The problem of specificity of psychological adaptation mechanisms at the patients with Type 2 Diabetes Mellitus (T2DM) is extremely actual. The aim is to investigate gender psychological characteristics associated with T2DMIn the comparative study 62 patients  with T2DM  and visceral obesity (Grade 2) were included.Research methods: the Depression Scale of Zung, the Spielberger trait scale anxiety, Toronto Alexithymia Scale and MMPI testT2DM-female-patients in comparison with T2DM-male showed significantly higher personal anxiety scores , depression scores , while alexithymia scores were higher at T2DM-males . In MMPI test  46,8% of patients demonstrated profiles with elevated scale 1 score  regardless of gender differences. However, the T-scores for T2DM-male patients were on the average by 1,07 higher than for T2DM-female , that indicated more higher concern related to own physical health condition. The female T2DM-patients significantly more often demonstrated profiles with scale 6 peak (exceeding 65 Т-scores): 79,4% vs. 21,4%, which indicated the more higher accentuation of personality traits The patient’s gender has to be taken into consideration at development of clinical, diagnostic and prevention activities of patients with T2DM and visceral obesity.


SLC20A1 expression (SLC20A1high) exhibited poorer clinical outcomes in those with tumor stage I luminal A breast cancer. In addition, this SLC20A1high subgroup of patients exhibited less responses to endocrine therapy, specifically in those with the luminal A and luminal B subtypes of breast cancer. However, patients with SLC20A1high showed good clinical outcomes following chemotherapy. Patients tested to be in the SLC20A1high group at the time of diagnosis also showed a higher incidence of recurrence compared with those with lower expression levels of SLC20A1, at >15 years for luminal A breast cancer and at 10–15 years for luminal B breast cancer. Therefore, we conclude that SLC20A1high can be used as a prognostic biomarker for predicting the efficacy of endocrine therapy and late recurrence for ER+ breast cancer.Estrogen receptor-positive (ER+) breast cancer intrinsically confers satisfactory clinical outcomes in response to endocrine therapy. However, a significant proportion of patients with ER+ breast cancer do not respond well to this treatment. Therefore, to evaluate the effects of endocrine therapy, there is a need for identification of novel markers that can be used at the time of diagnosis for predicting clinical outcomes, especially for early-stage and late recurrence. Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate symporter that has been proposed to be a viable prognostic marker for the luminal A and luminal B types of ER+ breast cancer. In the present study, we examined the possible association of SLC20A1 expression with tumor staging, endocrine therapy and chemotherapy in the luminal A and luminal B subtypes of breast cancer. In addition, we analyzed the relationship between SLC20A1 expression and late recurrence in patients with luminal A and luminal B breast cancer following endocrine therapy. We showed that patients with higher levels of  Breast cancer is the most common malignancy among women and the leading cause of cancer-associated mortality in women worldwide . In geneEstrogen receptor-positive (ER+) breast cancer is a major breast cancer subtype that accounts for 70–80% of all types of breast cancers . Breast SLC20A1 expression (SLC20A1high) in the claudin-low and basal-like subtypes have been reported to show inferior clinical outcomes. Radiation therapy against SLC20A1high claudin-low and basal-like subtypes of breast cancer tumors has been demonstrated to be insufficient [SLC20A1 expression for predicting late recurrence in patients with ER+ at early tumor stages, its relationship with tumor stage or endocrine therapy outcomes, and late recurrence remain to be clarified.Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate (Pi) symporter , 27. Thefficient . In addifficient . SLC20A1fficient . AlthougSLC20A1 expression in detail, we evaluated its association with tumor stage and endocrine therapy outcomes in patients with luminal A and luminal B breast cancers in the present study. Additionally, we analyzed the possible relationship between late recurrence and SLC20A1 expression in patients with luminal A and luminal B breast cancer after endocrine therapy.To assess the potential prognostic implications of http://cbioportal.org) [SLC20A1high and low-expression (SLC20A1low) groups through receiver operating characteristic (ROC) analysis. ROC was performed by SLC20A1 expression gene and disease-specific survival (DSS) or relapse free status (RFS) for each group divided, and calculated Youden’s index  dataset , 30 was tal.org) , 32 on Jtal.org) , 33, 34.’s index . PatientSLC20A1high and SLC20A1low groups using the log-rank (Cochran-Mantel-Haenszel) test. Kaplan-Meier survival curves were produced using BellCurve for Excel version 3.00 .Survival curves based on DSS and RFS were plotted using the Kaplan-Meier method. The curves were compared between the SLC20A1 gene expression on patient outcome and to estimate the SLC20A1high group’s adjusted hazard ratios (HRs) to SLC20A1low group for DSS or RFS. The ages at the time of diagnosis were adjusted as a confounding factor to remove the effect of age. We set the level of significance to be at 5% (two-sided). Multivariate Cox regression analyses were carried out using BellCurve for Excel version 3.00 .Multivariate Cox regression analysis was performed to evaluate the influence of high and low p-value was calculated from the statistic based on normal distribution and corrected using the Holm method. Incidence rate ratio was calculated as the ratio of the recurrence incidence rate of SLC20A1high group to that in the SLC20A1low group.The recurrence incidence rate was calculated using the number of recurrences divided by the observation term of the patients with luminal A and luminal B breast cancer after endocrine therapy. The observation term was divided every 5 years, where the number of recurrences was then counted during that term. The observation term of the patients was the total observation time (year) during that period. The SLC20A1 expression is associated with poorer clinical outcomes [SLC20A1 expression in ER+ breast cancer subtypes in detail, we analyzed a METABRIC dataset that included the gene expression data from 1904 breast cancer patients. We first compared the levels of SLC20A1 expression in tissues among the various tumor stages. Box-plot analysis revealed no statistical difference in SLC20A1 expression among the tumor stages    ; however, this was not the case with patients at tumor stage III    and stagp<0.001) . At tumo = 0.19) . To veri42–1.18) . These rSLC20A1high and SLC20A1low groups at tumor stages I, II or III. Kaplan-Meier analysis showed that patients with SLC20A1high luminal A breast cancer at tumor stages I, II and III showed poorer clinical outcomes    . However, patients with SLC20A1high luminal B breast cancer at tumor stage III showed longer survival   ; however, this was not the case, in patients at tumor stage III  . However, patients at tumor stage III exhibited longer survival rates   . On the  = 0.21) . Followi52–4.30) . By cont65–4.11) . These rSLC20A1high and SLC20A1low groups in patients with luminal A and luminal B breast cancer who did or did not receive endocrine therapy. Patients with SLC20A1high luminal A breast cancer showed poorer clinical outcomes      . Patients with SLC20A1high who received endocrine therapy exhibited poorer clinical outcomes   . Althoug = 0.58) , patient= 0.022) . Subsequ98–1.88) . Patient05–1.97) . To concSLC20A1high and endocrine therapy at each tumor stage, we performed Kaplan-Meier analysis of DSS and RFS between the SLC20A1high and SLC20A1low groups for both patients with luminal A and luminal B breast cancer without or with endocrine therapy at tumor stages I, II or III. At tumor stage I, patients with SLC20A1high luminal A breast cancer showed poor clinical outcomes regardless of whether they received endocrine therapy         (To determine the relationship between the prognoses of patients with  0.0091) . At tumo = 0.15) , those w= 0.023) . At tumo= 0.023) . At tumo = 0.34) . By cont = 0.79) . At tumo = 0.11) . At tumo = 0.11) . Patient = 0.27) .SLC20A1high luminal A breast cancer exhibited poor clinical outcomes regardless of endocrine therapy        . Patient37–1.85) . At tumo07–3.14) . Similar91–2.15) . At tumo63–4.64) . Taken tSLC20A1high claudin-low and basal-like breast cancers [SLC20A1high luminal A and luminal B breast cancer who did not undergo chemotherapy showed poorer clinical outcomes      . In addition, we previously reported that chemotherapy was sufficient for  cancers . Therefo 0.0095) . Interes = 0.24) .SLC20A1high luminal A and luminal B breast cancer without chemotherapy showed poorer clinical outcomes      . Accordi29–1.43) . Taken tSLC20A1high and SLC20A1low groups from 175 months onwards   . In particular, patients with SLC20A1high showed higher recurrence incidence rates compared with those with SLC20A1low at >15 years   .SLC20A1high group showed similar recurrence incidence rates compared with those in the SLC20A1low group at year 0–5 and year 5–10  . Furthermore, patients in the SLC20A1high group showed higher recurrence incidence rates compared with those in the SLC20A1low group at year 10–15 . Since none of patients with SLC20A1high recurred at >15 years, the incidence rate ratio could not be analyzed for this time period   . These r15 years . Among par 10–15 . The difSLC20A1high who underwent endocrine therapy showed poorer prognoses and shorter intervals to recurrence. By contrast, patients with luminal A who received chemotherapy showed good clinical outcomes. These results indicate that endocrine therapy for patients with stage I SLC20A1high luminal A is less effective, such that other treatment options, such as chemotherapy, would be necessary.In stage I luminal A tumors, patients with 0/G1 phase and cell cycle arrest [high on late recurrence in ER+ breast cancer require further study. SLC20A1high may reduce the efficacy of endocrine therapy against ER+ breast cancer from early tumor stages onwards with regards to late recurrence.It has been proposed that the main cause of late recurrence is dormancy , 23. Dore arrest , 36. SLCe arrest , 37, 38.e arrest . Therefoe arrest , 35, 36.e arrest . Therefoe arrest . TherefoSLC20A1high and SLC20A1low groups   was shorw groups . The pat dataset . In lumi dataset . This dihigh was identified to be a prognostic marker using information-theoretical analysis [Since SLC20A1analysis , this inSLC20A1high showed poorer clinical outcomes at early tumor stages and tend to be less responsive to endocrine therapy for luminal A and luminal B breast cancer. However, patients with SLC20A1high who underwent chemotherapy showed good clinical outcomes. In addition, patients with SLC20A1high at the time of diagnosis showed higher recurrence incidence rates compared with those in the SLC20A1low group at >15 years in those with luminal A and at 10–15 years in those with luminal B. Therefore, SLC20A1high can be used as a prognostic biomarker for predicting the effect of endocrine therapy and the likelihood of late recurrence in ER+ breast cancer.In the present study, we revealed that patients with S1 FigSLC20A1 at each tumor stage. (A) All stages. (B) Luminal A. (C) Luminal B.(A-C) Box graphs of showing the expression levels of (TIF)Click here for additional data file.S2 Fighigh) and a low SLC20A1 expression (SLC20A1low) at each stage. (A-F) Kaplan-Meier analyses comparing disease-specific survival (DSS), disease-free status (DFS) and progression-free status (PFS) in patients with luminal A breast cancer between the SLC20A1high and SLC20A1low groups. (A) DSS in tumor stage I. (B) DSS in tumor stage II. (C) DSS in tumor stage III. (D) DFS in tumor stage I. (E) DFS in tumor stage II. (F) DFS in tumor stage III. (G) PFS in tumor stage I. (H) PFS in tumor stage II. (I) PFS in tumor stage III.Kaplan-Meier analyses of patients with luminal A breast cancer with a high solute carrier family 20 member 1 (SLC20A1) expression (SLC20A1(TIF)Click here for additional data file.S3 FigSLC20A1high) and a low SLC20A1 expression (SLC20A1low) at each stage. (A-F) Kaplan-Meier analyses comparing disease-specific survival (DSS), disease-free status (DFS) and progression-free status (PFS) in patients with luminal B breast cancer between the SLC20A1high and SLC20A1low groups. (A) DSS in tumor stage I. (B) DSS in tumor stage II. (C) DSS in tumor stage III. (D) DFS in tumor stage I. (E) DFS in tumor stage II. (F) DFS in tumor stage III. (G) PFS in tumor stage I. (H) PFS in tumor stage II. (I) PFS in tumor stage III.Kaplan-Meier analyses of patients with luminal B breast cancer with a high solute carrier family 20 member 1 (SLC20A1) expression ((TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 Table(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


N6 methylation of adenosine (m6A), N1 methylation of adenosine (m1A), N7-methylguanosine (m7G), 5-methylcytosine (m5C), 2′O-methylation (2′-O-Me or Nm), pseudouridine (5-ribosyl uracil or Ψ), and adenosine to inosine RNA editing (A-to-I editing). We then show how they relate to different types of lung disease. This paper hopes to summarize the mechanisms of RNA modification in chronic lung disease and finds a new way to develop early diagnosis and treatment of chronic lung disease.Chronic lung diseases are highly prevalent worldwide and cause significant mortality. Lung cancer is the end stage of many chronic lung diseases. RNA epigenetics can dynamically modulate gene expression and decide cell fate. Recently, studies have confirmed that RNA epigenetics plays a crucial role in the developing of chronic lung diseases. Further exploration of the underlying mechanisms of RNA epigenetics in chronic lung diseases, including lung cancer, may lead to a better understanding of the diseases and promote the development of new biomarkers and therapeutic strategies. This article reviews basic information on RNA modifications, including  Chronic lung diseases include chronic obstructive pulmonary disease (COPD), pneumonia, idiopathic pulmonary fibrosis(IPF), asthma, and lung cancer . Despite1A, and then detects m1A on the mRNA . Am. AmN6 mecytosine . The m6Aversible ,42. The function . The m7Gnd tRNAs ,45,46. Rn miRNAs . The m7Gd ncRNAs . It is iigration ,50,51. 2igration ,53. Of aigration . Ψ can cigration . Ψ couldhttps://www.ncbi.nlm.nih.gov/geo, accessed on 27 November 2022). The data related to chronic lung disease in the GEO database is mainly related to m6A modification. The m6A levels in cisplatin-resistant A549 cells were up-regulated compared to A549 cells . In addition, multiple data sets were compared and screened for m6A markers in LUAD cells . There are also RNA-seq expression profiles associated with writer/eraser perturbation. For example, expression profiles after METTL3 knockdown in A549 and H1299 cells (GSE76367), ALKBH5 knockdown in PC9 cells (GSE165453), and YTHDF1 or YTHDF2 knockdown PC9 cells (GSE171634).High-throughput sequencing data has a key impact in the study of RNA modification. These sequencing data are available on public websites, including the NCBI-Gene Expression Omnibus database (NCBI-GEO) . Specialized reversible RNA modification databases include m6Avar [6A-TSHub [6A [6ASNP [6A modification. The m6AVar database is a comprehensive database for studying m6A-related variants that may affect m6A modification. It will explain the influence of variants through the function of m6A modification. The m6AVar can be used to predict potential modification sites of m6A in chronic lung disease. According to the m6AVar database, three m6A-related genes  are reliable prognostic indicators for lung adenocarcinoma (LUAD) patients and are potential therapeutic targets [6AVar in that it mainly collects related variants affecting m6A modification. However, RMVar has more comprehensive functions than m6AVar. RMVar also contains other RNA-modified variants. The databases of genetic variation in RNA modification also include RMDisease and m6A- TSHub. RMDisease integrates the predictions of 18 different RNA modification prediction tools and a large number of experimentally validated RNA modification sites and identifies single nucleotide polymorphisms (SNPs) that may affect eight types of RNA modification. Most of the m6A-associated cancer variants are tissue- and cancer-specific. The m6A-TSHub consists of four core components, namely m6A-TSDB, m6A-TSFinde, m6A-TSVar, and m6A-CAVar. The m6A-TSDB platform can be used to retrieve m6A sites in normal lung tissue and lung cancer cell lines. Then, m6A-TSVa can be used to explore the influence of lung tissue variation on m6A by integrating tissue-specific m6A. Finally, m6A-CAVar can be used to screen for cancer variants affecting m6A in lung tissue. Through these databases, we can screen out the gene variants associated with RNA modification in chronic lung disease for further verification.With the further study of RNA modification, databases containing various information are emerging. These databases, which have received widespread interest and use, in turn, provide the basis for further research about the function of RNA modification. The existing RNA modification databases can be divided into biochemical RNA modification databases, comprehensive reversible RNA modification databases, specialized reversible RNA modification databases, and RNA editing databases . Biochem6A-Atlas , m6A2tarA2target , m5C-Atl5C-Atlas , m7GHub s [7GHub  and RNA s [7GHub . RMBase e m6Avar , m6A-TSH6A-TSHub , CVm6A [SHub [6A , RMVar [SHub [6A  and RMDiSHub [6A . RNA ediSHub [6A , RigorouSHub [6A , DatabasSHub [6A , and REDSHub [6A . There aA [6ASNP  and ConsA [6ASNP . Genetic targets . RMVar i6A is regulated by three types of proteases, methyltransferases , RNA-binding motif protein 15/15B (RBM15/15B), zinc finger CCCH type containing 13 (ZC3H13), virlike m6A methyltransferase associated , cbl proto-oncogene like 1 (CBLL1), and Wilms’ tumor-associated protein (WTAP), demethylases  and alkB homolog 5 (ALKBH5)), and readers (including YTH domain family 1/2/3 (YTHDF1/2/3), YTH domain containing 1/2 (YTHDC1/2), insulinlike growth factor 2 mRNA binding protein 1/2/3 (IGF2BP1/2/3), and heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1)), and is reversible and can be dynamically regulated [6A levels. Reversible m1A methylomes are achieved by the dynamic modulation of m1A RNA-modifying proteins, including m1A methyltransferases such as tRNA methyltransferase 6 noncatalytic subunit (TRMT6)-TRMT61A complex, TRMT10C, TRMT61B and nucleomethylin (NML), m1A demethylases such as ALKBH1, ALKBH3 and FTO, and m1A-dependent RNA-binding proteins such as YTHDF1/2/3 and YTHDC1 [7G in mammals is catalyzed by the compounds METTL1 and WD repeat domain 4 (WDR4), a complex that facilitates the installation of m7G in tRNA, miRNA, and mRNA [7G. Among them, RNMT is the catalytic subunit, and RAM is the regulatory subunit, which plays the role of activation. Williams–Beuren syndrome chromosome region 22 (WBSCR22) and TRMT112 are responsible for regulating m7G in rRNA. The main function of these regulatory mechanisms is to add m7G to the target RNAs, thereby mediating many key biological processes by modulating RNA production, structure, and maturation [5C is reversibly regulated by methyltransferases, including DNA methyltransferase  and NOP2/Sun RNA methyltransferases (NSUNs), and demethylases, including ten-eleven translocation (TETs), and reader proteins including YTHDF2, Aly/REF Export Factor(ALYREF) and Y-box binding protein 1(YBX1) [Three proteins have been found to regulate RNA modification. The first is an enzyme that introduces modified nucleotides into RNA during post-transcriptional RNA modification; the second protein interacts with the modified nucleotides; the third protein removes the modification labels . The metegulated ,80. Thesd YTHDC1 . The modand mRNA ,83. RNA turation . The m5C 1(YBX1) ,86. Ther 1(YBX1) ,88. In a 1(YBX1) . Fibrill 1(YBX1) . Ψ is pr 1(YBX1) . A-to-I  1(YBX1) .6A can affect the RNA stability, localization, turnover, and translation efficiency of genes, thereby regulating cellular processes such as cell self-renewal, differentiation, invasion, and apoptosis, and is even critical for skeletal development and homeostasis [6A. This complex enhances m5C methylation, which synergistically promotes p21 expression and affects oxidative stress-induced cellular senescence [6A-related regulators promote the progression of atherosclerosis by regulating macrophage polarization and inflammation. WTAP and METTL14 also can affect the phenotypic regulation of vascular smooth muscle cells (VSMCs) via m6A modification [6A RNA methylation generates compensatory cardiac hypertrophy, whereas decreased m6A causes cardiomyocyte remodeling and dysfunction [6A modification plays an important role in controlling the generation and self-renewal of hematopoietic stem cells and in mediating the development and differentiation of T and B lymphocytes from hematopoietic stem cells [6A, can maintain the homeostasis and maturation of natural killer (NK) cells, and positively regulate the antitumor and antiviral activities of NK cells. Its deletion significantly impairs NK cell antitumor and antiviral activity in vivo [6A methylation is also associated with NK cell homeostasis and antitumor immunity [6A is associated with apoptosis, autophagy, pyroptosis, ferroptosis, and necrosis [5C mediates cell proliferation, differentiation, apoptosis, and stress response [RNA modifications have a key influence in the regulation of many fundamental biological processes associated with chronic lung disease . The meteostasis ,94. The nescence . In the fication . Enhancefunction . An increm cells . YTHDF2, in vivo . METTL3-immunity . In addinecrosis , and m5Cresponse .6A modification plays an important role in tumorigenesis, proliferation, and metastasis. Active m6A regulators in lung cancer are related with the activation of multiple signaling pathways, such as DNA replication, RNA metabolism, epithelial–mesenchymal transition (EMT), cell cycle, cell proliferation and apoptosis, energy metabolism, inflammatory response, drug resistance, tumor metastasis and recurrence [6A regulates tumor metabolism by directly regulating nutrient transporters and metabolic enzymes or indirectly by controlling key components in metabolic pathways [6A modification contributes to the progression of malignant tumors and affects patient prognosis. Some m6A-related mRNA markers have also been shown to be independent prognostic biomarkers in patients with different types of cancer [6A methylase METTL3 is abnormally activated in cisplatin-resistant nonsmall-cell lung cancer (NSCLC) cells. METTL3 enhances YAP mRNA translation by introducing YTHDF1/3 and eIF3b into the translation initiation complex and increasing YAP mRNA stability by regulating the MALAT1-miR-1914-3p-YAP axis. This induces NSCLC drug resistance and metastasis [6A demethylase discovered to promote NSCLC cell growth by demethylating USP7 mRNA or MZF1 mRNA transcripts and increasing their stability and transcriptional levels [6A-modified PDK4 mRNA positively regulates the glycolysis of cells by binding to IGF2BP3, thereby promoting the development of cancer. While METTL14 knockout can reverse the function of IGF2BP3 and play a tumor suppressor role [6A modification also affects the mediating tumor metastasis process. When lung cancer brain metastasis (BM) occurs, miR-143-3p was upregulated in paired BM tissues compared with primary lung cancer tissues. METTL3-mediated m6A modification induces the maturation of miR-143-3p, which induces lung cancer invasion and angiogenesis via suppressing the expression of the target gene vasohibin-1 [6A modification in LUAD. METTL14-mediated HCG11 inhibits the growth of LUAD by targeting large tumor suppressor kinase 1 (LATS1) mRNA through IGF2BP2 [7G methyltransferase METTL1 and WDR4 complex is significantly elevated in lung cancer, which can promote lung cancer cell growth and invasion and negatively correlate with patient prognosis. Impaired m7G tRNA modification in the absence of METTL1/WDR4 results in decreased proliferation, colony formation, cell invasion, and tumorigenicity of lung cancer cells [7G-let-7 miRNA can inhibit lung cancer cell metastasis by reducing the expression of target oncogenes, including high mobility group AT-hook 2 (HMGA2), RAS, and MYC, at the posttranscriptional level [5C levels can be used as a cancer marker. For example, in lung squamous cell carcinoma (LUSC), the upregulation of m5C-related NSUN3 and NSUN4 is related to poor patient prognosis [5C levels can be found in circulating tumor cells from lung cancer patients [FAK mRNA by binding to FAK. FAK blocks the ADAR-induced invasiveness of LUAD cells [RNA epigenetic modifications have been broadly reported in lung cancer development and other chronic lung diseases . The moscurrence . Reprogrpathways . Aberranf cancer ,106. Rectastasis . FTO is l levels ,109. Thesor role . In addisor role . The m6Aohibin-1 . The lnc IGF2BP2 . The m7Ger cells . Highly al level . Studiesrognosis . In LUADrognosis . Abnormapatients . The stupatients . The rs9patients . FinallyAD cells . A-to-I AD cells .6A RNA methylation regulators is abnormal in COPD, among which the mRNA expressions of IGF2BP3, FTO, METTL3, and YTHDC2 show a tight association with the occurrence of COPD. IGF2BP3, FTO, METTL3, and YTHDC2 have obvious correlations with various important genes enriched in signaling pathways and biological processes that promote the development and progression of COPD [2.5) is an important cause of COPD. METTL16 may regulate sulfate expression through m6A modification, thereby participating in PM2.5-induced microvascular injury and promoting the development of COPD [5C modification may negatively affect normal lung metabolic activities by upregulating gene expression levels in the lungs of mice exposed to PM2.5 [STAT2 mRNA m6A [6A modification was upregulated in the bleomycin (BLM)-induced pulmonary fibrosis mouse model, FMT-derived myofibroblasts, and lung samples from IPF patients. Silencing METTL3 can inhibit FMT by reducing m6A levels. KCNH6 is involved in the m6A-regulated FMT process. m6A modification regulates KCNH6 expression through YTHDF1 [6A methylation has also been implicated in the pathogenesis of asthma, and YTHDF3 has an effect on eosinophils for severe asthma, which can guide future immunotherapy strategies [7G has an important value in predicting the prognosis of IPF patients and early diagnosis of IPF patients [In addition to studies on lung cancer, the effects of RNA modification in other chronic lung diseases such as COPD, pneumonia, asthma, and pulmonary fibrosis have also been explored . Exposur of COPD . Exposur of COPD . In addito PM2.5 . LncRNA mRNA m6A . Myofibrh YTHDF1 ,130. ALKh YTHDF1 . The m6Arategies . Increaspatients .6A modification plays a multifunctional role in normal and abnormal biological processes, and its regulatory proteins can act as therapeutic targets for cancer and are expected to be biomarkers for overcoming drug resistance [6A modification. METTL3 facilitates the translation of a large subset of oncogenic mRNAs and has direct physical and functional interactions with translation initiation factor 3 subunit h (eIF3h). METTL3-eIF3h interaction is required for oncogenic transformation. The depletion of METTL3 inhibits tumorigenicity and sensitizes lung cancer cells to bromodomain-containing protein 4 (BRD4) inhibition [METTL3 mRNA, reduce its expression, and further inhibit NSCLC cell proliferation [6A methylome is a new mechanism for drug resistance in cancer, such as tyrosine kinase inhibitors (TKIs) [MZF1 mRNA level, and MZF1 gene silencing significantly inhibited the cell viability and invasion of LUSC [6A. Knockdown of ALKBH5 under this condition significantly inhibited cell invasion by upregulating the m6A level of FOXM1 mRNA and reducing its translation efficiency [6A modification destabilizes SLC7A11 mRNA and accelerates mRNA decay upon recognition by YTHDC2, thereby affecting cystine uptake and contributing to antitumor activity [6A modification-related inflammatory cytokine interleukin 37 (IL-37) has received extensive attention for the treatment of lung cancer. IL-37 inhibits the proliferative capacity of LUAD cells by regulating RNA methylation. Meanwhile, overexpression of IL-37 decreased the expression of ALKBH5 and thus can also be used to treat NSCLC patients [6A modification reduces gefitinib resistance (GR) in NSCLC patients via the FTO/YTHDF2/ABCC10 axis [In recent years, RNA modification has been identified as a novel regulatory mechanism in controlling cancer pathogenesis and treatment response/resistance. The msistance . METTL3 hibition . METTL3 feration . The dyns (TKIs) . FTO is  of LUSC . Two FTO of LUSC . Under tficiency . Downregactivity . The m6Apatients . GefitinC10 axis . The dev6A regulators are upregulated in cancer and play a role in promoting tumor growth. These regulators, including METTL3, METTL14, and WTAP, and their key targets are associated with the clinical characteristics of various cancer patients, which may provide new possibilities for early cancer diagnosis [6A targeted drugs with traditional chemotherapy drugs or PD-1/PD-L1 inhibitors has great therapeutic prospects [6A methylation [5C deletion of mitochondrial RNA in tumor cells can reduce the metastasis and invasion of cancer cells. This means that when cancer patients undergo clinical treatment, they can inhibit the metastasis and spread of cancer cells by inhibiting m5C modification in mitochondria, thus increasing clinical benefits. As an enzyme responsible for modifying RNA, NSUN3 is only used to modify the formation of m5C, so it is a very promising drug target [In conclusion, this article reviews the recent advances in the function and molecular mechanism of RNA epigenetics in the progression of chronic lung diseases. RNA epigenetics is expected to be a research tool for the development of new diagnostic biomarkers with clinical value. Enzymes involved in regulating RNA modification can be new targets for the treatment of chronic lung diseases. Most of the miagnosis . The comrospects . The mechylation . YTHDF1 hylation . The m5Cg target . RNA modIn the future, precision medicine based on RNA epigenetics may target individual patients with chronic lung diseases for diagnosis and treatment. However, the function of these chemical modifications in both coding and noncoding RNAs remains in its infancy, and collaborative efforts are still needed to establish a clear link between RNA epigenetics and chronic lung diseases. At present, many new methods of RNA modification have emerged, among which Direct RNA sequencing is the representative one. Through the unique way of the library building and relying on the algorithm, the collection of RNA modification information at the single base level can be achieved. The development of RNA modification quantitative map databases (such as DirectRMDB) based on this sequencing method also undoubtedly opens another direction for RNA modification detection . In brie


Subsequently, the PS/C16mimCl/Pd beads were used as a macroporous templating agent and C16mimCl was used as a mesoporous templating agent to prepare Pd-loaded hierarchical porous silica. A systematic study was carried out addressing the influence of the amount of C16mimCl and the mass ratios of m(Pd)/m(PS) on the PS/C16mimCl/Pd beads and the Pd-loaded hierarchical porous structures. The samples were characterized by electrophoresis experiments, SEM, TEM, small-angle XRD, and N2 adsorption–desorption experiments. It was found that the coverage of citrate-coated Pd nanoparticles (Pd NPs) onto the PS beads can be simply tailored by changing the amount of C16mimCl and the mass ratios of m(Pd)/m(PS). The resultant Pd-loaded hierarchical porous silica possessed a 3D ordered macroporous skeleton with a specific surface area of up to 967 m2 g−1, ordered mesoporous silica walls (SBA-3 type) and well-dispersed Pd NPs anchored on the inner walls of the spherical macroporous hollow. Importantly, the obtained Pd-loaded hierarchical porous silica exhibited an enhanced catalytic activity for the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2.Long-chain ionic liquid, 1-hexadecyl-3-methylimidazolium chloride (C 16mimCl) can be used as “bridges” to prepare PS/C16mimCl/Pd beads, and Pd-loaded hierarchical porous silica was synthesized using dual templates of the PS/C16mimCl/Pd beads and C16mimCl.1-Hexadecyl-3-methylimidazolium chloride (C Increasing efforts have been made to explore the self-organized behavior of the LILs in aqueous solution.2 Various characterization methods, such as surface tension, electrical conductivity and fluorescence measurement, have been used to determine the micelle formation and the micelle aggregation numbers of the LILs in aqueous solution.3–6 The results indicated that the surface activity of the LILs is slightly superior to that of the typical cationic surfactants. Moreover, the LILs have been demonstrated to exhibit the behavior of both lyotropic and thermotropic liquid crystals,7,8 which offer a partially ordered microenvironment for organic reactions.9 These advantages of the LIL derivatives have been broadly applied in the introduction of ordered self-organized structures for synthesis of functional nanoparticles and other inorganic nanostructures.10,11 Particularly, the supramolecular templating function of the LILs has been used to create various ordered porous materials. For example, Adams et al. reported the synthesis of mesoporous silica with 2D hexagonal structure by using the LILs as templating agent.12 Zhou et al. employed long-chain 1-hexadecyl-3-methylimidazolium chloride (abbreviated as C16mimCl), as template to prepare a supermicroporous lamellar silica by so-called “nanocasting” technique.13,14 Our group has also contributed much effort to successfully synthesize the mesoporous silica with high-quality cubic gyroid and 2D hexagonal mesoporous structures by using C16mimCl as template.15 These results indicated that the LILs have significantly stronger tendency toward self-aggregation and supramolecular templating in the preparation of ordered mesoporous structure. However, their potential as modifying agent, especially the formation of LIL-functionalized polymer microspheres or their multifunctional combination for the synthesis of nanostructured materials is less commonly known.Long-chain ionic liquids (LILs), especially imidazolium-based LILs, have been most extensively studied for both fundamental research and practical applications.16mimCl) was used as a surface functional molecule to modify polystyrene (PS) beads, which can effectively connect small citrated-coated palladium (Pd) nanoparticles (Pd NPs) to construct PS/C16mimCl/Pd beads. Secondly, based on a dual templating method of the PS/C16mimCl/Pd beads and C16mimCl, a more facile access to prepare Pd-loaded silica with hierarchical porous (macropore-mesopore) structures and catalytic activities is presented.The objective of the present work is twofold: firstly, long-chain ionic liquid, 1-hexadecyl-3-methylimidazolium chloride  on the surface of PS beads. Before the deposition, the non-cross-linked PS beads were functionalized by using carboxylic acid- and amino-groups to offer an effective connection of small noble-metal NPs.21 Here, the C16mimCl is designed to modify PS beads, which forms the C16mimCl-functionalized PS beads for the deposition of the preformed citrated-coated Pd NPs.Recently, loading Pd NPs on suitable supports have attracted great attention due to their unique catalytic properties for the carbon–carbon cross-coupling reactions.22,23 Particularly, bimodal mesoporous–macroporous inorganic materials possess increased mass transport through its macropores as well as retaining a specific surface area on the level of fine mesoporous structures.24 In previously reported preparation of Pd NPs-loaded ordered porous silica, the commonly used method is simple impregnation with metal salt solutions into porous channels and further reduction to form Pd NPs in the porous silica channels. However, this method often causes the partial plugging and destruction of the porous system because of crystallization of the metal salt during the calcination process to remove the pore-forming templates. Some improved approaches have been reported to enhance the stabilization of the Pd NPs-loaded hierarchical porous structure. For instance, Zhou et al. reported the cyclodextrin (CD)-based homogeneous incorporation of Pd NPs into silica with bimodal pore structure.25 In the present synthesis of the Pd-loaded hierarchical porous silica, the prepared PS/C16mimCl/Pd beads and long-chain C16mimCl were employed as dual templates to create 3D macroporous silica skeleton with ordered mesoporous walls. The inverse replicas of the PS/C16mimCl/Pd beads resulted in spherical voids, in which Pd NPs were anchored on the inner walls of the spherical voids, and the inverse replicas of the C16mimCl leaded to ordered mesoporous silica channel walls. The method that employs the LILs not only as a connector but also as a templating agent in one system should provide important guidelines for the multifunctional applications of the LILs in the synthesis of nanostructured materials.Ordered porous materials can also be considered as an ideal scaffold for loading Pd NPs.16mimCl) was prepared according to a route reported in the literature.15,26 All chemicals were purchased from Acrös and used as received. As a typical synthesis, 1-methylimidazole  was mixed with an excess of 1-hexadecylchloride . The mixture was put into a 250 mL flask, refluxed at 90 °C for 24 h, and then cooled down to room temperature. The product was further purified by recrystallization in tetrahydrofuran (THF). After washing several times with THF, the white crystalline powder of C16mimCl was collected by vacuum filtration, and dried in air at room temperature. The structure of the obtained C16mimCl was identified by IR spectrum  as emulsifier. Typically, 21 mL of styrene was washed in a separatory funnel four times with 20 mL of 0.1 M NaOH, then four times with 20 mL water. 0.005 g of SDBS was dissolved in 180 mL water in a round-bottomed flask. The washed styrene was added, and nitrogen was bubbled to deaerate the mixture for 20 min. 0.2328 g of potassium persulfate initiator was added to the mixture, and the mixture was then heated to 70 °C for 12 h with rapid stirring. The PS beads in emulsion were collected by centrifugation.16mimCl aqueous solution (2.985 g L−1) was added dropwise and slowly to 10 mL of the PS bead dispersion (4.686 g L−1) with stirring for 4 h at room temperature to obtain the dispersion (0.781 g L−1) of the C16mimCl-coated PS beads (PS/C16mimCl). In order to explore the effect of the amount of adding C16mimCl on the surface charge of the PS/C16mimCl beads, the different volumes of the C16mimCl solution (V(C16mimCl) = 1, 25, 75 and 100 mL) were also added dropwise to the PS bead dispersion.The PS bead solid was redispersed in water under ultrasound. 50 mL of C27 Briefly, 7.5 mL of PdCl2 solution (0.99 g L−1), 15 mL of sodium citrate solution (60 g L−1), and 52.5 mL of water were mixed with stirring. The mixture was heated to boil, and kept boiling and stirring for 6 h. The color of the citrate-coated Pd NP (Pd/Cit.NP) dispersion (0.0599 g L−1) turned from yellow to brown.Pd nanoparticle (Pd NP) dispersion was prepared by using sodium citrate as a reduction agent as well as a stabilizing agent according to a reported method.16mimCl/Pd beads were synthesized using the PS/C16mimCl beads as nuclei for subsequent adsorption deposition of Pd/Cit.NPs according to the compositions listed in V(Pd/Cit.NP) = 15.62, 31.24, 46.86 and 62.48 mL) (0.0599 g L−1) were added dropwise to 20 mL of the PS/C16mimCl dispersion (0.781 g L−1), respectively. The mixture was sufficiently stirred for 4 h to obtain the PS/C16mimCl/Pd beads. The mass ratios of m(Pd)/m(PS) were calculated based on the mass of Pd in the Pd/Cit.NP dispersion and the mass of PS in the PS/C16mimCl dispersion to be 6%, 12%, 18% and 24%, respectively. The four PS/C16mimCl/Pd bead samples were coded as PS/C16mimCl/Pd(x), x = 6%, 12%, 18% and 24%, respectively.The PS/C16mimCl/Pd beads and C16mimCl as macroporous and mesoporous structural templating agents, respectively. In a typical synthesis procedure, C16mimCl and HCl were added to the PS/C16mimCl/Pd dispersion under mild magnetic stirring. After homogenization of the mixture, TEOS was added dropwise at room temperature. The molar compositions of the starting mixtures were n(TEOS)/n(C16mimC)/n(HCl)/n(H2O) = 0.23/0.2/56/504. The resulting mixtures were stirred at room temperature for 2 h and then aged for 12 h. After the sol–gel treatment, the mixtures were filtered, washed with deionized water, dried under atmosphere at room temperature, and finally calcined by a temperature programmed route at 300 °C for 2 h, and then, 550 °C for 2 h with a temperature ramp of 2 °C min−1 under static air condition to remove the templates. The resultant sample based on the PS/C16mimCl/Pd(12%) beads and C16mimCl dual templates is coded as Pd–SiO2(12%), and the sample based on the PS/C16mimCl/Pd(18%) beads and C16mimCl dual templates is coded as Pd–SiO2(18%).The Pd-loaded hierarchical porous silica was prepared in acid medium by a sol–gel procedure using tetraethylorthosilicate (TEOS) as silicon source, the prepared PS/C2(12% or 18%)), the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 was employed as a model reaction. Briefly, in each of glass tubes, 200 μL of TMB aqueous solution (10 mmol L−1), 200 μL of H2O2 solution (250 mmol L−1) and 1560 μL of PBS buffer solution (pH = 5) were added with stirring. Then, the Pd-loaded hierarchical porous silica catalysts (1 mg) were added to the above 1960 μL solutions. With constant magnetic stirring, the mixture color changed from colorless to blue. After reacting at different time intervals, the mixtures were scanned by UV-vis absorption spectrograph. The control experiment was also carried out by mixing the solution of TMB and H2O2 in the absence of the Pd-loaded hierarchical porous silica catalyst. For comparison, the catalytic activities of PS/C16mimCl/Pd(12%), PS/C16mimCl/Pd(18%), and PS/C16mimCl/Pd(24%) beads were studied respectively under identical reaction conditions. According to the amount of PS/C16mimCl/Pd bead dispersion used for the synthesis of 1 mg Pd–SiO2(12% or 18%), the volume of the bead dispersion tested for the catalytic activity was controlled to be 2 μL. Moreover, the reusability of the prepared Pd–SiO2(12%) and Pd–SiO2(18%) catalysts were also studied by tracking the changes in UV-vis absorption spectrograph of the reaction system under different cycles.In order to test the catalytic activities of the Pd-loaded hierarchical porous silica  was used to collect absorption spectra of the solution in catalytic reactions. A TENSOR27 Fourier transform infrared (FT-IR) spectrometer was employed for recording IR spectra.A field emission scanning electron microscopy (FEI Nova NanoSEM 450) was used to observe the morphology of the samples. Transmission electron microscopy (TEM) images were obtained by using a JEM-2100 electron microscope at an acceleration voltage of 200 kV. The specimens for TEM were prepared by dropping a small drop of the solutions onto a carbon-coated copper grid. Electrophoresis experiments were performed on a Nanjing Sangli DYL-3 electrophoresis apparatus with 15 V of external voltage. X-ray diffraction (XRD) patterns were measured on a TTR III powder X-ray diffractometer using Cu Kα radiation (wavelength 0.154 nm) at a rate of 0.05° 216mimCl beads were prepared by adding the C16mimCl aqueous solution (2.985 g L−1) to PS bead dispersion (4.686 g L−1). It was found that a drop by drop and slow adding way for the C16mimCl aqueous solution is especially important for preventing the micelle formation of C16mimCl in the dispersion. The cmc of C16mimCl at 298 K was reported to be 1.21 mM,28 and the concentration of one drop of the C16mimCl aqueous solution in the suspension is about 0.043 mM, which is well below the cmc of C16mimCl. Hence, this adding way is favorable to coat the individual C16mimCl molecule onto the PS efficiently. Moreover, we found that C16mimCl is freely soluble in water at room temperature, which increases the possibility of the direct interaction between individual C16mimCl molecule and PS bead. In order to understand the effect of the amount of C16mimCl, different volumes of the C16mimCl aqueous solution (V(C16mimCl) = 1, 25, 50, 75, and 100 mL) were added dropwise and slowly to the PS dispersion (10 mL). ζ) as a function of the V(C16mimCl). Two features should be noted. Firstly, the uncoated PS bead dispersion produced a zeta potential of about −7 mV. The presence of a small quantity of C16mimCl (1 mL) caused a reversal in zeta potential (+14.5 mV). The multiple tests of adding small quantity of C16mimCl, even 0.2 mL of C16mimCl, leaded to the zeta potential reversal from negative charge to positive charge, implying a hypothetical structure of the exposure of imidazole head groups of C16mimCl toward the surrounding solution, and the hydrophobic long alkyl chain extending to the surface of PS bead. This result showed that the hydrophobic interaction between the PS beads and the alkyl chain of C16mimCl is stronger than the electrostatic interaction between the PS beads and the imidazole head groups of C16mimCl. Secondly, the increase in the feeding V(C16mimCl) increases the zeta potential of the resultant PS/C16mimCl beads. When the V(C16mimCl) is 50 mL, the highest value of zeta potential (+60 mV) can be obtained, which is consistent with the value of zeta potential measured from the PS latex particles with poly (PAH) or poly (PDADMAC) as outer shell prepared by LbL method.29 The high zeta potential onto the PS/C16mimCl beads suggests that a close-packed array of the C16mimCl molecules can form onto the PS beads. Sastry et al. considered that the increase in the alkyl chain length of ionic liquids leads to the parallel packing of chains in the interior of aggregates and the cationic rings with delocalized π-electron clouds favor high aggregation number.30PS/CV(C16mimCl) to 75 mL, the value of the zeta potential for the PS/C16mimCl beads is obviously decreased (18.5 mV). When the V(C16mimCl) is 100 mL, corresponding zeta potential value becomes 13.6 mV. This is because when the concentration of C16mimCl is too high, the excess of C16mimCl in the dispersion increase the viscosity of the colloidal solution, and reduce the fluidity of PS/C16mimCl beads in the electrophoresis experiments, resulting in the decrease of the zeta potential values. Therefore, we confirm that when the amount of C16mimCl is the appropriate value (V(C16mimCl) = 50 mL), the optimal value of zeta potential (+60 mV) can be obtained.However, as shown in 16mimCl/Pd(x) beads with x = 6%, 12%, 18% and 24% (x is different mass ratios of m(Pd)/m(PS)) were prepared by mixing corresponding volumes  of citrate-coated Pd NPs suspensions  with 20 mL of PS/C16mimCl dispersion , respectively. 16mimCl/Pd(18%) beads. Compared with the uncoated PS beads with a narrow size distribution and a smooth particle surface  exhibited a coarse surface in each bead, indicating that a layer of Pd coating with a few clusters was deposited on the surface of PS/C16mimCl beads. These SEM images are similar to the images of PS spheres covered with dense and uniform Au NPs.31In order to reveal the effect of the amount of Pd NPs, PS/C16mimCl/Pd(x) bead with different coverage. Compared to PS/C16mimCl/Pd(6%) with an extremely sparse coverage, PS/C16mimCl/Pd(12%) (16mimCl/Pd(18%) /m(PS) from 18% to 24%, the Pd NPs on the PS beads became more denser (16mimCl beads. The best matching ratios of the mass ratios of m(Pd)/m(PS) are 12% and 18%./Pd(12%)  and PS/C/Pd(18%)  exhibitee denser , and som16mimCl/Pd beads. As shown in the 16mimCl-functionalized PS beads by a hydrophobic interaction between the PS beads and the alkyl chain of C16mimCl to expose imidazole head-groups of C16mimCl molecules to the surrounding solution. In the second step, the PS/C16mimCl beads with positive charge can serve as active sites to link the negatively charged citrate-coated Pd NPs by means of an electrostatic interaction. Thus, the C16mimCl are applied as ‘bridges’ to connect the PS beads and citrate-coated Pd NPs. It is possible to manipulate the uniformity and coverage of the Pd NPs on the PS beads by controlling the amount of C16mimCl and the matching mass ratios of m(Pd)/m(PS).In brief, there are two steps for the synthesis of the PS/C2), PS/C16mimCl/Pd beads were used as a macroporous structure templating agent. The inverse replicas of PS/C16mimCl/Pd beads resulted in spherical voids, in which Pd NPs were anchored on the inner walls of the spherical voids. The C16mimCl was used as a mesoporous structure templating agent. The inverse replicas of the C16mimCl micelles leaded to ordered mesoporous silica channel walls.As shown in the 2 adsorption–desorption isotherm of the calcined Pd–SiO2(12%) prepared using PS/C16mimCl/Pd(12%) beads and C16mimCl as dual templates. The sample exhibited one well-resolved diffraction peak (100) in the 2θ range between 2 and 6 ,32–34 indicating a mesoporous structure with short range order. This may be due to a thin wall in the macroporous silica skeleton, thus, only one diffraction peak can be observed. The sample displayed a type IV adsorption isotherm with an obvious hysteresis loop at a relatively high p/p0 values according to IUPAC,35 indicating the presence of open pores  are 967 m2 g−1, 0.68 cm3 g−1, and 3.0 nm, respectively. 2 and 6 , which ien pores . A steep2(12%) (2(18%) has similar TEM images  appeared some characteristics of cubic phase , PS/C16mimCl/Pd(18%) beads have more Pd particles compared to PS/C16mimCl/Pd(12%) beads, which can provide a denser reaction system. Meanwhile, PS/C16mimCl/Pd beads possess a certain hydrophobic property, which can be regard as a ‘cosolvent organic molecules’. When the beads are close to the hydrocarbon chains of long-chain ionic liquid, the volume fraction of the hydrocarbon chain should increase. The increase in V results in the increase of the value of g. Thus, it is possible that the transformation from hexagonal p6mm to atypical cubic Ia3d structure can be observed in the small-angle XRD curve of Pd–SiO2(18%).TEM image of the calcined Pd–SiO2(12%)  reveals 2(12%)  can be as Fig. S3, but Pd e Fig. S4. In clas2(12%) and Pd–SiO2(18%), we employed the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 as a model system. In the previous publication, the oxidation of TMB by H2O2 can produce diazo dianiline, which exhibited two typical absorption peaks at 370 nm and 652 nm, and induced a color change from colorless to blue in solution.37,38 In comparison with the control experiment of the oxidation of TMB by H2O2 in the absence of catalyst (2(12%) and Pd–SiO2(18%) used for the oxidation reaction can facilitate the reaction effectively within certain time (2(18%) is faster than that of Pd–SiO2(12%), suggesting that increasing the amount of Pd NPs in the catalyst is beneficial to promote the catalytic performance for the oxidation of TMB by H2O2. A color change from colorless to blue in the solution (2(12%) or Pd–SiO2(18%), indicating that Pd–SiO2(12%) and Pd–SiO2(18%) have enhanced catalytic activity. For comparison, parallel experiments were performed to monitor the catalytic behavior of the synthesized PS/C16mimCl/Pd(x) beads. 2O2 catalyzed by adding PS/C16mimCl/Pd(12%), PS/C16mimCl/Pd(18%), and PS/C16mimCl/Pd(24%) beads, respectively. It was observed that the obtained PS/C16mimCl/Pd(x) beads exhibited significant absorption peaks at 370 nm and 652 nm, indicating that the composite beads had an enhanced catalytic activity compared with the control experiment without catalyst (16mimCl/Pd(12%) beads (2(12%) (16mimCl/Pd(18%) beads showed higher absorption peak intensity (16mimCl/Pd(12%) beads (2(18%) (2(18%) compared to PS/C16mimCl/Pd(18%) beads may be attributable to its relatively distinctive hierarchical porous structure. Furthermore, of the three composite beads, PS/C16mimCl/Pd(24%) had the lowest absorption peak intensity  beads  is simil (2(12%) . PS/C16mntensity  than PS/%) beads , but had (2(18%) . The higntensity . This mantensity , leading2O2, the concentration of H2O2 was greatly higher than that of TMB, so it is consider as a constant during the reaction process, and the pseudo-first-order kinetics with respect to TMB can be applied as shown by:c0 is the initial concentration of the TMB solution, c is the reactant concentration at time t and k is the rate constant. Fig. S5A/A0 and reaction time for the oxidation reaction of TMB by H2O2 . For the pseudo first-order kinetics, the c/c0 can be measured from the relative intensity of absorbance A/A0 because A/A0 is in proportion to c/c0. Thus, according to the linear relationship of ln(A/A0) versus time (t), the rate constant k can be calculated. The calculated rate constants are 0.0562 min−1 (Pd–SiO2(18%)), 0.0436 min−1 (Pd–SiO2(12%)), and 0.0274 min−1 , respectively. It can be clearly seen that the addition of Pd–SiO2(18%) and Pd–SiO2(12%) significantly fast the oxidation reaction. It is accepted that for the effective promotion of the oxidation reaction, TMB should be concentrated and adsorbed on the surface of the Pd-loaded hierarchical porous silica catalyst due to the high surface area and ordered mesoporous channels. Meanwhile the macroporous spherical voids can increase TMB transport, facilitating access to reactive sites of Pd NPs anchored on the inner walls of the spherical voids. After the completion of the catalytic reaction, the Pd–SiO2(12%) and Pd–SiO2(18%) catalysts were recovered simply by centrifugation and then dried at room temperature, and finally calcined at 550 °C for 2 h under static air conditions to remove the catalytic reaction products. The recovered catalysts were used again in successive cycles (four times) under identical reaction conditions. 2(12%) and Pd–SiO2(18%) after the three cycles, indicating that there is no loss of catalytic activity. However, after the fourth cycle, the rate constants of the recovered Pd–SiO2(12%) and Pd–SiO2(18%) were decreased, which suggests their catalytic activity was reduced. The phenomenon may be caused by partial collapse of the hierarchical porous structure of the recycled catalysts.In the oxidation reaction of TMB by H16mimCl/Pd beads using long-chain 1-hexadecyl-3-methylimidazolium chloride (C16mimCl) as a connector. The amount of C16mimCl and the mass ratios of m(Pd)/m(PS) were systematically studied to optimize the coverage of Pd NPs. When m(Pd)/m(PS) = 12% and 18%, the citrate-coated Pd NPs are well-dispersed onto PS beads to form well-defined PS/C16mimCl/Pd beads. Moreover, the Pd-loaded hierarchical porous silica with a 3D ordered macroporous skeleton and ordered mesoporous silica walls (SBA-3 type) was prepared using the dual templates of PS/C16mimCl/Pd beads and C16mimCl. The inverse replicas of the PS/C16mimCl/Pd bead template led to well-dispersed Pd NPs anchored on the inner walls of the spherical macroporous voids. Furthermore, the obtained Pd-loaded hierarchical porous silica with a high surface area of up to 967 m2 g−1 exhibited enhanced and Pd loading capacity-dependent catalytic efficiencies.In summary, we report a flexible approach for the controllable synthesis of PS/CThere are no conflicts to declare.RA-008-C8RA03884H-s001


Semaphorin 6D (SEMA6D),a member of the class 6 semaphorin family,is a membrane-associated protein that plays a key role in the developmentof cardiac and neural tissues. A growing body of evidence suggeststhat SEMA6D is also involved in tumorigenesis. In breast cancer, highSEMA6D levels are correlated with better survival rates. However,very little is known about the functional significance of SEMA6D inbreast tumorigenesis. In the present study, we aimed to investigatethe effects of SEMA6D expression on the normal breast cell line MCF10Aand the breast cancer cell lines MCF7 and MDA MB 231. We demonstratedthat SEMA6D expression increases the proliferation of MCF10A cells,whereas the opposite effect was observed in MCF7 cells. SEMA6D expressioninduced anchorage-independent growth in both cancer cell lines. Furthermore,migration of MCF10A and MCF7 cells and invasion of MDA MB 231 cellswere elevated in response to SEMA6D overexpression. Accordingly, thegenes related to epithelial-mesenchymal transition (EMT) were alteredby SEMA6D expression in MCF10A and MCF7 cell lines. Finally, we providedevidence that SEMA6D levels were associated with the expression ofthe cell cycle, EMT, and Notch signaling pathway-related genes inbreast cancer patients’ data. We showed for the first timethat SEMA6D overexpression has cell-specific effects on the proliferation,migration, and invasion of normal and cancer breast cell lines, whichagrees with the gene expression data of clinical samples. This studylays the groundwork for future research into understanding the functionalimportance of SEMA6D in breast cancer. Further studies revealed that semaphorin familymembers are involved in tissue homeostasis as well as tumorigenesisthrough the regulation of cell proliferation, cell survival, celladhesion and migration, immune response regulation, and angiogenesis.8 Beyond its function in developmental processes, accumulating evidencesuggests that SEMA6D also plays a part in tumorigenesis. In osteosarcoma,SEMA6D was identified as a candidate oncogene through transposon mutagenesisscreening. The functional analysis supported the oncogenic potentialof SEMA6D in osteosarcoma, where its overexpression increased softagar colony formation, cell proliferation, and in vivo tumor formationthrough ERK phosphorylation.9 More recentresearch also presented evidence for an active role of SEMA6D in thecisplatin resistance mechanism in osteosarcoma.10 Likewise, SEMA6D showed an oncogenic behavior in mesotheliomaby protecting the cells from apoptosis and increasing soft agar colonyformation.11 Supporting these findings,in gastric and esophageal cancers, SEMA6D expression was found tobe increased in tumors compared to normal tissues.12 On the other hand, in lung adenocarcinoma, SEMA6D expressionwas downregulated, and lower SEMA6D levels were associated with shorteroverall survival indicating a tumor suppressor role in the lung.13 Altogether, the data suggest a context-dependentrole for SEMA6D in tumorigenesis.SEMA6D research extensively focused ondevelopmental aspects highlightingits role in axonal extension, cardiac development, neural tube closure,anti-inflammatory macrophage polarization, oligodendrocyte positioning,and crossing of retinal ganglion cells.14 To develop better diagnostic and therapeutic approaches,the fundamental issue is understanding the molecular mechanisms underlyingbreast cancer. In this context, the role of SEMA6D in breast tumorigenesisremains largely unexplored. What we know is based on the two previousstudies that examined breast cancer patient samples.16 The first study analyzed SEMA6D expression in 1100 samples throughThe Cancer Genome Atlas (TCGA) database and reported that a high SEMA6Dexpression is correlated with better survival, which is more significantlypronounced in the triple-negative subgroup.15 More recently, single nucleotide variations in SEMA6D were reportedin two of the 37 young patients with early onset luminal breast cancer.16 Moreover, in line with the previous data, overallsurvival and relapse-free survival rates were better in the patientgroup with a higher SEMA6D expression.16 Research to date has not yet thoroughly determined the functionalsignificance of SEMA6D expression in breast cancer.Breast cancer is the mostcommonly diagnosed cancer type and theleading cause of cancer-related deaths in women.Here, weaimed to analyze cellular and molecular changes in responseto SEMA6D overexpression in normal breast and breast cancer cell lines.We showed that although SEMA6D overexpression induced a pro-tumorigenicphenotype in the normal cell line, it negatively affected tumorigenictraits of the cancer cell lines while promoting a more migratory andmesenchymal phenotype in both normal and cancer cell lines. The experimentalwork we present here provides the first evidence showing that SEMA6Dhas cell-specific effects in breast cancer, which might reflect onthe different clinical outcomes in different breast cancer patientgroups.2 at 37 °C. Overexpression ofSEMA6D was performed by a lentiviral expression system. Viruses wereprepared, and infections were carried out as explained previously.17 pLX304-SEMA6D plasmid was obtained from Dharmacon(OHS6085-213576758) (GenBank reference: BC150253). LacZin pLX304 (pLX304-LacZ) was a gift from William Hahn (Addgene plasmidno. 42560).18 Stable cell lines were generatedby 2 μg/mL blasticidin  selection. MCF10Acells that have increased Notch activation upon overexpression ofNotch1 intracellular domain (NICD) were described previously.17MCF10A, MCF7, MDA MB231, and HEK293T cells were obtained from American Type Culture Collection,ATCC. MCF10A cells were cultured in high glucose DMEM-F12  supplemented with 5% horse serum ,1% penicillin/streptomycin ,20 ng/mg EGF , 0.5 μg/mg hydrocortisone , 100 ng/mL choleratoxin , and 10 μg/mginsulin . MDA MB 231, MCF7, and HEK293T cell lines weremaintained in high glucose DMEM  supplemented with10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin. All cell lines were maintained in a humidifiedincubator with 5% COTable S1.TotalRNA was isolated usingthe Pure-link RNA mini kit . cDNA was synthesizedusing the RevertAid first-strand cDNA synthesis kit . PCR amplification and detection were done on Roche LightCycler96 Real-Time PCR Detection System using FastStart Essential DNA GreenMaster . TATA-box binding protein (TBP) was usedas the internal control for normalization. Relative mRNA levels werecalculated using delta–delta Ct method. Each experiment isnormalized to its own control condition. At least three independentexperiments were done. The primer pairs are listed in 5 MCF10A cells, 4 × 105 MDA MB 231, and MCF7 cells seeded on a 6-well plate after2 days of incubation with serum-free medium. Protein isolation fromCM was done by methanol/chloroform precipitation as described previously.19 The amount of protein was quantified with BradfordAssay. The total amount of protein of 60 μg from cell lysatesand 20 μg from CM were separated via SDS-gel and transferredonto polyvinylidene fluoride (PVDF) membranes. After blocking with5% skim milk, the membranes were incubated with the following antibodies:anti-β-actin , anti-N-cadherin , anti-SNAI2, anti-SEMA6D  , and anti-SEMA6D (CM) . Following secondary antibody incubation, the detectionwas done using Vilber Fusion SL Imaging System.Total proteinwas extracted using RIPA lysis buffer. Conditioned medium (CM) wascollected from 3.5 × 104 cells/well were seededon a 24-well plate. Cells were treated with methylthiazolyldiphenyltetrazoliumbromide (MTT)  for 4 h on the second, fourth, sixth,and eighth days after plating. Following the addition of DMSO as thesolvent, absorbance was read at 570 and 650 nm by using Thermo MultiskanSpectrum.6 ×105 MCF10A and 3.5 × 105 MDA MB 231 and MCF7cells/well were grown on coverslips on a 6-well plate for 48 h. Forthe BrdU assay, cells were incubated with 20 μM bromodeoxyuridine(BrdU) for 4 h. Cells were gently washed with PBS 1X and fixed with1 mL of 4% Paraformaldehyde (PFA) in PBS 1X for 20 min at room temperature.Then the cells were permeabilized with 0.1% TritonX-100/PBS for 15min and blocked with 5% BSA in 0.1% TritonX-100/PBS for 30 min atroom temperature. Anti-BrdU for BrdU, anti-V5  for V5 tag, and DAPI  for nucleus staining were used. Afterward, the cellswere rinsed with PBS 1X  and mounted on IbidiMounting Medium . Fluorescent images were capturedby using Olympus IX83 fluorescent microscope.2.5 × 104 cells were mixed with 0.35%noble agar and seeded on top of solidified 0.5% noble agar on 6-well plates.The complete medium was added after the solidification of the toplayer, and the medium was changed twice a week for 6 weeks. Afterstaining colonies with 0.05% crystal violet, the images focused onthree different Z layers were captured for each well under a LeicaDMI8 confocal microscope. The sizes of colonies that are higher than30 μM in diameter were counted using ImageJ. Total colony numberswere normalized to the control condition in each of three independentexperiments.3 ×105 cells/wellwere seeded on a 12-well plate. The following day, cells were incubatedwith 10 μg/mL Mitomycin C  for 2 h. Thenthe scratch was introduced with a 10 μL pipet tip, and 1% serumand 1% penicillin/streptomycin-containing medium were added. The gapswere monitored at 37 °C with 5% CO2 under a LeicaDMI8 confocal microscope supplemented with the incubation chamber.Open area percentages were calculated for each position in three independentexperiments.7.5 × 1017 Briefly, cells were labeled transiently witha green fluorescent dye using Green Cell Tracker CMFDA .The stock solution of Green Cell Tracker (25 mM) was diluted in serum-freeDMEM to 5 μM final concentration. Following incubation with5 μM CMFDA dye at 37 °C for 30 min, cells were washed withPBS and supplemented with the culture medium until loading. Then thegrowth factor reduced Matrigel  was mixed with theserum-free medium in a 1:1 ratio and loaded into the middle channel.After polymerization, 20% serum-containing medium was loaded to thelower channel, and labeled cells at the concentration of 1 ×106 cells/ml in serum-free medium were loaded to the upperchannel. The chips were incubated at 37 °C with 5% CO2 in a humidified incubator and visualized under Leica DMI8 confocalmicroscope for 3 days. Quantification was done as previously explained.17Three-channel lab-on-a-chipsystem (Initio)was used for invasion analysis, as explained previously.http://xena.ucsc.edu/) was usedto analyze mRNA expression of the SEMA6D gene in addition to cellcycle-related genes, epithelial-mesenchymal transition (EMT) genes,and Notch pathway genes (Table S2) usingthe TCGA database (https://www.cancer.gov/tcga) for breast-invasive carcinoma (1247 patients).20 The entire population was stratified based on SEMA6D mRNAexpression quartiles, and the highest (0–25th %) Sema6D expressingsamples were labeled as Sema6D-High and the lowest (75th %–100th%) Sema6D expressing samples were labeled as Sema6D-Low. Expressionof the cell cycle, EMT, and Notch-related genes were compared betweenSema6D-Low and -High populations. Results were presented as means± SD. Data were analyzed for statistical significance by theWelch test . Furthermore,cell cycle, EMT, and Notch-related genes were clustered based on uniformmanifold approximation and projection (UMAP) for dimension reduction(arXiv:1802.03426) using BioVinci software and presented with respectto SEMA6D expression with a scoring threshold of 0.4.UCSC Xena  Student’s Figure S1A). In accordance with previous results, a higher SEMA6D protein signalwas detected in total cell lysates . Furthermore, we detected SEMA6D in the conditioned media of allthe cells that overexpress SEMA6D . Consistent with the decrease in BrdU incorporation in responseto SEMA6D overexpression, the number of alive cells in the MCF7-LacZgroup was increased by 11.9-fold, which was reduced to 9.9-fold inMCF7-SEMA6D group . In MDA MB231 cells, SEMA6D overexpression did not change either the growthcurve pattern or the BrdU incorporation rate (S2).Next, we analyzed whether SEMA6D overexpression affects the proliferationrate of the cells by BrdU incorporation assay. SEMA6D overexpressionincreased BrdU incorporation from 11.9% to 39.9% in MCF10A cells 1C. On thOverall, these data showed that SEMA6D overexpression has variableeffects in different cell lines that it induces proliferation in nontumorigenicMCF10A cells while reducing it in MCF7 cancer cells and showed noeffect on MDA MB 231 cancer cells.Anchorage-independent growth is correlatedwith the tumorigenic potential of the cancer cells. Thus, we analyzedhow SEMA6D affects anchorage-independent growth potential using softagar assay. SEMA6D overexpression reduced the number of colonies formedin soft agar both for MCF7 and MDA MB 231 cells 1D. SpeciMigration and invasion potential of the cancer cells are closelyrelated to the aggressive phenotype of tumors. Thus, we analyzed whetherSEMA6D has an effect on migration and invasion. Migration potentialwas assessed by wound healing assay, in which MCF10A and MCF7 cellsthat overexpress SEMA6D covered a larger area compared to the controlcells 24 and 48 h after scratching, respectively 2A. The i17 On each LOC, the Matrigel channel is separatedfrom the cell channel by three gates, through which cells receivechemoattractant signals and invade Matrigel. Invasion of MDA MB 231cells that overexpress LacZ or SEMA6D was observed for 3 days  system in which cells labeled with green fluorescent dye invadethrough Matrigel loaded channel toward serum-rich medium.r 3 days 3A. The dr 3 days 3B. Moreor 3 days 3C. On thTaken together, these results demonstrate that SEMA6D induces migrationin the normal cell line MCF10A and the cancer cell line MCF7, whereasalthough it cannot trigger a migratory phenotype per se, SEMA6D canaugment the invasive potential of MDA MB 231 cancer cells.N-cadherin was increased, while mesenchymal marker SNAI2and epithelial marker E-cadherin was downregulatedupon SEMA6D overexpression  markers to investigatewhether SEMA6D overexpression affects the EMT phenotype. In MCF10Acells, mRNA expression of mesenchymal markers SNAI1, vimentin, and pression 4A. In th31 cells4A.21 and was previouslyshown to be correlated withSEMA6D in breast cancer;15 thus, we analyzedSNAI2 protein (Slug) expression  and the lowest expressing quartile  as subgroups. Compared to SEMA6D-Low samples, SEMA6D-Highsamples had 81% higher SEMA6D gene expression . mRNA expressions of cell cycle-related geneswere mostly downregulated in the SEMA6D-High group compared to SEMA6D-Lowsamples ). The UMAPalgorithm reduced the dimension of cell cycle genes in six clustersbased on expressions .Finally, we analyzed whether the effects of SEMA6D overexpressionwe demonstrated in the cell lines had clinical relevance in breastcancer using The Cancer Genome Atlas (TCGA) data. For this purpose,we sorted all samples with respect to SEMA6D expression and selectedthe highest expressing quartile . CDH1 (−5%)and SNAI1 (−4%) expressions were significantly lower in SEMA6D-Highsamples, while the remaining EMT markers showed significantly higherexpressions. The UMAP algorithm detected nine clusters for EMT markergenes . Still, once this dimensionallyreduced expression data was stratified based on SEMA6D status, SEMA6D-Lowsamples showed a clear separation from the SEMA6D-High group basedon downregulation of ZEB1, ZEB2, and SNAI2 genes with cutoff valuesof 0.95, 0.58, and 0.44, respectively  5. Genes 17 Thus, welater asked whether SEMA6D status is associated with Notch signaling.The majority of the genes related to Notch signaling were significantlyupregulated in SEMA6D-High samples, except DLL3, which had a 47% (p < 0.001) reduction . UMAP dimensional reduction identified seven clusters ; however, stratification based onSEMA6D status showed a distinction based on NOTCH4 and DLL4 upregulation,with the cutoff values of 0.94 and 0.53, in SEMA6D-High samples  and protein  expressions were upregulated in response to Notch activation suggestingthe Notch pathway as a potential upstream regulator of SEMA6D.Notch signaling is one of thesignaling pathways that is deregulatedin breast tumorigenesis. We and others have shown that in additionto affecting proliferation and transformation, Notch activation alsoinduces EMT in breast tissue.eduction S3B. UMAP16 Thus, the aim of this study was to develop a better understandingof how SEMA6D affects the molecular and cellular properties of normalbreast and breast cancer cell lines.The greater part of the research on SEMA6D investigated its rolein developmental processes, while the comparatively small number ofstudies explored whether and how SEMA6D is involved in tumorigenesis.Specifically, the two studies focusing on SEMA6D in breast cancerare limited to the analysis of expression and mutation data of breastcancer patients and suffer from the lack of experimental evidence.8 Onthe other hand, a more recent study noted that proliferation of prenatalcardiomyocytes was reduced in SEMA6D knockout mouse model, which alsohad less Cyclin D1 and Cyclin D2 levels.7 In line with this result, SEMA6D overexpression was reported toinduce proliferation in osteosarcoma cell line HOS, which contributesto its oncogenic role.9 In accordance withthe previous observations, our results also demonstrate a cell-specificeffect of SEMA6D on the proliferation of breast cells. SEMA6D overexpressioninduced proliferation in the normal breast cell line, MCF10A, hintingat a tumor-initiating role. However, the inability to induce anchorage-independentgrowth of MCF10A cells indicates that SEMA6D per se could not actas a strong oncogene activating these two traits simultaneously innontumorigenic breast cells. On the other hand, SEMA6D overexpressionreduced proliferation in MCF7 cells, illustrating an antitumorigenicrole, which is also supported by reduced anchorage-independent growthof both breast cancer cell lines. Our results differ from the positivecorrelation demonstrated between SEMA6D expression and anchorage-independentgrowth in malignant mesothelioma and osteosarcoma cells11 highlighting the importance of tissue and cell context in tumorigenesis.In accordance with the experimental data, TCGA analysis revealed anassociation between SEMA6D levels (high vs low) and the expressionof cell cycle-related genes. Although CCND2 and CCND1 were among theupregulated genes in the SEMA6D-High subpopulation, the majority ofthe positive regulators of the cell cycle, including CCNE1, CDC25C,CDC20, E2F1, and E2F2, were downregulated at a greater magnitude,which might reflect the growth-inhibitory effect of SEMA6D in MCF7cells. Whether increased proliferation due to SEMA6D overexpressionobserved in MCF10A cells represents an early stage of breast tumorigenesisremains unknown due to the lack of relevant clinical sample groupin the data set.First, we investigatedthe effect of SEMA6D overexpression on proliferation.Prior studies have not established a consistent association betweenSEMA6D and proliferation. In different regions of the developing heart,SEMA6D was shown to regulate the migration of mesenchymal and endothelialcells but had no effect on the proliferation.8 Likewise, BMP-inducedmigration during atrioventricular cushion development was dependenton SEMA6D upregulation.5 SEMA6D was reportedas a repulsive regulator for dorsal root ganglion, the crossing ofretinal ganglion cells, and pericyte–endothelial cell interaction,all of which are related to migratory behavior of cells.22 A recent study that focused on osteosarcoma noted that SEMA6D overexpressionrescued the migration and invasion potential, which was decreasedin response to knockdown of cicUBAP2, a positive regulator of SEMA6Dexpression.10 Here, we showed that SEMA6Dinduces migration in MCF10A and MCF7 cell lines but does not affectMDA MB 231 cells. Considering that the latter could be explained bythe extensively migratory nature, we also analyzed invasion and demonstratedthat SEMA6D promoted the invasion potential of MDA MB 231 cells. Together,in addition to confirming the previous findings, these results presentedthe first evidence that in breast cells, SEMA6D can promote migrationand invasion.A number of studies described a link betweenSEMA6D and migrationmainly during embryonic development. SEMA6D was demonstrated as anessential molecule involved in cardiac development and neural tubeclosure, during which its major effect was on the migration of mesenchymaland endothelial cells.E-cadherin and increased SNAI2, ZEB1, and ZEB2, suggesting a correlationbetween SEMA6D and mesenchymal transition.15 In gastric cancer, colocalization of SEMA6D with SNAI1 protein (Snail)was positively correlated with invasion and lymph node metastasis.23 Here, we showed that SEMA6D regulated mRNA expressionof the EMT markers, mainly shifting toward a more mesenchymal patternin MCF10A cells. On the other hand, in breast cancer cells, therewas no change in the mRNA levels of EMT markers other than a decreasein N-cadherin in MCF7, which was not anticipatedconsidering the shift toward a more migratory and invasive phenotypein response to SEMA6D overexpression. On the other hand, SNAI2 (Slug)protein was increased in MCF7 cells, consistent with a more mesenchymalphenotype. Supporting the experimental data, SEMA6D levels were associatedwith the expression of EMT markers in the TCGA data set. Consistentwith the previous study,15 in the SEMA6D-Highgroup mesenchymal markers, including ZEB1, SNAI2, ZEB2, and TWIST,were increased. Collectively, the data we present here suggest a correlationbetween SEMA6D expression and a migratory/invasive phenotype supportedby a mesenchymal shift in the EMT markers’ expression, whichare in accordance with the previous studies. Although both our experimentaldata and TCGA data analyzed by us and others converge on SNAI2 infocus, the existing data fail to provide clear evidence of a directtranscriptional and translational regulation of EMT by SEMA6D.Although SEMA6D was implicated in migration inthe context of developmentand cancer, its role in EMT, which is related to the migratory phenotype,has not been investigated thoroughly. In breast cancer, TCGA analysisshowed that the SEMA6D-high patient group has decreased SEMA6D induced a similar trend in breast cancer cell lines, suchas a decrease in anchorage-independent growth and an increase in mobility.However, the two cell lines showed distinct phenotypes in migrationand expression profile of EMT markers. Although the cell lines usedin this study represent the characteristic properties of luminal (MCF7)and basal (MDA MB 231) breast cancer subtypes, the data cannot beextrapolated to subtype-specific roles of SEMA6D. This issue needsto be explored in further research using a panel of cell lines representingdifferent subtypes.Finally, we showed an association betweenNotch pathway-relatedgenes and SEMA6D levels in the TCGA data set and induction of SEMA6Dexpression upon Notch activation in MCF10A cells. Although our datasuggest that Notch signaling might be an upstream regulator of SEMA6Din the breast, the functional importance of Notch-SEMA6D relationin breast tumorigenesis remains to be answered.9 Although PlexinA1 was reported as the receptor of SEMA6D, other cell-surface proteinssuch as vascular endothelial growth factor receptor 2 (VEGFR2), Off-Track(OTK), Ng-CAM related cell adhesion molecule (Nr-CAM), and PlexinA4 were also shown to interact with SEMA6D.11 Thus, differentialrepresentation of SEMA6D interacting proteins on the cell surfacecould also explain cell-specific effects that we observed. Anotherreason for the cell-specific effect could be the endogenous expressionprofile of SEMA6D. Although we observed a single band in the conditionedmedia, different bands of SEMA6D were visible in whole cell lysatesof each cell line, which might indicate distinct endogenous SEMA6Disoforms. As SEMA6D has nine isoforms,24 we cannotexclude the possibility of SEMA6D overexpression interfering withendogenous SEMA6D isoforms and contributing to the manifestation ofdifferent phenotypes. Although the issue of different isoforms isan intriguing one, to date there is no data on the functional importanceof different SEMA6D isoforms in breast cancer, and it requires furtherresearch.In summary,here, we showed a cell-specific effect of SEMA6D innormal and tumorigenic cell lines of the breast. A possible explanationfor this observation is the different downstream mechanisms activatedupon SEMA6D overexpression in different cell lines. A notable examplein this context comes from osteosarcoma, where only two out of fourcell lines had increased ERK phosphorylation to different extentsupon SEMA6D overexpression.In this study, we provided the first experimentalevidence on thefunctional role of SEMA6D in normal and tumorigenic breast cell lines.However, there is an abundant room for further investigations to answerthe questions such as interacting partners on the cell surface, upstreamregulators, and downstream mediators of SEMA6D to develop a full pictureof SEMA6D’s role in breast cancer.


Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 μg/ml. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats and in humans, with the symptoms restricted to the skin of immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts  such as Ag Sporothrix spp. are dimorphic, saprophytic and geophilic fungi of the Sporothrix complex and responsible for sporotrichosis, a chronic infection of the skin and subcutaneous tissue of humans and animals , Ag3[PMo12O40], Ag6[SiW10V2O40], and Ag4[SiW12O40]) were analyzed by X-ray diffraction using an X-ray diffractometer URD 65 model-SEIFERT/GE and an X-ray tube with a copper filament. Samples were treated previously at 90°C for 30 min, sieved through 200-mesh netting and maintained in a desiccator.Ag-HPA salts  obtained from Instituto Oswaldo Cruz (IOC), Rio de Janeiro, Brazil. Our clinical strains were isolated from infected cats  by the clinical practitioner of the Veterinary Hospital of Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil. Further, all samples were stored in plates with potato dextrose agar (PDA) , which were supplemented with chloramphenicol (50 mg/ml)  and cycloheximide (400 mg/ml) (Sigma-Aldrich), and stored at 28°C. To obtain the yeast cells, new cultured cells were grown in Brain Heart Infusion (BHI) Agar (India) followed by incubation at 37°C.Some selected strains present different levels of melanization, which, according to the literature, could have some influence on drug response mechanisms . The strThe Ag-HPA salts were suspended in sterile distilled water to a ratio of 51.2 mg/ml. The antifungal drugs miconazole nitrate (Sigma-Aldrich), itraconazole and terbinafine (Sigma-Aldrich) were diluted in dimethyl sulfoxide (Sigma-Aldrich), and amphotericin B was diluted in sterile distilled water to obtain stock solutions of 12.8 mg/ml, which were maintained at -70°C. All reagents used as controls in this step were purchased from Sigma-Aldrich, USA.Sporothrix spp. . To obtain yeast cell cultures, from each stock culture, a small fragment of agar containing colonies was transferred and spread onto Petri dishes containing Brain Heart Infusion Agar and allowed to grow at 37°C for 5 days. To access yeast forms, the isolates were maintained by transferring the cultures into the same medium weekly. A suspension of inoculum was obtained from yeast cells in a tube with 10 ml of sterile 0.85% saline, corresponding to 0.5 McFarland suspension of 5 × 105 CFU/ml carried out in a photometer OD550nm , as established by the manufacturer. To assay the effect of each Ag-HPA salt (32 μg/ml) on yeast growth, 0.1 ml of fresh inoculum and 0.1 ml of each Ag-HPA salt were added to 1.8 ml BHI broth Infusion Broth (BHI) and incubated at 37°C. Optical density readings at 550 nm were taken at zero hour, and then readings were conducted every 24 h for the following 14 consecutive days. For cell growth, itraconazole (500 μg/ml), and medium plus HPA acid, and HCl (32 μg/ml) were used as positive and negative controls, respectively. The latter was used to evaluate the effect of H+ on fungal growth. The optical densities were plotted as a function of the incubation time for each fraction. Experiments were performed in triplicate and repeated two times. The inhibition rate was defined as the median value.The activity of the Ag-HPA salts on fungal growth was tested against three strains of 5 CFU/ml, which was determined with a densitometer at OD550nm and diluted 1:1000 in RPMI 1640 medium. Each well was filled with 0,1 ml of Sporothrix spp. inoculum and 0,1 ml of compound suspensions to obtain a final Ag-HPA salt concentration of 0.0625-256 μg/ml. All microtitration plates were incubated in a humid chamber at 37°C for 96 h. The MIC values were defined as the lowest concentration that imposed complete inhibition of Sporothrix spp. growth, which was revealed by adding 30 μl/well resazurin (100 μg/ml) reagent (Sigma-Aldrich). The tests were performed in triplicate on two different days.Minimum inhibitory concentrations (MIC) for the Ag-HPA salts were determined using the broth microdilution technique, according to Clinical Laboratory Standards Institute M27-A3 recommendations . The com5 CFU/ml, which was determined with a densitometer at OD550nm and spread by using a sterile swab. Antifungal disks were placed in the center of each Petri dish and incubated for 96 h at 37°C. The inhibition zone was measured with the aid of a digital caliper  and compared with controls without HPA salt. The mycelial forms of CMDB/IOC 01980599 and the clinical strain LSASs01 were evaluated for synergistic-like effects after seven days in the presence of AMB (100 μg) and ITC (10 μg) individually and in combination with Ag-HPA salts at a final concentration of 128 μg/ml by the disk diffusion method in Potato Dextrose Agar. The conidia inoculum was prepared according to a previously reported procedure , H3[PMo12O40], H4[SiW12O40], and H6[SiW10V2O40] were obtained as large and brightly-colored crystals after two crystallization steps from water solutions, as shown in 6[SiW10V2O40]. The salts obtained were subjected to FTIR analysis, and their structures were compared with the literature data [3[PMo12O40], silver dodecatungstophosphate (Ag3[PW12O40]), silver dodecatungstosilicate (Ag4[SiW12O40]) and silver decatungsto-vanadosilicate (Ag6[SiW10V2O40]). The obtained spectral results are summarized in The heteropolyacids Hure data , 25. The3[PW12O40] described previously  showed the highest activity, with 93% inhibition of growth, followed by Ag3[PW12O40] (80%), and Ag4[SiW12O40] (68%)  and Ag3[PW12O40] (69%) (6[SiW10V2O40] inhibited cells the least at approximately 57% (ATCC 32285), 48% (CMDB/IOC 01980599) and 36% (LSASs01), respectively. The positive control in the presence of 500 μg/ml itraconazole presented total (100%) inhibition of growth against all strains tested. The negative controls, HPA acid in the form of H3[PMo12O40] and HCl presented a weak effect and were ineffective, respectively, on the growth of the strains tested when compared to the other treatments.  . Toward ectively . Toward 0] (69%) . Of the atments. -2C.Sporothrix spp. and on the five standard strains as shown in 3[PMo12O40]) with MIC values varying from 8 to 16 μg/ml. The dodecatungstophosphate of silver (Ag3[PW12O40]) also presented expressive results with MIC values varying from 8 to 32 μg/ml. The MIC of dodecatungstosilicate of silver (Ag4[SiW12O40]) also varied from 16 to 32 μg/ml, and the MIC of divanadodecatungstosilicate of silver (Ag6[SiW10V2O40]) varied from 16 to 128 μg/ml. All antifungal drugs used as controls presented activity superior to Ag-HPA salts. Miconazole nitrate and terbinafine exhibited MIC values as low as 0.125-0.5 μg/ml and 0.5-2 μg/ml, respectively, which were even better than the MIC values of amphotericin B (1 to 8 μg/ml) and itraconazole (1 to 32 μg/ml), the recognized agents for the treatment of sporotrichosis  presented considerable alterations after Ag-HPA salts and the antifungal drugs ITC and AMB were combined, as shown in entation 4 and 4C4entation 1 and 4A1Membrane permeabilization of the yeast after 96 h incubation with Ag-HPA salts was observed on a light microscope by using vital dye staining. As shown in 3[PMo12O40], yeast cells presented large abnormal vacuoles and less dense microfibrillar material , leading to significant disorganization of membrane components, cytoplasm disorder, vacuolation, and the presence of vesicles in the space formed between the membrane and the vacuole (white star), despite the preserved external microfibrillar material  showed a normal morphology with budding and regular surfaces . After tPW12O40]  or Ag3[PMo12O40] , cells png cells , in cont3[PW12O40] is representative of the Ag-HPA salt profile used in the present study. A similar diffractogram of the POM H3[PW12O40] was described [et al. [3[PMo12O40]) and three polyoxotungstates  on yeast and mycelial forms of Sporothrix spp., and their interactions with antifungal drugs associated with cell damage. The results presented in HPAs have been discussed as efficient, reusable, green and cost effective catalyst compounds that can be quickly synthesized , 27. Theescribed  that res ) and tungsten (Ag3[PW12O40]) presented a higher inhibition, with 78-93% and 69-85%, in the yeast form, respectively, compared with salt silicon-containing heteroatoms and metals tungsten (Ag4[SiW12O40]) and vanadium-substituent (Ag6[SiW10V2O40]), which inhibited yeast growth by 68-76% and 36-57%, respectively. In the MIC assay, P-containing Ag-HPA salts appeared to present overall lower MICs, although the values were higher than those presented by traditional antifungal agents  were significantly more active against the macroscopic melanized clinical strain LSASs01. ITC and AMB were less active against the mycelial form of the CMDB/IOC 010980599 and LSASs01 strains when tested alone; however, when combined with Ag-HPA salts, their growth was retarded, and restricted pigmentation production suggested interference in melanization impairment after 15 days of incubation at room temperature. It is noteworthy to mention that ITC is a drug commonly used against this fungus in routine human and veterinary practice  and Ag6[SiW10V2O40]) were significantly more active against the CMDB/IOC 01980599 strain. Fungi also respond to stress stimuli by activating the SOS defense system by DNA repair, providing a survival mechanism , and against relevant mycotic diseases caused by other yeasts, such as albicans . The Ag-