wholly and independently explain asthma etiology, heterogeneity, and phenotypes . ResearcHistorically, DNA methylation in mammals was documented just after the discovery of DNA as a genetic component . MethylaBisulfite conversion (sequencing) of DNA is the most commonly used technique for DNA methylation. Other assessment techniques include high-performance liquid chromatography, microarrays or bead chips, mass spectrometry, methyl sensitive cut counting (MSCC), and antibodies or protein that bind to methylated DNA . To dateDNA methylation is the most studied mechanism in asthma. Alteration in DNA methylation status results in differential gene expression related to cytokines and transcription factors, resulting in various and distinct phenotypic presentations in asthma .ALOX15 (genetic loci: 17p13.2) and POSTN (genetic loci: 13q13.3) genes were hypomethylated in the nasal epithelium and associated with asthma [ALOX15 promotes eosinophilic inflammatory diseases, such as asthma [POSTN gene is upregulated in asthmatic airway epithelium by Th2 cytokines and can be used as a biomarker for long-term predictability in the management of severe asthma [ALOX12 in blood, at genetic loci 17p13.1 [FOXP3 (12q15) and IFN-γ (Xp11.23) results in impaired T cell function along with the repression of Tregs and T effector cell genes in the blood [IL-4, RUNX3, and TIGIT in blood, at genetic loci 5q31.1, 1p36.11, and 3q13.31, respectively [h asthma . ALOX15 s asthma , and asps asthma . The POSe asthma . The inc 17p13.1 . Hypermehe blood . Asthma IL-2 Site1 (4q27) has been found in the cord blood of children with severe asthma who were followed up for 8 years [IL5RA (3p26.2) in the blood is associated with asthma in teens [GATA3 (location 10p14), a significant regulator of Th2 differentiation, is associated with reduced asthma risk in cord blood [ZPBP2 (Zona Pellucida Binding Protein 2) hypomethylation results in asthma in a lymphoblastoid cell line at genetic location 17q12-q21 [CYP26A1 (10q23.33), a regulator of the allergic immune response, was found to be hypermethylated in blood samples of patients with asthma [TET1 (10q21.3) from the nasal epithelium results in asthma in children exposed to traffic-related air pollution [TET1 converts 5-methylcytosine to oxymethylcytosine along with 5-formylcytosine, 5-carboxylcytosine, and 5-hydroxymethylcytosine, resulting in DNA demethylation. IL-36, IL-38 [IL1R2 (2q11.2) in blood shows asthma-related phenotypes [Hypermethylation of  8 years . Hypometin teens , and IL-in teens . Hypermerd blood , while Z7q12-q21 . CYP26A1h asthma . Hypometollution . TET1 co6, IL-38 , and IL-enotypes . These sAXL gene (19q13.2) in CpG islands in newborn blood spots [Increased risk of wheezing, especially in girls, is associated with hypermethylation of the od spots . Hypermeod spots , and airod spots .ADRB2 gene (5q31-33) sampled from blood is hypermethylated and associated with severe asthma, but nitrogen dioxide (NO2), a known air pollutant, has been shown to alter this association [ADRB2 hypomethylation sampled from blood and saliva [PCDH20) has been found in the sputum of asthmatic adult smokers at the genetic locus 13q21.2 [ADRB2) gene play a vital role in the phenotypic presentation of asthma.The ociation . Interesd saliva . Hyperme 13q21.2 . EpigeneCDH26 and CDHR3  from the epithelial airway are hypomethylated in children with a history of atopy and can easily differentiate atopy with atopic asthma [C7orf50 and ZAR1 , which are sampled from cord blood and are associated with IgE levels [STAT5A genes are downregulated, differently methylated, and associated with different asthma levels [STAT5A genes in the region 17q21.2 are hypermethylated and associated with enhanced Th1 response and decreased eosinophil recruitment in the airway epithelium [Differently methylated regions are considered the beginning of development stages and with reprogramming progress . Cadheric asthma . The notE levels . STAT5A a levels . STAT5A ithelium . These mWNT signaling gene in neutrophilic asthma and purine and calcium metabolism gene in eosinophilic asthma. Hypermethylation of WNT2 genes in the region 7q31.2 from blood samples is associated with neutrophilic asthma [ORMDL3 from endobronchial airway epithelial cells at the genetic locus 17q12-21 results in asthma in adults [Research has shown that asthmatics showed distinctive methylation differences associated with the c asthma . Similarc asthma . Hypermen adults . Hypermen adults . From thThe environment is considered a significant influencer of DNA methylation and asthma. Maternal tobacco smoking, air pollution, stress, heavy metal exposures, pesticides, microbes, and possibly some foods are the epigenetic influencer. Therefore, the epigenetic mechanism, through the influence of environmental factors, affects the gene transcription and results in a more complex phenotypic pattern and presentation of asthma and allergy .2 from cord blood samples and a dose-dependent NO2 connection with pregnancy [iNOS by particulate matter 2.5 (PM2.5) and particulate matter 10 (PM10) [ACSL3 by traffic-related air pollution (TRAP) [γ CpG sites by exhaust particulate matter (DEP) [Prenatal and early childhood maternal tobacco smoking is considered a risk factor for asthma, and epigenome-wide studies have shown variations in DNA methylation in many genes involved in asthma  along wiregnancy . Mixtureregnancy . Researc0 (PM10) , methylan (TRAP) , and hyper (DEP) . DEP is er (DEP) . TogetheA study in 2019 showed that lead and cadmium are associated with asthma and other allergic conditions, and an interestingly significant degree of airflow obstruction is associated with the serum level of these metals . VanadiuSMAD3 [Environmental microbial agents play an immune-modulating role under many conditions. The clinical asthma characteristics and airway microbiota have shown a significant correlation between asthma control and various pathogens . RhinoviSMAD3 .Chlamydia muridarum, Rotavirus 1 (RV1), and Haemophilus influenzae for the induction of non-Th2 inflammation. HDM exposure has been found to induce DNA methylation of various potentially important genes for allergic asthma development [FOXO1, RUNX1, SP1, and APP) are potentially crucial for the development of asthma in the OVA model [Ovalbumin (OVA) and house dust mites (HDM) are the most common allergen used in the mouse model of allergic asthma for the induction of the Th2 pathway. Other asthma models use paramyxovirus, elopment . In alleelopment . DNA metVA model .The role of diet, folate, vitamin B12, vitamin C, calcium, vitamin D, fish oil, and antibiotics in pregnancy and asthma has been established –80. The Histones are a highly alkaline principal fundamental protein component of chromatin that acts as a spool for DNA winding. The unwound DNA length to width ratio would be beyond 10 million to 1. Therefore, it is considered a crucial element for genetic stability, packaging, and gene expression.Histone modification, an epigenetic mechanism, is an influencing factor in the pathogenesis and development of asthma, affecting the maturation and differentiation of cells involved in asthma  Figure . HistoneAllfrey et al. have reported the first documented case of histone acetylation in 1964 from calf thymus , and theThe addition of phosphate, phosphorylation, is mediated by kinases, while phosphatases remove phosphate. Similarly, histone ubiquitination is mediated by ubiquitin ligases and opposed by ubiquitin-specific peptidases, also known as deubiquitinating enzymes. Histone sumoylation is mediated by the histone sumoylation proteins, also known as the small ubiquitin-like modifier (SUMO) protein. Interestingly, although the biological process of DNA methylation and histone methylation differs, they mutually interact with each other .Notch gene is crucial for Th2 response in asthma, and Notch 1 dysregulated signaling of T cells is observed through the hyperacetylation of H3K9, H3K14, H3K18, H3K27, and H3K16 and trimethylation of H3K4 and H3K79 [Notch1 expression by HAT inhibitors reduced Th2 cytokines, especially IL-4, IL-5, and IL-13 [LAT is a protein-coding gene of T cells, and its deficiency enhances Th2 proliferation. LAT hypoacetylation results in the inhibition of LAT in an allergic asthma model [SOX2, and HDAC1 mediates the remodeling of the asthmatic epithelium, while its inhibition prevents airway remodeling through SOX2 [HDAC2 reduction signifies the possibility that the poor effectiveness of corticosteroids and inflammatory genes may be regulated by deacetylation of the glucocorticoid receptor through HDAC2 . Activatnd H3K79 . Suppresnd IL-13 , and it ma model . Self-reugh SOX2 .IFN-γ, IL-4, IL-17A, IL-17F, and transcriptional factors of Th17 and Treg cells (RORγt and Foxp3) have been observed with H3K4 trimethylation (H3K4me3) [Foxp3 and IL-13 gene acetylation is associated with childhood asthma [ORMDL3 is associated with asthma [ORMDL3 expression is associated with airway remodeling and Ca2+ oscillations. EGFR plays an important role in the growth and maintenance of airway epithelium. Delta Np63 induces epithelial shedding, and STAT6 is involved in chronic inflammation of the airway mucosa. TSSs of EGFR, STAT6, and Delta Np63 show increased acetylation of H3K18 in the airway epithelium of asthmatics [Increased expression levels of H3K4me3) . Foxp3 ad asthma . Histoneh asthma , and incthmatics .CCR4 and CCL5 dimethylation (H3K4me2) at single nucleotide polymorphisms is associated with Th2 differentiation [Chemokine ligand-8, a neutrophil activator produced by macrophages, and H3K18 acetylation leads to increased secretion of this activator from airway smooth muscles . CCR4 isntiation .Studies have documented environment-induced effects on histone modification and asthma risk. Smoking induces increased acetylation at H3 and H4, and transmission of asthma in subsequent generations has been studied in an animal model , 97. HDAAcinetobacter lwoffii F78 administration to experimental mice during the prenatal period has shown beneficial effects and prevented the asthma phenotype. In this case, H4 acetylation of IFN-γ and deacetylation of IL-4 promoter genes result in the downregulation of Th2 cytokines, showing transmaternal asthma protection [Environmental microbes may play roles in the immune response's modulation of immune responses. Histone H4 acetylation of essential Th2 genes is modified by nematode infections, such as ascariasis and HDM . Interesotection . Gut micotection , 107. Inotection .Nickel, a silver-colored metal found in a large amount in some grains, vegetables, and fruits, can cause allergies in susceptible people and are associated with asthma and wheezing . Nickel-In 1993, Victor Ambros' laboratory discovered miRNAs and simultaneously identified the miRNA target gene. miRNAs are approximately 22-25 nucleotide single-stranded small noncoding RNA molecules transcribed from the DNA; however, they are still not translated into proteins and have a role in gene expression either by blocking or by altering mRNA translation stability . miRNAs Although the discovery of miRNA is approaching thirty years, its role in asthma became a topic of investigation in the last decade. Many mouse and human studies have been conducted to identify the role of microRNAs via epigenetic changes in genes, and altered miRNA expression has been found in various lung diseases, including asthma , 114 Fi. In the A recent study published in 2020 has shown that circulatory miRNA levels correlate with the clinical status in patients with allergic and nonallergic asthma , and cirγ and IL-12 from dendritic cells with reduced IFN-γ production from CD4+ T cells [miR-21 prevents the expression of IL-3, IL-5, and IL-12, while the removal of miR-21 induces the production of IFN- T cells . miR-21  T cells –120. A s T cells . Th2 cyt T cells , and miR T cells . Similar T cells . TogetheThe increased expression level of miR-1248 is believed to be involved in the pathogenesis of asthma. miR-1248 increases IL-5 expression by interacting with the 3′UTR of the gene . Upregulβ2-agonist responses are altered by miR-16 through ADRB2 [STAT3 inhibition [MMP-16 and ATG7 by miR-192-5p [In asthma, miR-16 and lung function parameters are negatively correlated, and inhaled gh ADRB2 . Circulagh ADRB2 . Pulmonagh ADRB2 . The Th2gh ADRB2 , and exagh ADRB2 . VEGF-A gh ADRB2 . Airway hibition  and MMP-R-192-5p . This meR-192-5p . Thus, rSYK and EGFR genes are targets of miR-27-3p that influence the PI3K-AKT pathway and cytokine production [β suppression [miR-27-3p is an important player in experimental pediatric asthma that mediates immune reactions. oduction . An invepression . hsa-miRpression .Taken together, these studies have shown the role of multiple miRNAs in Th1/Th2 polarization, disease presentation, correlation of clinical status, severity, remission, noninvasive biomarker, therapeutic potential, and response to therapy in asthma.in vivo and in vitro studies have identified an association between environmental factors, miRNAs, and asthma. Different types of allergens, such as cigarette smoking, air pollution, agriculture pesticides, metals, and various microbes, induce epigenetic changes in genes through the miRNA and show various phenotypic presentations and even remission of asthma. This section reviews the association between environmental factors, miRNAs, and asthma. However, some of the associations have already been described in miRNAs and Asthma.Some NCX1 downregulation and miR-212-5p activation, as shown by the significant restoration of blood pressure after the knockdown of miR-212-5p in asthmatic mice [OVA induces experimental asthma by downregulating let-7 miRNAs and IL-13 expression . Exogenotic mice . OVA, HDmiR-335-5p inhibits inflammatory cytokines, and cigarette smoking lowers the expression of miR-335-5p in parenchymal lung fibroblast . The uprFOXP3 through downregulation of miR-34a [Recent findings have shown that indoor air pollution changes serum miR-155 levels and aggravates asthma . As an a miR-34a . Indoor β pathway via inhibin-β A [The association between serum levels of heavy metals, such as cadmium, mercury, and lead, and asthma and allergic rhinitis has been established. miR-148a, miR-211, miR-520c-3p, and miR-572 are associated with lead poisoning and are biomarkers of lead susceptibility in the Chinese worker population . miR-211ibin-β A . Cadmiumibin-β A . miRNA-3ibin-β A , 161. Ocibin-β A . These sin vivo viral respiratory infection has shown increased IFN-γ production, Th1 polarization (IFN-γ/IL-4 ratio), and proinflammatory responses [The influenza virus, rhinovirus, and respiratory syncytial virus (RSV) cause respiratory illness and exacerbate asthma , 72, 74.esponses . Rhinoviesponses . Reducedesponses . In seveesponses , which mesponses . These vAsthma predominantly occurring during childhood is childhood asthma, while asthma occurring during adulthood and old age is termed adult-onset/old age asthma. The T2 phenotypes of asthma (T2 high) include early-onset allergic and late-onset nonallergic eosinophilic asthma. The common Th2 biomarkers used in clinical practice are mainly blood eosinophils, FeNO, and IgE levels. Interestingly, the majority of people with T2 asthma respond well to standard therapy with inhaled corticosteroids , whereasHowever, we should not forget that these profiles are not strictly age-limited , and specific and useful signature biomarkers are lacking for non-T2 asthma apart from the absence of T2 high inflammation. Together, these observations suggest the two phases of asthma in human life as childhood asthma and adult-onset/old age asthma.Environmental factors are asthma epigenetic influencers that induce epigenetic changes in genes through DNA methylation, histone modifications, and miRNAs, as reviewed in Sections in utero nicotine exposure, maternal E-cigarette exposure, and even secondhand smoke (ETS) during pregnancy are associated with increased risk and rate of childhood asthma, elevated IgE levels, wheezing, alteration of childhood respiratory symptoms, and increased bronchial activity [Chlamydia muridarum, Rotavirus 1, and Haemophilus influenzae induce non-Th2 inflammation [Acinetobacter lwoffii F78 prenatal administration prevents childhood asthma in an animal model [Maternal tobacco smoking, activity , 149. Practivity . Similaractivity . Viral rammation , 72, 129ammation . Interesal model . TogetheMultiple environmental factors are also associated with adult-onset/old age asthma. Occupational exposure to lead, cadmium, mercury, nickel, and pesticides results in asthma and symptomatic airflow obstruction in adults , 68. RhiOverall, human asthma studies and experimental asthma models have shown the involvement of various environmental factors in the pathogenesis of childhood and adult-onset/old age asthma , 154 by Is there a link between childhood asthma and adult-onset/old age asthma? Does adult-onset/old age asthma represent the persistence or relapse of childhood asthma? How does increased IgE, eosinophil-predominant, and steroid-sensitive T2 asthma change into neutrophilic/paucigranulocytic, steroid-insensitive non-T2 severe asthma with age? At this point, through the review of components of epigenetic mechanisms  and genetics and environmental factors, we strongly believe that the epigenetic mechanisms are involved in the cellular transformation of childhood asthma to adult-onset/old age asthma .in vivo, in vitro, and human studies are needed, we believe that the asthma epigenetic mechanism can be a novel process for the identification of unsolved pathogenesis, new asthmatic loci, heterogeneity, and treatment options through the discovery of new drugs. Identification of epigenetic markers is vital for identifying asthma endotypes, phenotypes, personalized treatments, and prevention. In the future, we expect that the study of asthma epigenetics will guide us towards gene therapy, T cell therapy, and even provide directions for treating severe uncontrolled drug-insensitive asthma and predict long-term outcomes.Asthma epigenetics play an important role in immune response and upregulation of various cellular functions, including T cell differentiation, Th cell balance, changes in the expression of inflammatory genes resulting in asthma, and remission or protection in some cases. Asthma epigenetic studies can be a valuable approach to identify the increasing incidence of asthma and equally crucial for the study of environmental factors involved in asthma pathogenesis. Although more clinical, epidemiological, environmental, cellular, and interventional Given the queries raised, we believe that the epigenetic mechanisms play vital roles in the cellular transformation of childhood asthma  to adult-onset/old age asthma . However, we expect further epigenetic studies on this aspect in the future.Although epigenetic studies of asthma are still at early stages, we hope that the mystery surrounding asthma will be solved to some extent via the integration of omics data via novel findings for improved phenotyping, diagnoses, and asthma treatments, as well as the mechanism that will eventually prevent its development. Together, in view of all asthma epigenetic studies, we can conclude that asthma epigenetics are a valuable, novel approach to solve the mysteries surrounding asthma and will guide us to the discovery of new therapeutic agents and prediction of long-term outcomes in severe cases and aid in the understanding of the cellular transformation of childhood asthma to adult-onset/old age asthma.