Therefore, Gpc4 is a key regulator of Aβ enrichment in NSCs. Inhibiting Gpc4 rescued the Aβ-induced toxic effect and attenuated the nontoxic Aβ enrichment into intracellular toxic concentrations. Gpc4 contributed to Aβ internalization and toxic/physical concentrations of Aβ-induced mitochondrial membrane potential damage and cell apoptosis, partly via LRP1. These findings suggest a potential role of Gpc4 in treating Alzheimer’s disease at an early stage, by targeting NSCs.Neural stem cell (NSC) damage has been reported in patients with Alzheimer’s disease. Intracellular Aβ plays a vital role in NSC damage. Heparan sulfate proteoglycans are potent mediators of Aβ enrichment in the brain. We hypothesized the heparan sulfate proteoglycan glypican 4 (Gpc4) regulates Aβ internalization by NSCs. We evaluated Gpc4 expression in NSCs from P0–P2 generations using immunofluorescence. Adenovirus and lentivirus were used to regulate Gpc4 expression in NSCs and APP/PS1 mice, respectively. Co-immunoprecipitation was used to determine the relationship between Gpc4, Aβ, and low-density lipoprotein receptor-related protein 1 (LRP1). Intracellular Aβ concentrations were detected using enzyme-linked immunosorbent assay and immunofluorescence. The role of Gpc4/LRP1 on toxic/physical Aβ-induced effects was evaluated using the JC-1 kit, terminal deoxynucleotidyl transferase dUPT nick end labeling, and western blotting. Gpc4 was stably expressed in NSCs, neurons, and astrocytes. Gpc4 was upregulated by Aβ in NSCs and regulated Aβ internalization. Gpc4 attenuation reduced Aβ uptake; Gpc4 overexpression increased Aβ uptake. Gpc4 regulated Aβ internalization through LRP1 and contributed to Aβ internalization and toxic/physical concentrations of Aβ-induced mitochondrial membrane potential and cell apoptosis, partly  Neural stem cell (NSC) damage has been reported in the brains of Alzheimer’s disease (AD) patients  aggregation occurs before the extracellular deposition and plaque formation Bloom, . IntraceHeparan sulfate proteoglycans accumulate abundantly in conjunction with Aβ  and apolipoprotein E , according to the manufacturer’s instructions . NSCs were treated with AdV for 24 h and then the medium with AdV was changed to complete medium without AdV. After 72 h of infection, Aβ was added. Internalization was detected after 6 h, and toxic effects were detected after 12 h.NSCs were isolated from mouse embryos (E14.5) as previously described  assay. Cells were plated in 96-well plates at 20,000 cells/well density, 24 h before the experiments. The cells were then treated with different concentrations of Aβ  for 12 h. Next, 20 μl of CCK-8 were added to each well and the cells were incubated for 2 h at 37°C. Absorbance was measured at 490 nm using a microplate spectrophotometer . Triplicate parallel wells were examined in each experiment, and data were collected from three independent experiments. The results are presented as a percentage of the absorbance compared to the control group.To investigate the toxic effect of Aβ on NSC apoptosis mechanism, a terminal deoxynucleotidyl transferase dUPT nick end labeling (TUNEL) assay was performed according to the manufacturer’s instructions . Cells were fixed on cover slides in 4% paraformaldehyde for 15 min. The cells were then permeabilized in 0.3% Triton X-100 in phosphate-buffered saline for 5 min on ice. Cells were incubated in the TUNEL reaction mixture for 1 h at 37°C. Subsequently, the cells were counterstained with 4′,6-diamidino-2-phenylindole  before mounting. Images were taken within 2 h. Nine random fields were counted for each group with a 40× objective. The ratio was calculated as the percentage of TUNEL-positive cells compared to that of DAPI-positive cells.6 cells/ml. The cells were incubated with PI/Annexin V for 15 min in the dark. Subsequently, the cell solution was analyzed using FACSCalibur (BD Biosciences) for PI/Annexin V. The percentage of apoptotic cells in this experiment was calculated as the ratio of Annexin V-positive and PI-negative cells from the lower right quarter.Flow cytometry analysis was used for the apoptosis tests in this study. The propidium iodide (PI)/Annexin V apoptosis detection kit  was used according to the manufacturer’s instructions. Cells were dissociated, washed in cold phosphate-buffered saline, and resuspended in 1× binding buffer to obtain a concentration of 1 × 10For immunohistochemistry performed in APP/PS1 mice, mouse brains were fixed with 4% paraformaldehyde and frozen coronal slices (30 μm) were obtained using a frozen section machine . NSCs were fixed in 4% paraformaldehyde. The sections were treated with 0.3% Triton for 30 min. The cells were then blocked with 5% bovine serum albumin for 30 min. The sections were incubated with primary antibodies overnight at 4°C. The cells were then incubated with the secondary antibodies, goat-anti-rabbit-IgG-488 and goat-anti-mouse-IgG-594 , for 2 h at room temperature. Slides were cover-slipped using a mounting medium containing DAPI (blue). The primary antibodies used in this experiment were: mouse polyclonal anti-nestin antibody , rabbit polyclonal anti-Gpc4 antibody , rabbit polyclonal anti-sox2 antibody , rabbit polyclonal anti-Ki67 antibody , mouse polyclonal anti-sox2 antibody , mouse polyclonal anti-LRP1 antibody , goat-anti-rabbit-IgG-488, goat-anti-mouse-IgG-594 , and DAPI . For 3,3’-diaminobenzidine staining, we followed the manufacturer’s instructions using a secondary antibody staining kit .g) for 30 min at 4°C. The protein concentration was quantified using the BCA assay . Protein (10 μg) was added to each sample, electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels, and transferred to a polyvinylidene fluoride membrane . The membranes were then blocked in 10% skimmed milk for 2 h and incubated with the following primary antibodies: rabbit anti-Gpc4 , rabbit anti-Cyt c (1:500), rabbit anti-Bax (1:500), rabbit anti-Bcl 2 (1:500), rabbit anti-GAPDH , or β-actin  . The primary antibody was dissolved in 10% skimmed milk, and the membrane was incubated at 4°C overnight. The membrane was then washed and incubated with horseradish peroxidase-conjugated secondary antibody. Membranes were developed using enhanced chemiluminescence and exposed to photographic films. All results were obtained from three independent experiments. The results were analyzed using ImageJ software  to infect NSC cell cultures. The overGpc4 sequence was obtained from Gene_ID:14735, GenBank: NM_008150. The forward siGpc4 sequence was 5-GGAUGGCAGU GGAUGACUUTT-3 and the reverse was 5-AAGU CAUCCACUGCCAUCCTT-3. The forward and reverse siLRP1 sequences were 5-GCCCAUUGGAUGAGUUUCATT-3 and 5-UGAAACUCAUCCAAUGGGCTT-3, respectively. The lentivirus vector for Gpc4 overexpression was GV367 Ubi-MCS-SV40-EGFP-IRES-pruromycin , and the lentivirus vector for siGpc4 was hU6-MCS-Ubiquitin-FGFP-IRES-puromycin . AdV was plated into 6-well plates at a density of 2 × 106 cells/ml; 10 μl of 2 × 1012 μg/ml AdV was added to each well in NSC complete medium . Twenty-four hours after infection, the medium was replaced with a complete medium without AdV. The cells were used for the experiment 72 h after infection.siGpc4, overexpressed Gpc4 (overGpc4), and siLRP1 were coated with AdV (4 × 1012 μg/ml AdV (overGpc4 and si Gpc4) was plated into 6-well plates at a density of 2 × 106 cells/ml . Twenty-four hours after infection, the medium was replaced with a complete medium without AdV. Aβ was added into each 6-well plates cells at a working concentration of 200 nM or 2 μM, 72 h after Adv infection. NSC samples were collected 12 h after Aβ treatment. Mouse Aβ enzyme-linked immunosorbent assay (ELISA) Kit  and PS1dE9 on a C57BL6SJL background. Acute crossing of these two lines produces an accelerated mouse model of AD on a B6J/B6SJL background combining cognitive and amyloid pathologies starting at 4 months old. Genotypes were confirmed by a polymerase chain reaction from a tail biopsy. All wild type mice in this study are of B6J/B6SJL littermates. All APP/PS1 mice were APP-and PS1 positive. The animals were obtained by Yang Wei-Na from Xi’an Jiaotong University, housed in an animal facility with two mice per cage in temperature- (22 ± 1°C) and humidity-controlled (50 ± 10%) conditions under a 12 h light/dark cycle. The mice had access to food and water via stereotaxic surgery under deep isoflurane anesthesia (n = 6). Gpc4 siRNA lentivirus was injected using stereotaxic coordinates and a microsyringe. Virus was injected into the lateral ventricles and the CA1 hippocampal subfield  of both sides . The injection speed was 0.2 μl/min, and the total volume/side was 1 μl, 2 × 108/μl. The control virus was also injected into APP/PS1 mice (n = 5). After surgery, the animals were given 2 months for recovery and lentivirus expression.Gpc4 siRNA lentivirus was injected into each mouse’s brain After the brain was perfusion-fixed and sectioned at 30 μm on a freezing microtome, the selected series were mounted onto gelatine-coated glass slides and air-dried. The tissue was hydrated by passing it through a series of ethyl alcohol solutions  before staining with 1% Thioflavin-S for 30 min  were pre-cleared for 1–4 h at 4°C with 100 μl of 80–100 protein G. Precleared supernatants were incubated overnight at 4°C with the desired IP antibody  and 200 μl of protein G beads. IP complexes were washed five times with wash buffer and eluted twice with 40 μl elution buffer. Protein complexes (80 μl) were obtained and used for western blotting.Immunoprecipitation  was performed as previously described . JC-1 is a membrane-permeable lipophilic dye that exists as JC-1 aggregates in the mitochondrial matrix (red) or as monomers in the cytoplasm (green). During mitochondrial depolarization, the red JC-1 aggregates form green monomers due to a change in proton motive force (ΔΨ). Thus, depolarization can be measured as an increase in the green fluorescent/red fluorescent intensity ratio. The JC-1 assay was performed as follows. Solutions containing 5 μg/ml JC-1 were added for AdV and Aβ treated NSCs for 30 min. The stained cells were rinsed twice with the culture medium. After the fresh media was added, the cells were analyzed under a green fluorescence microscope (480/525 nm).P-value <0.05 was regarded as significant. t-test was used to compare between two groups. All in vitro experiments have been independently repeated at least three times. For in vivo experiments, in each group at least five mice were used for statistical analysis.All data were analyzed with GraphPad Prism version 6.0 . For normal distribution data, one-way analysis of variance (ANOVA) followed with Tukey’s multiple comparisons test were performed. Neural stem cells derived from the cortex of embryonic 14.5 (E14.5) mice were observed. Approximately 5 days after primary culture (P0), the isolated NSCs were passed for the next generation (P1) and then for P2 . We obseThis experiment aimed to determine the toxic dose of Aβ in NSCs. We found that 200 nM Aβ induced cell viability of NSCs, but that 1–20 μM Aβ was toxic to NSCs . TherefoTo identify the role of Gpc4 in NSCs in AD, we examined Gpc4 expression patterns in NSCs. Gpc4 (green) colocalized with cells positive for Ki67 or nestin (red), which proves the expression of Gpc4 in NSCs. Gpc4 was widely expressed in NSCs. Double-positive Gpc4/Ki67 represents NSCs undergoing cell division .Co-IP was performed to detect the relationship between Gpc4 and Aβ. We found that Gpc4 forms a protein complex with Aβ in the APP/PS1 mouse brain . To furtNext, cells were monitored to detect whether Gpc4 induced Aβ production. No significant differences were observed between the siGpc4 and overGpc4 adenovirus groups . overGpcLRP1 is an Aβ receptor that plays a role in Gpc4-induced Aβ internalization. We first identified the expression of LRP1 in NSCs by double staining NSCs with Ki67 and LRP1. LRP1 colocalized with Ki67-positive cells . Double-To more precisely define the relationship between Gpc4 and LRP1, co-IP was performed on the cell lysis of APP/PS1 mice. Gpc4 was able to bind to LRP1, forming a protein complex in the brains of AD mice . These oWe next aimed to reveal the action of Gpc4 and LRP1 in Aβ internalization. NSCs were transfected with control, siLRP1, siLRP1+siGpc4, and siLRP1+overGpc4 AdV for 3 days, and then Aβ was added for 6 h before cells were harvested for ELISA. siLRP1 remarkably decreased Aβ uptake by NSCs. In the siLRP1 and siGpc4 groups, the amount of internalized Aβ was much less compared to the siLRP1-treated group. Furthermore, in the siLRP1+overGpc4-treated group, the internalized Aβ level was increased compared to the siLRP1+siGpc4 group .Control, siLRP1, overGpc4, and overGpc4+siLRP1 AdV were transfected into NSCs for 3 days, and then Aβ was added for 12 h. The JC-1 kit was used to detect the MMP in NSCs. JC-1 monomers (green) and aggregates (red) were compared and normalized to those of the control group. In 2 μM Aβ experiments, JC-1, TUNEL, and western blotting were used to determine whether Gpc4/LRP1 regulates the toxic effect of Aβ on NSCs. siLRP1 significantly decreased the ratio of JC-1 monomers/aggregates compared to the control group. Moreover, overGpc4 increased MMP damage compared to siLRP1. overGpc4+siLRP1 significantly increased the MMP compared to siLRP1; however, the MMP damage in the overGpc4+siLRP1 group was lower than that in the overGpc4 group . MoreoveIn 200 nM Aβ experiments, JC-1 and TUNEL assays were used to determine whether Gpc4 induces toxic effects by enriching the nontoxic effect of Aβ on NSCs. Initially, 200 nM Aβ had no toxic effect on NSCs, but overGpc4 in 200 nM Aβ-treated NSCs increased MMP damage . FurtherThe dysregulation of adult neurogenesis in AD occurs at an early stage of the disease and the mechanism has not been completely elucidated (Boese et al., −10–10−9 M; Ma and Qian, in vitro studies have suggested that the critical concentration for spontaneous aggregation of Aβ is in the μM range. Therefore, Aβ concentrations in vivo would have to be increased by at least three orders of magnitude for spontaneous aggregation. Several mechanisms have been proposed to explain this large concentration gap. The most likely hypotheses are that the concentration could be increased through membrane association (Gorbenko and Kinnunen, Heparan sulfate proteoglycans have been proposed to be responsible for Aβ enrichment. We found that Gpc4 expression was increased in Aβ-treated NSCs in a concentration-dependent manner. Nevertheless, considering neurogenesis deficiency is an important aspect of memory loss, Gpc4 may regulate Aβ enrichment in NSCs. The average concentration of Aβ42 in the brain white matter of patients with AD is 60 pM/L (Collins-Praino et al., The role of Gpc4 in regulating different concentrations of Aβ internalization in NSCs is Aβ dose-dependent. siGpc4 decreased NSC uptake of 2 μM Aβ, and overGpc4 increased NSC uptake of 200 nM Aβ. This different role of Gpc4 on 2 μM Aβ and 200 nM Aβ transportation indicates that Gpc4 is functional at nanomolar Aβ concentrations; however, the transportation efficiency of Gpc4 reaches its saturation point at micromolar Aβ concentrations. Gpc4 is a key regulator of Aβ enrichment at nanomolar concentrations of Aβ. This illustrates why siGpc4 lentivirus had no effect on β-amyloid plaque numbers. Therefore, Gpc4 is a key regulator of Aβ enrichment during the early stages of AD, but not in the later stages.Heparan sulfate, the extracellular portion of Gpc4, has a high affinity for Aβ and its receptors (Yamada and Hamaguchi, via Gpc4 overexpression, leading to an upregulation of intracellular Aβ levels. Second, ERK1/2, as the downstream target of both LRP1 and Gpc4, may also regulate the expression of other Aβ receptors. siLRP1 decreased the expression of ERK1/2; however, Gpc4 overexpression activated ERK1/2, subsequently activating other receptors related to Aβ internalization. Lastly, Gpc4 is responsible for many cellular growth processes, such as cell division and cell metabolism (Farhy-Tselnicker et al., LRP1 is an important receptor for the induction of Aβ internalization in astrocytes. Previous studies have shown that LRP1 clears 73.8% of Aβ injected into the brain towards blood circulation through the blood-brain barrier (Ma et al., in vitro and in vivo (Yang et al., via LRP1. We used the JC-1 kit to detect the role of internalized Aβ on MMP, which is also called mitochondrial depolarization. We found that in toxic Aβ-treated groups, attenuating Gpc4/LRP1 expression decreased intracellular Aβ, MMP damage, and NSC apoptosis. In the physical Aβ-treated groups, Gpc4 overexpression caused MMP damage but had no effect on cell apoptosis. These results indicate that internalized Aβ is harmful to both mitochondrial function and cell viability.We have previously shown that internalized Aβ is transported into the mitochondria via the well-known Aβ receptor, LRP1. However, many questions remain unanswered. Heparan sulfate on the extracellular surface of Gpc4 is important for receiving and binding to Aβ and its receptors. Gpc4 was initially reported to have a major effect on synapse formation and neurogenesis; therefore, it is also important to determine whether Gpc4 mediates NSC-derived neurogenesis by regulating Aβ internalization in NSCs.We demonstrated that Gpc4 regulates Aβ internalization at an early stage of AD. This process is dose-dependent, and occurs partly The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Xi’an Jiaotong University.KM performed most of the experiments, analyzed data, and wrote the rough draft. SX performed experiments, data analysis, and redrafting. YLu and YiLi performed NSCs cell culture. CZ performed the animal housing. ZZ and YF took part in the virus infection experiment. ZZ and YoLi participated in the experiment design. XC designed the research and finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The solute carrier family has been reported to play critical roles in the progression of several cancers; however, the relationship between solute carrier family 12 member 8 (SLC12A8) and bladder cancer (BC) has not been clearly confirmed. This study explores the prognostic value of SLC12A8 for BC and its correlation with immune cell infiltration. We found that the expression of SLC12A8 mRNA was significantly overexpressed in BC tissues compared with noncancerous tissues in multiple public databases, and the result was validated using real-time PCR and immunohistochemistry (IHC). The Kaplan-Meier method and Cox proportional hazards models were used to evaluate the prognostic value of SLC12A8 for BC. The high expression of SLC12A8 led to a shorter overall survival time and was an unfavorable prognostic biomarker for BC. The mechanisms of SLC12A8 promoting tumorigenesis were investigated by Gene Set Enrichment Analysis (GSEA). Moreover, the correlations of SLC12A8 expression with the tumor-infiltrating immune cells (TICs) in BC were explored using TIMER 2.0 and CIBERSORT. SLC12A8 was associated with CD4+ T cells, dendritic cells, neutrophils, and macrophages infiltration. The expression of SLC12A8 was positively correlated with crucial immune checkpoint molecules. In conclusion, SLC12A8 might be an unfavorable prognostic biomarker in BC related to tumor immune cell infiltration. The incidence of BC ranks 10th, and mortality ranks 13th in cancers globally; among men, BC ranks 7th in incidence and 9th in mortality . NeoadjuSLC12A8 belongs to the solute carrier (SLC) transporters family. The most significant transporters in the body have essential roles in regulating the transport of substances inside and outside cells . A recenThis study aimed to explored the prognostic value of SLC12A8 in BC using the public database TCGA and GES13507. To further confirm the clinical diagnostic value of SLC12A8, qPCR and immunohistochemistry (IHC) methods were performed in the collected clinical tissue samples. The possible molecular mechanisms and signal pathways by which SLC12A8 participates in BC were analyzed. Finally, Timer2.0 and CIBERSORT were used to evaluate the effect of SLC12A8 on immune cell infiltration and its relationship with immune checkpoint protein expression in BC.TCGA-BLCA includes gene profile data of 414 BC samples and 19 noncancerous samples. Oncomine and GES13507 were used to verify the differential expression of SLC12A8 mRNA in BC tissues (n = 188) and normal tissues (n = 68) ,21. ClinTo further evaluate the prognostic value of SLC12A8 in BC, patients were divided into two groups according to the median expression of SLC12A8. The Kaplan-Meier method was used to explore 5-years overall survival or cancer-specific cancer specific survival time in TCGA and GSE13507. Univariate and multivariate Cox regressions were used to evaluate proportional hazards for overall survival . The pathttps://biit.cs.ut.ee/mem/index.cgi), a gene expression query, and visualization tool [https://string‐db.org/) [Genes that are co-expressed with SLC12A8 were obtained from the Multi Experiment Matrix (MEM)  .http://www.broad.mit.edu/gsea/) was used to determine significant and concordant differences between a set of biological processes or signaling pathway genes from the Molecular Signatures Database (MsigDB) and SLC12A8 high expression groups based on TCGA with the cut‐off criteria of FDR < 0.25 and nominal p < 0.05 [GSEA v3.0 , and all patients gave verbal consent. RB approval number: Y (2021) 039.2.One normal human urinary tract epithelial SV-HUC-1 and five urinary BC cell lines  were obtained from FuHeng Biology. T24, 5637, and EJ-1 cells were cultured in RPMI 1640 medium, UMUC3 was cultured in DMEM medium, J82 was cultured in MEM medium, and SV-HUC-1 was cultured in F-12 K medium. All media contained 10% fetal bovine serum. The cell culture conditions were 37 °C and 5% COΔΔCT method was used to quantify relative SLC12A8 mRNA expression [Total mRNA of BC tissues or cell lines were extracted using TRIzol . Reverse transcription was performed using Fasting gDNA Dispelling RT SuperMix Kit . The reverse transcription conditions were as follows: 42 °C for 15 min, 95 °C for 3 min. qPCR was performed using the TL988 Real-Time PCR System (TIANLONG TECHNOLOGY). According to SYBR Green Kit , the reaction conditions were 95 °C for 60 sec, 95 °C for 10 sec, and 63 °C for 30 sec by 40 cycles. The sequence of SLC12A8 primer pairs were 5′-AGAAAGCTCCCAGTTACGGC-3′(forward), and 5′-CTGGGCTGGCTACTCTCAAG-3′ (reverse) and GAPDH were 5′-AGTCCACTGGCGTCTTCAC-3′ (forward) and 5′-GAGGCATTGCTGATGATCTTGA-3′ (reverse). 2 pression .The protein expression levels of SLC12A8 in tissue samples were determined using IHC. Briefly, 4-μm paraffin tissue chips were baked at 65 °C for 1 hour before dewaxing. The staining procedure was operated according to the IHC kit protocol (Maxim Biotechnology) using the SLC12A8 antibody . The sections were subjected to DAB color development, hematoxylin staining, dehydration, and sealing. The sections were taken photographs under a microscope with ×100 and ×200 magnification. IHC profiler, a plug-in of Image J, was used to analyze the immunohistochemical results. The percentage of positive cells was divided into five grades according to the following scoring rules: less than 10% = 0, 10%–25% = 1, 26–50% = 2, 51–75% = 3 score, and more than 75% = 4. The staining intensity was divided into four grades: negative = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. The positive proportion score was multiplied by the staining intensity score; according to the corresponding expression classification, 0–3 points indicated low expression and 4–12 points indicated high expression .The proportion of 22 immune cells in BC samples were calculated using CIBERSORT ,31. TIMETo evaluate the response and therapeutic outcomes of immune checkpoints through SLC12A8 gene expression, we further analyzed the correlation between SLC12A8 expression and important immune checkpoints gene expression according to TCGA-BLCA expression profile data . TIMER2.Statistical processing and analysis were performed using R (version 3.6.2) and SPSS Statistics 26.0 in this study. Independent T-tests or non-parametric tests were used to compare SLC12A8 mRNA expression between groups. The chi-squared test was to determine relevance between clinicopathologic characteristics and SLC12A8 expression levels . The KapThe expression of the SLC12A8 gene was significantly increased in BC tissues identified by bioinformatics analysis and experiment determination. Overexpression of SLC12A8 was correlated with poor outcome as an unfavorable biomarker in BC. We also found that SLC12A8 was related to immune cell infiltration and positively correlated with crucial immune checkpoint molecules in BC. The pathway analysis indicated the mechanism of SLC12A8 involved in the tumorigenesis.The expression levels of SLC12A8 mRNA in normal and BC tissues were evaluated using BC gene expression profiles from TCGA-BLCA. The expression of SLC12A8 mRNA was significantly greater in BC tissues than in normal tissues , pathological stage (p < 0.001), histological grade (p < 0.001), N stage (p < 0.001) and M stage (p < 0.001) in TCGA-BLCA cohort. In the GSE13507 dataset, the expression of SLC12A8 was closely associated with age (p < 0.001), pathological grade (p < 0.001), T stage (p < 0.001), and N stage (p < 0.05) .Table 1.In TCGA-BLCA cohort, Cox univariable analysis showed pathological stage (hazard ratio (HR) = 1.7618, p < 0.001), T stage , N stage , M stage  and SLC12A8 expression  influenced overall survival. Multivariable Cox analysis showed that only SLC12A8 expression  independently influenced overall survival among BC patients . In the IHC was performed on 29 paired BC tissues and adjacent normal tissues to examine SLC12A8 protein expression levels further. Representative IHC staining image indicated that SLC12A8 was located in the cytoplasmic and plasma membrane. IHC scores suggested that levels of protein expression of SLC12A8 in BC tissues were significantly higher than in adjacent tissues . AccordiThe genes co-expressed with SLC12A8 were found in MEM and cBioPortal databases. There were 50 co-expressed genes of SLC12A8 in the MEM (P < 0.001) and cBioPortal databases. Spearman correlation coefficient > 0.4 was used as the criteria for inclusion (P < 0.001). The GO and KEGG analyses of 483 co-expressed genes after eliminating duplicates showed that they were enriched in extracellular matrix tissue, cell adhesion, inflammatory response, and signal transduction in terms of biological process . The co-The PPI network of SLC12A8 described by STRING to detect the interacting protein showed 11 nodes, 15 edges, and an average local clustering coefficient of 0.919 . CystatiWe used GSEA to identify associated genes enriched in response to SLC12A8 high expression based on TCGA-BLCA. The chemokine signaling pathway, cell adhesion molecules, ECM receptors, and the cancer pathway related to tumorigenesis, invasion, and metastasis were significantly enriched in the group of high SLC12A8 expression. GSEA analysis indicated that SLC12A8 might play an essential role in BC development .Figure 7The compositions of 22 immune cells in BC samples calculated by CIBERSORT were filtered using the criterion P < 0.05, and the filtered results of 11 normal samples and 238 tumor samples were displayed using a barplot . The sevThe effect of gene expression level on the potential efficacy of immunotherapy was further investigated. We found that the expression of SLC12A8 was positively correlated with the expression of immunotherapy-related markers, PDL-1 (p < 0.001), CTLA-4 (p < 0.001), LAG-3 (p < 0.001), TIM-3 (p < 0.001) and TIGIT (p < 0.001), stimulatory checkpoint molecules, GITR (p < 0.001), ICOS (p < 0.001) and CD27 (p < 0.001) . SpearmaDue to the lack of confirmed specific molecules, BC diagnosis and targeted therapy are greatly restricted . TherefoFurthermore, SLC transporters regulate energy metabolism by glucose uptake mediation and then affect cell proliferation and tumor microenvironment ,43. SLC1https://portals.broadinstitute.org/ccle). The expression of SLC12A8 protein was localized to the cell membrane. The staining of normal and low-grade tissues was almost negative, and the high-grade presented weak, moderate, or intense staining.In the present research, we used public cancer databases for data mining and found SLC12A8 mRNA overexpression in BC associated with poor prognosis. Real-time PCR further validated the finding in BC tissues and urinary tract cell lines. SLC12A8 mRNA expression was significantly elevated in BC tissues. However, the fold change of SLC12A8 mRNA in T24, UMUC3, and EJ-1 was negative, consistent with published data in Broad Institute Cancer Cell Line Encyclopedia (To further explore the possible mechanism of SLC12A8 in the progression of BC, we performed GO, KEGG, and PPI analyses. GO & KEGG pathway analysis and GSEA results revealed that SLC12A8 mainly affects tumor progression by acting on tumor-related pathways, including NF-kb, PI3K-Akt, and Toll-like receptors. Meanwhile, SLC12A8 may regulate ECM-receptor interaction, extracellular matrix organization, cell adhesion, and immunoglobulin binding to impact tumor microenvironment and immunity. Cystatin A, a protein that interacts with SLC12A8, is a putative tumor suppressor that modulates extracellular matrix remodeling, cell adhesion, tumor invasion, and metastasis ,46. By cThe tumor microenvironment plays an integral part in various processes of tumorigenesis as well as immune responses . In receIn addition, we found that the expression of SLC12A8 was significantly positively correlated with the expression of common immune checkpoint biomarkers, suggesting that SLC12A8 may inhibit the immune response by inducing the expression of immune checkpoint molecules. Some studies confirmed that high expression of immune checkpoint molecules is associated with a higher response rate. We speculate that the expression of SLC12A8 may h potentially predict the response of immune checkpoint therapy for BC.In summary, we proposed a prognostic model of SLC12A8 expression level and survival rate in patients with BC. We confirmed overexpression in BC tissues in many databases, clinical samples, and cell lines. More importantly, we first used IHC to analyze SLC12A8 protein expression in normal tissues and bladder tumors, providing evidence for SLC12A8 as a potential diagnostic marker for BC. Furthermore, we found that SLC12A8 may affect the tumor microenvironment and may be a potential marker of immunotherapy response. However, there are some limitations to our study. Our sample size was insufficient and lacked overall survival information. The molecular mechanism of SLC12A8 and research on how it activates the signaling pathway for BC development will be further explored. In addition, we will further investigate the value of SLC12A8 in immunology, such as the effect of SLC12A8 gene expression on immune checkpoint inhibitor response.This study confirmed that SLC12A8 was significantly overexpressed in clinical BC tissues using bioinformatics, real-time PCR, and IHC methods. The high expression of SLC12A8 is related to poor outcomes in BC. Subsequently, we found that SLC12A8 was associated with multiple tumor immune cell infiltration and positively correlated with immune checkpoint molecules in BC. SLC12A8 may be a potential biomarker and immunotherapy-related target.


Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely. Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in leaves, fruits, and barks, with phloridzin (phloretin 2′-O-glucoside)  to the dihydrochalcone phloretin -labelled substrates were synthesized as described previously -substrate  1.5–3 µL recombinant enzyme preparation, 5 µL NADPH (0.83 mg/mL H2O), and 55 µL 0.1 M Tris/HCl . The reaction mixture was incubated for 10 min at 25 °C. Thereafter, the reaction was stopped by mixing with 70 µL ethyl acetate and 10 µL 100% acetic acid. After centrifugation for 5 min at 10,000× g for phase separation, the organic phase was transferred to a precoated cellulose plate  and substrate and products were separated by thin-layer chromatography (TLC) in chloroform/acetic acid/H2O . The conversion rates were determined with a TLC linear analyzer . The optimized reaction conditions are summarized in Protein determination was performed by a modified Lowry procedure with crystalline BSA as the standard . Enzyme Malus x domestica flavonoid 3′-hydroxylase (MH468789)), CsCH3H  and CrCPR (NADPH-cytochrome P450 reductase from Catharanthus roseus (X69791)). The reaction mixtures contained in a final volume of 100 µL: 40 µL Saccharomyces cerevisiae INVSc1 microsomal preparation of recombinant produced enzyme, 1.55 mM NADPH, 10 µM substrate in 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5. The mixture was incubated for 30 min at 30 °C and the reaction was stopped with 20 µL of 80% acetonitrile/20% acetic acid. After centrifugation at 16,000× g for 5 min, the reaction solution was filtered through a 0.22 µM PTFE membrane.For LC-MS analysis, three recombinant enzymes were tested: MdF3′HII , equipped with a 1290 Infinity Binary Pump , a 1260 Infinity II Diode Array Detector HS , a 1290 Infinity II Multisampler , and a 1290 1290 Infinity II Multicolumn Thermostat .2O and eluent B was acetonitrile, both with 0.1% formic acid. Solvent gradient was as follows : %B: 0.00: 15%; 0.50: 15%; 8.50: 60%; 10.50: 98%; 15.50: 98%; 15.75: 15%; 19.00: 15%, (Post Run Time: 6 min for Equilibration).One µL of extract was injected onto a ZORBAX Eclipse Plus C18 Rapid Resolution column , with a length of 150 mm, an internal diameter of 2.1 mm and a particle size of 1.8 µm at a column temperature of 35 °C and a flow rate of 0.3 mL/min. Eluent A was Millipore™ Hm/z 100 to 1,000, product ion scan range from m/z 50 to 350, capillary voltage 3.5 kV for; gas temperature 350 °C; gas flow 10L/min, nebulizer 40 psi, sheath gas temperature 350 °C, sheath gas flow 12 L/min, fragmentor 180 V; skimmer 75 V. Nitrogen was used as nebulizer and auxiliary gas. Data acquisition was carried out using the Agilent Mass Hunter Workstation Data Acquisition  and evaluated using Agilent MassHunter Qualitative Analysis 10.0. Identifications were based on chromatographic elution time, Accurate Mass, MS/MS fragmentation pattern, and comparisons with available standards.After separation, dihydrochalcones were detected by the Agilent 1260 Infinity II Diode Array Detector HS at 287 nm with a bandwidth of 4 nm. Scanning range was 190–600 nm. Identification was performed using an Agilent High-Resolution-y MS 6545 Q-TOF with Electrospray Ion Source Dual AJS ESI, both supplied by the company Agilent . The main instrumental conditions were as follows: negative ionization mode, MS scan range was from Experiments for determination of kinetic parameters of the recombinant enzymes were performed by varying the substrate concentrations from 0.12 µM to 2.5 µM at a fixed concentration of 0.5 mM NADPH. The amounts of crude microsomal preparations used of MdF3′HI was 5 µg for naringenin, 3 µg for DHK and 1.5 µg for kaempferol and of MdF3′HII 3 µg for naringenin, 2 µg for DHK and 1.5 µg for kaempferol. Data analysis was carried out by nonlinear regression mean values, and standard deviations were calculated based on three repetitions. Calculations and graphs were carried out employing the program OriginPro 2018 (OriginLab).Our studies showed that F3′H from apple have a relatively narrow substrate specificity, as they accept, under in vitro conditions, only the most common substrate classes, flavanones, dihydroflavonols, and flavonols. This also confirms that F3′H from apple is not a suitable candidate for metabolic engineering of the dihydrochalcone pathway in microbial strains. On the other hand, the recent case of the gm-orange petunias clearly demonstrated that even if a compound is such a weak substrate, in vitro conversion at standard conditions is not observed, but accumulation of its converted metabolites can be nonetheless observed in a suitable physiological background .Malus F3′Hs and testing the effect on the levels of constitutively present 3-hydroxyphloridzin.In the case of F3′H, a competition with the glucosyltransferases for phloretin as a substrate would be decisive. A high conversion of phloretin into phloridzin, as becomes obvious from the fact that the majority of dihydrochalcones are present in apple in glucosylated form, corresponds well with the very low amounts of 3-hydroxyphloridzin present in apple, and the fact that usually the concentration of free phloretin is higher than that of 3-hydroxyphloridzin  could re


DYNC2H1 (OMIM: 603297) have been reported to cause SRTD3.Short-rib thoracic dysplasia 3 with or without polydactyly  is an autosomal recessive disorder. SRTD3 presents clinically with a narrow thorax, short ribs, shortened tubular bones, and acetabular roof abnormalities. Clinical signs of SRTD3 vary among individuals. Pathogenic variants of We performed a detailed clinical prenatal sonographic characterization of a foetus with SRTD3. Trio whole-exome sequencing was used to identify causative variants in the family. The identified variants in the families were validated by Sanger sequencing and mass spectrometry. Multiple computational tools were used to predict the harmfulness of the two variants. A minigene splicing assay was carried out to evaluate the impact of the splice-site variant.DYNC2H1 (NM_001377.3:c.11483T > G (p.Ile3828Arg) and c.2106 + 3A > T). Various statistical methods predicted that the variants would cause harmful effects on genes or gene products. The minigene assay findings suggested that c.2106 + 3A > T caused the skipping over exon 14, producing an exon 14 loss in the protein.We evaluated prenatal sonographic images of the foetus with SRTD3, including abnormal rib curvature, narrow thorax, bilateral hypoplastic lungs, bilateral polydactyly, syndactyly, and foetal visceral situs inversus with mirror-image dextrocardia. We revealed novel compound variants of DYNC2H1) variants, expanding the phenotypic spectrum of SRTD3. The minigene study of c.2106 + 3A > T was predicted to cause an inframe exclusion of exon 14, which was predicted to have important molecular functions. Our findings strongly supported the use of WES in prenatal diagnosis and helped to understand the correlation of genotype and phenotypes of DYNC2H1. The specific sonographic findings and the molecular diagnosis helped add experience to further our expertise in prenatal counselling for SRTD3.This study identified a foetus with SRTD3 with situs inversus totalis with mirror-image dextrocardia in a Chinese family, revealing two novel compound heterozygous dynein cytoplasmic 2 heavy chain 1 (The online version contains supplementary material available at 10.1186/s12920-022-01205-z. Short-rib thoracic dysplasia 3 with or without polydactyly  is an autosomal recessive disorder. SRTD3 presents clinically with a narrow thorax, short ribs, shortened tubular bones, and acetabular roof abnormalities . Some foA 22-year-old woman was referred to our department for routine sonographic examination at 22.4 gestational weeks (GW). Systematic prenatal sonographic examinations, including two- and three-dimensional ultrasound and echocardiography, were obtained to a final diagnosis by at least two chief physicians. GE Volusion E10 was used to perform the sonographic examination with a 1–5 MHz probe. These sonographic scans were all performed using a GE Volusion E10 machine with a 1–5 MHz sector transducer.Trio whole-exome sequencing (WES) of the family was performed at BGI company, Wuhan, China. In this method, genomic DNA was collected from blood from the parents and umbilical cord blood from the foetus. First, DNA was broken into 100- to 500-bp fragments. The 200- to 300-bp fragments were collected by magnetic beads . Base “A” was added at the 3' overhangs, which could pair the “T” base with a special adapter. After elution and postcapture amplification, the library was enriched for 16 h at 65 °C by array hybridization . The magnitude of enrichment of the product was estimated. The qualified products were pooled, quantified, and prepared for circularization. Finally, the MGISEQ-2000 sequencing platform was used for variant detection. The average sequencing depth of the target region was ≥ 180X, and the percentage of loci with an average depth of > 20X in the target region was > 95%. After the sequencing was completed, raw reads from the machine were evaluated for sequencing quality, and low-quality and joint-contaminated reads were removed for further analysis [https://phoenix.bgi.com/autocnv/). To predict the effects of missense variants, we used dbNSFP, which contains seven well-established in silico prediction programs , Polyphen2, LRT, Mutation Taster, and PhyloP) [Sequenced fragments were aligned with the UCSC hg19 human reference genome by BWA (Burrows Wheeler Aligner) to remove duplicates . Base qu PhyloP) . To predDYNC2H1 variant c.2106 + 3A > T (NM_001377.3) was validated by Sanger sequencing. Primers were designed upstream and downstream of the segment. Polymerase chain reaction (PCR) amplification and Sanger sequencing were performed using the forward primer TTTGCCAGACTGCCTTATCA and the reverse primer AGACTGAGGAGGGGAGGCTA. The DYNC2H1 c.11483T > G (NM_001377.3) validation was performed by using the MassARRAY system (Agena Bioscience company) according to a previously described method [To verify the results of gene chip capture and high throughput sequencing, the two suspected variants were validated by Sanger sequence and mass spectrometry. The DYNC2H1-AF, AAGCTTGGTACCGAGCTCGGATCCGTGGCACATTTTTATAATTCTATTGATC; DYNC2H1-AR, AAGAAGGCAGACAGCCATGGGGTTTCACCATGTTGG; DYNC2H1-BF, ATGGCTGTCTGCCTTCTTCATTCTTCCTTTCCG; DYNC2H1-BR, TTAAACGGGCCCTCTAGACTCGAGCTGTGCTTCTACAGTTGCTAAGCCAGTT. The correct wild-type and mutant Minigene plasmids were transfected into HEK293T cell line for 48 h. Total RNA was extracted, and cDNA synthesis was performed . Real-time PCR was performed using the primers F-GGCTAACTAGAGAACCCACTGCTTA and R-CTGTGCTTCTACAGTTGCTAAGC, and the products were sequenced.A genomic segment encompassing the variant c.2106 + 3A > T sequence along with flanking intronic sequences, including exon 13, part of intron 13–14, exon 14, intron 14–15 and exon 15, was PCR-amplified from patient genomic DNA. The segments containing the wild-type  and mutant-type  sequences were separately cloned into the minigene vector pMini-CopGFP . The following primers were used to construct the vectors using the ClonExpress® II One Step Cloning Kit : A 22-year-old woman was referred to our department for routine sonographic examination at an ultrasonic gestational age of 20.6 weeks and 22.4 weeks from the last menstrual period (LMP). This was the second pregnancy of the woman. She had an early pregnancy loss in her first pregnancy. The couple was healthy and non-consanguineous. The sonographic examination detected multiple anomalies. The measurement value (− 2 standard deviation (SD)/+ 2 SD) of the biparietal diameter (BPD) was 5.7 cm 4.9 cm/6.1 cm); the head circumference (HC) was 21.7 cm (18.3 cm/21.8 cm); the abdominal circumference (AC) was 16.7 cm (15.7 cm/19.6 cm), the femur length (FL) was 3.3 cm (3.3 cm/4.1 cm); and the humerus length (HL) was 2.5 cm (3.21 cm/4.05 cm), respectively. . The thoracoabdominal transverse diameter ratio was 0.66. The size of the left lung was 2.7 * 1.1 cm, and that of the right lung was 1.6 * 1.0 cm. The two foetal kidney collecting systems were separated by approximately 0.4 cm. Echoes of 6 toes were seen on the left foot. The first and second toes of the right foot were widely spaced, and the fourth and fifth toes appeared to have syndactyly. The tibial length was 2.7 cm (− 2.8 SD); the ulnar length was 2.6 cm (− 3.2 SD). The final US diagnosis was short-rib thoracic dysplasia , situs inversus totalis, and mirror-image dextrocardia. The parents chose to terminate the pregnancy due to multiple anomalies.DYNC2H1 gene associated with the subject's phenotype in SRTD3 were detected, NM_001377.3: c.11483T > G (p.Ile3828Arg) and c.2106 + 3A > T. We did not detect rare or likely pathogenic homozygous or heterozygous variants in other STRD-causing genes in family trio WES. No pathogenic chromosomal CNV variants above 1 M and LOH variants above 5 M associated with the phenotypes of the proband were detected through the analysis of the high-throughput data. c.11483T > G (p.Ile3828Arg) was detected in the father and the foetus in family trio WES. The c.11483T > G variant was located in exon 78 of the DYNC2H1 gene and caused the alteration of isoleucine to arginine at amino acid position 3828. c.11483T > G had frequencies of 0.000013 in GnomAD and 0.00019 in GnomAD-EAS (East Asian population). This variant was not found in the control population in the ESP, 1000, and EXAC databases. Various statistical methods, including conservative and evolutionary predictions, predicted that the variant would cause harmful effects on genes or gene products. Computer software tools, including SIFT, Mutation Taster, and Condel, predicted that predicted the missense variant was pathogenic. All three tools  showed that c.11483T > G was conserved (Table Two heterozygous variants of the A minigene expression assay revealed the synthesis of a messenger RNA lacking exon 14, which generated a predicted in-frame deleted protein. The agarose gel electrophoresis result is shown in Fig. DYNC2H1, DYNC2LI1, WDR19, WDR34, WDR35, WDR60, IFT140, TTC21B, TCTEX1D2, IFT80, IFT172, EVC1, EVC2, KIAA0586, CEP120, INTU, ICK, NEK1, and C21ORF2 [. Among the above genes, DYNC2H1 is the most common gene associated with SRTD3 disease. DYNC2H1 encodes a dynein protein that participates in ciliary intraflagellar transport [DYNC2H1 will lead to the dysfunction of primary cilia, causing a heterogeneous spectrum of conditions such as skeletal dysplasias [DYNC2H1 is the pathogenic gene of SRTD3 disease. SRTD3 disease is clinically and genetically heterogeneous. There is significant variability in the course and severity of the thoracic phenotype, both between siblings with the same DYNC2H1 allele and between individuals with different alleles, suggesting that the DYNC2H1 phenotype may be influenced by modified alleles and nongenetic or epigenetic factors [Many genes have been reported to be related to SRTD3, such as  C21ORF2 . Among tsplasias . DYNC2H1SRTD3 generally presents prenatally with extremely shortened long bones and ribs, round metaphyseal ends and lateral spikes, a small and narrow thorax, and preaxial and postaxial polydactyly, sometimes presenting with bowing femora, genitals and multiple malformations . Among aDYNC2H1 compound heterozygous variants of the autosomal recessive inheritance pattern were consistent with SRTD3 disease. The foetal manifestation accorded with the SRTD3. No rare or likely pathogenic homozygous or heterozygous variant in other STRD-causing genes was found in family trio WES. We performed a minigene splicing study and confirmed that the variant caused a skipping over exon 14. Though no pathogenic variants were reported in this region previously, exon 14 was predicted to participate in many GO processes such as molecular_function, protein binding, catalytic activity and so on by NetGO 2.0 [DYNC2H1 are highly likely the cause of the SRTD3 with polydactyly in this patient.The etGO 2.0 . AccordiDYNC2H1. These findings expanded the spectrum of DYNC2H1 variants which was more diverse and predicted important molecular functions of exon 14 of DYNC2H1. The specific sonographic findings and the molecular diagnosis helped add experience to further our expertise in prenatal counselling for SRTD3. Our findings strongly supported the use of WES in prenatal diagnosis and further helped to understand the correlation of genotype and phenotypes of DYNC2H1 variants. Next-generation sequencing was an effective method of prenatal diagnosis of a genetic skeletal disorder.Here we presented a foetus with abnormal rib curvature, narrow thorax, bilateral hypoplastic lungs, bilateral polydactyly, syndactyly and foetal visceral situs inversus with mirror-image dextrocardia presentations caused by compound heterozygous variants in Additional file 1: Figure S1. The original image of figure 3A.


These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103  •Epithelial colonization by gut cDC2s leads to their transcriptional reprograming•Unlike lamina propria cDC2s, intraepithelial cDC2s show an immature-like phenotype•Intraepithelial cDC2s trigger T cell hyporesponsiveness•The phenotype of intraepithelial cDC2s is imprinted by both retinoic acid and mucus Gut cDC2s have been described to migrate into the epithelium, but whether this event modifies their phenotype is unknown. Rivera et al. show that upon epithelium colonization, cDC2s adopt an immature-like phenotype, revealing the existence of subtissular niches able to shape cDCs' fate and function. Conventional dendritic cells (cDCs) were initially described for their capacity to patrol peripheral tissues and transport the antigens collected to lymph nodes for presentation to T lymphocytes. This process constitutes the first step of adaptive immune responses. The cDCs that reside in most peripheral tissues are in the so-called “immature stage”: they exhibit a high antigen internalization capacity and express low amounts of costimulatory molecules, proinflammatory cytokines, and the chemokine receptor CCR7 . Upon dehiMHCIIhiCD103+CD11b-) and cDC2s (CD11chiMHCIIhiCD103-CD11b+), differentiating from pre-cDC1 and pre-cDC2 precursors, respectively , their number being 50% reduced in the small intestine of mice that do not synthesize this metabolite  cells  cells . Whether+CD11b+ cDC2s can migrate from the LP into the epithelium of the small intestine , CD103 (Itgae), and CD11b (Itgam) were expressed similarly in all clusters  showed that cluster 2 was enriched for cytokine signaling genes , including genes from the interleukin-1 (IL-1) pathway   A. This p1,24e-3) B. This c1,24e-3) A and 2C, subtype ; yet, iter mouse  , antimicrobial peptide genes , and phagolysosome maturation-associated genes  were not enriched for proinflammatory genes A and 2B.ed genes G. They r+CD11b+ cDC2s, we exploited the divergences observed between both samples to generate single-cell trajectories. For this, we used a pseudotime trajectory algorithm  to form a transitional pool of CD103+CD11b+ cDC2s (cluster 2), which then either remained in the LP to become proinflammatory mature cDC2s (cluster 3) or moved to the epithelium to generate a pool of immature cDC2s (clusters 0-1)  pre-DCs and adoptively transferred them into syngeneic recipients. Mice used for pre-DC purification were inoculated beforehand with Flt3l-producing tumor cells to increase the size of their pre-DC compartment . Analysihat time E. Intraehat time F. Of nottransfer D. These ers 0-1) G.Pilra and Pilrb genes and Amica1  leukocyte transmigration through endothelia , namely paired receptors d Amica1 A and 4A d Amica1 , (2) act+CD11b+ cDC2s in Pilra−/− mice (Abi3 gene (subunit of the WAVE complex), which activates branched actin nucleation by Arp2/3, was enriched in clusters 0-1 (Abi3−/− being not available). As for Pilra, we found fewer intraepithelial DCs in the Arpc4−/− mice  was not as strongly upregulated as Abi3 in intraepithelial cDC2s , which preferentially accumulates in the upper region of the small intestine  transcription factor, which induces the expression of Aldh1a2, the enzyme responsible for ATRA production from retinal  inhibitor bisdiamine B. We fountestine . Neverthved mice F, their ved mice C. Of notin vitro strategy. We sorted LP cDC2s, treated them or not with ATRA, and let them migrate into microfabricated channels containing small constrictions (1.5–2 μm). For these experiments, we used LP cDC2s from green fluorescent protein (GFP)-tagged Myh9 mice to assess the effect of ATRA on the actin motor. We found that the percentage of cells able to migrate through constrictions was considerably increased by the treatment, suggesting that ATRA promoted their passage through small holes  transmigration through the basement membrane that separates the LP from the epithelium could per se shape the phenotype of cDC2s, analogously to what was proposed for DC differentiation from monocytes ; (2) thiotential . To testpression A. In conal cells A. These To gain an insight into the epithelial cues involved, we prepared gut organoids from the duodenum D. Incubaex vivo , which can be secreted by epithelial cells and endow mouse bone marrow DCs with anti-inflammatory properties ex vivo . Additio of CD86 F and S5C+CD11b+ cDC2s with mucus protein 2 (Muc2) decreased their CD86 surface expression but did not affect CCR7  or OVA peptide (OVAp), and co-cultured with OT-II T cells. We found that LP cDC2s activated T cells efficiently, as shown by the upregulation of CD69, T cell proliferation, and interleukin-2 (IL-2) production , which has been associated with the induction of tolerance . Of note+CD11b+ cDC2 pool that resides in the LP, which exhibits a “mature-like” proinflammatory phenotype and is able to migrate to lymph nodes (CCR7+), and (2) a CD103+CD11b+ cDC2 pool located within the epithelium, which displays an “immature-like” phenotype and tolerogenic properties.We here show that epithelial colonization of cDC2s in the small intestine under homeostatic conditions contributes to the functional diversification of this cDC subset by promoting the formation of two functionally distinct pools of cells: (1) a CD103+CD11b+ cDC2s, as LP cDC2s, relied on the presence of Flt-3 ligand and expressed the same amounts of DNGR1, suggesting that they might differentiate from common pre-cDC precursors. These precursor cells move into the small intestine from blood vessels that are located within the LP. This implies that the precursor of intraepithelial cDC2s must maneuver through the LP before reaching their niche inside the epithelium  , which occurs upon Foxp3+ Tregs transmigration into the epithelial cell layer  . Second,ll layer . In linell layer . Third, +CD11b+ cDC2s modified upon epithelial colonization? Our results identified ATRA as an important regulator of this process: ATRA stimulated actomyosin contractility, then promoted cDC2 transmigration and epithelial colonization, and at the same time, it acted as an environmental cue, shaping the phenotype of these cells. Once cDC2s have reached the epithelium, the mucus protein Muc2 can decrease the surface expression of CD86 while increasing the expression of CD209a, which might help their antigen capture function. These data suggest the involvement of distinct imprinting cues for gut intraepithelial cDC2s to acquire their immature phenotype, with some of them having additive effects. They strongly suggest that the complexity of tissue-resident cDC populations most likely results from the concerted action of multiple cues to which they have access by physically reaching specific subtissular niches. Identifying these niches and cues within different tissues will thus be essential to understand cDC functional diversification and develop effective strategies to manipulate these cells in a pathological context.How is the phenotype and function of CD103Salmonella can trigger CCR7 expression at the surface of intraepithelial cDC2s ex vivo .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ana-Maria Lennon-Duménil  until use. Pilra-/- mice  and kindly provided by Pierre Guermonprez. Ccr2-/- mice were kindly provided by Emmanuel Gautier. Ccr7gfp reporter mouse    were ori2+, Mg2+-free Phenol Red 1X HBSS; H4385 Sigma-Aldrich, St. Louis, MO, USA diluted in filtered H2O) in the presence of 1 mM DTT , 5mM EDTA (15575-038 from Invitrogen) and 15mM HEPES (15630-056 from Gibco) at 37°C for 30 min to recover epithelial fraction, and subsequently incubated with 1 mM EDTA in 5% FBS/ PBS at 37°C for 10 min. This was followed by incubation with 15mM Hepes in 1% FBS/PBS at room temperature for 7min without agitation. The supernatants containing intestinal epithelial fraction were collected and analyzed by flow cytometry. Isolated tissues were further digested using 0.15 mg/mL Liberase (054010200001 from Roche) and 0.1mg/mL DNAse1 (10104159001 from Roche) in HBSS at 37°C for 45min with magnetic agitation. Tissues were then homogenized, filtered on 100μm cell strainer, and washed in HBSS. Gradient was performed on both epithelial fraction and lamina propria in 44% and 67% fractions of Percoll  prepared in 10mM Hepes in HBSS. Single-cell suspensions were stained with mouse antibodies and analyzed by flow cytometry.For the preparation of single-intestinal-cell suspension the small intestines were extracted from mice by separation from the mesentery. Peyer patches were removed, and intestines were opened with scissors along the intestinal length, then washed in PBS. Next, intestinal tissues were incubated on a magnetic shaker in a complete medium , LSR II (BD) and FACSAria (BD), and analyzed using FlowJo software version 10. Percentage values were charted with Graphpad Prism version 8.Cells were stained in 2mM EDTA, 5%FBS in PBS. RALDH activity in individual cells was measured using an Aldefluor kit according to the manufacturer’s protocol. 7.5 μM of DEAB was added in different tubes at 37°C for 15 min as Aldefluor fluorescent baseline control. Intracellular staining of cytokines was performed with BD Cytofix/Cytoperm™ Solution Kit, after obtention of cells and incubation with PMA (10ng/ml), Ionomycin (1μg/ml) and Brefeldin A (1μg/ml), during 4h at 37°C/ 5% CO+CD11b+ dendritic cells were sorted from the lamina propria and epithelium of 2 pooled small intestines of C57BL/6J mice. Cellular suspensions were loaded on a 10X Chromium instrument (10X Genomics) according to the manufacturer’s protocol based on the 10X GEMCode proprietary technology. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3’ v2 Reagent Kit (10X Genomics) according to manufacturer’s protocol as described in CD103cellranger mkfastq (with default parameter) was applied to generate fastq files for the Read1 for cell barcode and UMI and Read2 for transcript. After, cellranger count (with default parameters) aligned the Read2 to the mouse reference genome mm10 using STAR (version 2.5.1) (Cell Ranger (version 2.0.1) (from 10x genomics) was used to process Chromium single cell 3’ v2 RNA-seq output files. First, n 2.5.1) . Furthern 2.5.1) . For theFindClusters function with the following parameters: dims.use = 1:20, resolution = c), n.iter = 10000, force.recalc = T; all other parameters are default settings). Clustree analysis  was used by changing the resolution parameters from 0 to 1.5. Finally, we kept a resolution parameter at 1.0 and we defined nine clusters. After controlling the expression of some quality control genes, we excluded the clusters with cells expressing Cd8a or Mafb and cells that do not express Itgam. These clusters of cells were considered as contaminating cells, then we finally analyzed five clusters.For dimensionality reduction analysis, we first identified 2634 genes as highly variable genes across the single cells . Then we performed PCA using the variable genes as input and determined 20 PCs as significant PCs. These principal components were used as input for t-Distributed Stochastic Neighbor Embedding (tSNE) . ClusterFindAllMarkers from Seurat package . Heatmap, feature plots and violins plot were performed using Seurat package.Cell specific marker genes were identified by comparing cells in a specific cluster with cells in all other clusters using differentialGeneTest from Monocle2 . Cells were ordered along the inferred trajectory to indicate their differentiation progress. Then, the pseudotime trajectory was visualized on the reduced dimensional space.To construct single cell pseudotime trajectory we applied Monocle2 (version 2.6.4)  using 35Pathway analyses were performed using Enrichr .www.genosplice.com) using RNA Velocity  at 4°C overnight, and dehydrated in 20% sucrose in PBS for 4h at room temperature. After washing twice with 40 mM NaH2PO4, 160 mM Na2HPO4 in H2O, samples were embedded with OCT in tissue cassettes, snap frozen using liquid nitrogen, and stored at -80°C. Samples were incubated in permeabilization buffer (1% Triton X100 in PBS) for 1h, then in blocking buffer  for 1h. Tissue staining was performed by incubating gut slices with primary antibodies overnight in 0,2% Tx100 in PBS (100μL/3slices), using the following dilutions: 1/50 for anti-CD103; 1/100 for all other antibodies diluted. Samples were washed 3 times 0,2%Tx100 in PBS for 1h (each wash), with mild shaking rocking. When required, samples were incubated with secondary antibodies overnight (both diluted 1/100) and then washed as described before. Samples were kept at room temperature during all steps before mounting with Aqua Polymount medium. Z-stacks consisting of 1024∗1024 pixels (150nm pixel size) images spaced by 0.35μm were acquired using an inverted confocal microscope  equipped with a 63X oil immersion objective, pixel size 1024∗1024, z-step 0,35mm.The small intestine was extracted and washed by flushing the lumen with cold Leibovitz’s L-15 medium . 5mm fragments from the small intestine were obtained and tissue was fixed in the fixative solution  and RT-qPCR was performed with SYBR Green Master mix after single-stranded cDNA obtention using the high capacity cDNA synthesis kit (Thermo Fisher) according to manufacturer’s protocol. Oligonucleotides primers were synthesized by Eurogentec. Defa24 primers were designed as from CD1036 B16 Flt3l-producing tumor cells were injected subcutaneously in WT CD45.2 mice and after 15-20 days, preDCs were sorted from BM. 6.5 x 105 CellTrace Violet proliferation dye (eBioscience) labeled cells were injected intravenously into CD45.1 recipient mice, and their differentiation dynamic was followed in small intestine after 4, 7 and 10 days post-transfer by flow cytometry. Total epithelium sample was acquired by flow cytometry for the small intestine epithelium, corresponding to around 3-3.5 million events per sample. For LP, 4 million of cells were acquired by sample, to ensure data robustness.PreDC transfer experiments were performed as previously described . Briefly+ cells were detected after 1, 4 or 10 post single injection by flow cytometry following manufacturer’s protocol.C57BL/6J mice were injected I.P. with 25mg/kg of EdU (baseclick) and EdUImmunoblotting was performed as previously described . BrieflyC57BL/6 were gavaged during 10 days with 200μL per day of PBS or 0.5mg/mL Fluconazole (F8929) in PBS or antibiotic cocktail in PBS of Ampicillin A9393 1mg/mL+ Gentamicin sulfate G4918 1mg/mL+ Vancomycin 861987 0,5mg/mL+ Metronidazole M1547 1mg/mL+ Neomycin trisulfate salt N1876 1mg/mL .Mice were gavaged for 2 days with 230μL olive oil or 0,1mg/kg of Bisdiamine  diluted in olive oil. RALDH activity was assessed by flow cytometry with the ALDEFLUOR kit (StemCell).Micro-channels were prepared as previously described . Polydim+CD11b+ DCs were added on the top part of 96 well 3μm-pore permeable supports Transwell® and let them transmigrate overnight at 37°C with 5% CO2 in supplemented RPMI medium . DCs were obtained from the upper and lower compartments and analyzed by flow cytometry.Sorted lamina propria CD103+CD11b+ dendritic cells were sorted from small intestine lamina propria of 4 pool C57BL/6J mice and incubated with freshly obtained small intestinal epithelial cells in a ratio 1:10, with intestinal epithelial cell supernatant or alone, overnight at 37°C with 5% CO2 in supplemented RPMI medium and analyzed by flow cytometry. ATRA (1nM), Bisdiamine (45 μM) or antiTGF-beta 10μg/ml were added at the moment of co-culture. For mucus experiments, sorted CD103+CD11b+ dendritic cells were incubated overnight in the presence or absence of 50 μg/ml porcine MUC2 (Sigma Aldrich) at 37°C with 5% CO2, and analyzed by flow cytometry.CD103+CD11b+ dendritic cells were added to the culture. ENR medium was changed by supplemented RPMI medium to ensure dendritic cells viability during co-culture. After overnight incubation, dendritic cells were obtained from the supernatant and from inside the Matrigel by disrupting the Matrigel by strong pipetting and incubation with Trypsin for 6min. Cells were after stained and analyzed by flow cytometry. For imaging, LP CD103+CD11b+ dendritic cells were sorted from CD11c-YFP reporter mice, co-cultured overnight with membrane-fluorescent (mTmG mice) derived organoids in 8 well ibidi chambers and imaged in live using an inverted confocal microscope  equipped with a 40X oil immersion objective.Mouse intestinal crypts from duodenum were isolated as described in +CD11b+ dendritic cells were sorted from the lamina propria and epithelium of 4 pooled small intestines of C57BL/6J mice. For antigen presentation assay, after isolation, cells were plated in round bottom 96 well plates and pre-incubated with different concentrations of Ovalbumin: 2 mg/mL, 1mg/mL and 0.5 g/mL, or OVA peptide II at 10 μg/mL for 5 hours at 37°C with 5% CO2. After extensive washing, DCs were incubated with CFSE-labeled OT-II T cells in a ratio of 1:10. OT-II T cell activation was analyzed 18 hours after, and after 3 days, proliferation was measured by flow cytometry. Supernatants were collected after 3 days and the concentration of IL2 was analyzed by Luminex. For antigen presentation assay with anti-ICOS-L antibodies, the antibody or isotype was added at the moment of OT-II – DC co-culture at 50 μg/ml. For re-activation of T cells, 1 day after DC-T cells co-culture, OT-II T cells were plated on anti CD3 coated 96 well plates, and anti CD28 antibody was added soluble. For antigen capture assay, after sorting, cells were incubated with fluorescent Ovalbumin Texas Red  at 37°C with 5% CO2 for 1 hour, washed and analyzed by flow cytometry; negative control was incubated at 0°C during the same time.CD103+CD11b+ dendritic cells were centrifuged at 700rpm for 5 minutes in the cytocentrifuge. Cells were stained with May-Grünwald dye for 3 minutes, washed with neutral water and stained for 20 minutes with Giemsa dye diluted at 15% in neutral water.Purified gut CD103∗p <.05, ∗∗p <.01, ∗∗∗p <.001, ∗∗∗∗ p < ,0001).Number of mice and experiments, and statistical tests are reported in each figure legend. Analyses were performed using GraphPad Prism 8 software. Statistical significance was calculated using t test (paired or unpaired) or Mann-Whitney, one-way ANOVA or Kruskal-Wallis test, or two-way ANOVA according to test requirements. Error bars represent SEM and p values <.05 were considered statistically significant (


A multiscale structure is realized through layer-by-layer deposition with atom-scale precision via atomic layer deposition2O3 exhibits record-high absorption properties in low-frequency bands.FeSiAl@ZnO@AlThe corrosion resistance is improved by the unique multistage oxide barriers.The online version contains supplementary material available at 10.1007/s40820-021-00678-4. 2O3 as a robust barrier to FeSiAl core is introduced to mitigate corrosion resistance. The FeSiAl@ZnO@Al2O3 layer by layer hybrid structure is realized with atomic-scale precision through the atomic layer deposition technique. Owing to the unique hybrid structure, the FeSiAl@ZnO@Al2O3 exhibits record-high microwave absorbing performance in low-frequency bands covering L and S bands with a minimum reflection loss (RLmin) of -50.6 dB at 3.4 GHz. Compared with pure FeSiAl , the RLmin value and effective bandwidth of this designed novel absorber increased up to ~ 3.7 and ~ 3 times, respectively. Furthermore, the inert ceramic dual-shells have improved 9.0 times the anti-corrosion property of FeSiAl core by multistage barriers towards corrosive medium and obstruction of the electric circuit. This is attributed to the large charge transfer resistance, increased impedance modulus |Z|0.01 Hz, and frequency time constant of FeSiAl@ZnO@Al2O3. The research demonstrates a promising platform toward the design of next-generation MAs with improved anti-corrosion properties.Developing highly efficient magnetic microwave absorbers (MAs) is crucial, and yet challenging for anti-corrosion properties in extremely humid and salt-induced foggy environments. Herein, a dual-oxide shell of ZnO/AlThe online version contains supplementary material available at 10.1007/s40820-021-00678-4. Since the investigation into microwave absorbers (MAs) in the 1940s, developing highly effective MAs with “thin, light, wide and strong” characteristics has been a major challenge so far to deal with electromagnetic pollution and realizing strength for military and civil purposes . General2O4/rGO@PVP composite with a minimum reflection loss (RLmin) value of -56.8 dB with a matching thickness of 1.96 mm and an effective absorption bandwidth  of 6.8 GHz [2, and TiO2 shells), which achieved the RLmin value of -58.2 dB at 10.4 GHz [2O3 functional oxides better MAs can be developed due to their dipole relaxation and dielectric behaviors [2O3 in effect with impedance matching [2O3-ZnO through atomic layer deposition (ALD) gives RLmin of –50 dB at 9.44 GHz, much better than that of Ni-Al2O3 nanowires [min of -57.1 dB at 7.8 GHz [3O4-rGO hybrid-architectures, Fe3O4@C nanorings, Co1–xS hollow spheres, and 3D Fe/C porous nanofibers for improving microwave absorption performances due to their dielectric and crystalline anisotropies [Furthermore, microwave absorption bandwidth and efficiency are limited by Rozanov (The minimum thickness of absorbers should not less than 1/17 of the wavelength corresponding to the minimum operating frequency) and Snoek’s limit , therefore, single ferrite magnetic MAs are difficult to possess excellent microwave absorption performance because of their poor impedance matching –14. Unti 6.8 GHz . Similar10.4 GHz . Moreoveehaviors . For insmatching . Where ianowires . In the  7.8 GHz . The othotropies –26. This2O3, ZnO, TiO2, ZrO2, and HfO2 films on the Cu surface and verified the anti-corrosion properties in 0.1 M NaCl electrolyte. The results confirmed that ceramic oxides can protect the copper metal from Cl- ions and O2, and H2O molecules [2O3, ZrO2), and magnetic absorbers  [Nevertheless, traditional magnetic MAs  have limitations in the complex corrosive and oxidative environments , 11. Theolecules . In the olecules . They reNiFe2O4) –33. It i2O3 anchored on FeSiAl surface was designed in a layer-by-layer fashion with atomic-scale precision through the ALD process to demonstrate a useful strategy for the fabrication of highly efficient MAs with anti-corrosion properties. The results showed that the as-obtained absorbers exhibited dielectric-magnetic hybrid structure, well-matched impedance, and enhanced microwave attenuation ability with record-high absorption properties in low frequencies covering L and S bands. Especially, the FSA@ZnO@Al2O3 gradient structure achieves a high RLmin value of -50.6 dB at 3.4 GHz, covering the relatively low-frequency bands. The RLmin and EAB value of the novel absorber increased up to ~ 3.7 and ~ 3 times, respectively, than bare FSA alloy. Moreover, the ZnO/Al2O3 dual-oxide-shell also possesses 9 times better corrosion protection than that of a single ZnO or Al2O3 single-oxide-shell in 5.0 wt% NaCl solution. This technique provides a promising way for the designing of next-generation MAs with improved anti-corrosion properties.In this work, a dual-oxide shell of ZnO/AlFeSiAl alloy (FSA) with particle size 2–4 µm shown in Fig. S1b was purchased from Changsha Tianjiu metal materials company (AR 99.99%).2 as a carrier gas, using diethyl zinc 2) and deionized H2O as the Zn and O source, respectively. DEZn and H2O were introduced into the reaction chamber with the sequence of ABAB about 250 cycles, the dose and purging time were 0.02 and 8 s, respectively. Subsequently, the Al2O3 shell was deposited using trimethylaluminum 3) as an Al source, following the same steps and conditions as for ZnO deposition. Finally, the collected sample was transferred to a furnace and annealed at 500 ℃ under an N2 atmosphere for about 2 h. The sample was named FSA@ZnO@Al2O3. The single ZnO and single Al2O3 shells were deposited on the FeSiAl at 150 °C with the similar conditions of FSA@ZnO@Al2O3 as a comparison, named as FSA@ZnO and FSA@Al2O3, respectively. In addition, FSA alloy was transferred to a furnace and annealed at 500 ℃ under an N2 atmosphere for about 2 h as a control sample, named FSA-500.Dual-oxide-shell encapsulated FSA was prepared by the ALD technique. FSA particles were dispersed in ethanol by sonicating for 1 min, then dispersed onto an alumina substrate and air dried. Afterwards, the ZnO layer was deposited on FSA at 150 ℃ with N2O3@ZnO gradient structure were scanned by X-ray photoelectron spectroscopy (XPS). Besides, the magnetic properties of FSA and as-prepared materials were characterized using the physical property measurement system (PPMS) at room temperature. The related electromagnetic parameters were measured by an N5234A vector network analyzer in 0.5–18 GHz. The electrochemical behaviors of those samples were characterized using an electrochemical workstation in 5.0 wt% NaCl solution with a typical three-electrode system . The testing working electrodes were fabricated by filling the absorbers into polyvinylidene difluoride (PVDF) binder with a mass ratio of 4:1 in the form of plate-like samples. The open-circuit potential (OCP) was measured for ~ 1600 s to achieve a balanced state of the electrochemical systems, then the Tafel test and electrochemical impedance spectroscopy (EIS) were tested. The Tafel test was conducted in -500–500 mV vs. OCP with a scan rate of 1 mV s−1. The EIS measurements were carried out at OCP over 105–10−2 Hz with 5 mV perturbation potential. Also, the recorded data were fitted through the ZSimpWin software to achieve corresponding corrosion parameters.X-ray diffraction (XRD) was used to identify the crystal structure of FSA and FSA-based absorbers. The morphology, microstructure, and elements distribution were measured using field-emission scanning electron microscopy  and high-resolution transmission electron microscopy . The surface compositions and element valence state of FSA@Al2O3 core–shell hybrid is synthesized using ALD approach as described schematically in Fig. 2O3 core–shell structure is displayed in Fig. 2O3 shell with an average thickness of ~ 25.5 nm over the FSA core. The lattice spacings of 0.28 and 0.45 nm were observed in HREM analysis corresponds to (022) and (111) planes of Al2O3 shell, respectively [2O3 gradient structure having an average thickness of ~ 40.1 nm for ZnO/Al2O3 dual-oxide-shell as shown in Fig. 2O3 as shown in Fig. 2O3 shells was observed, assuring the construction of a very fine heterojunction. The elemental maps of FSA@ZnO@Al2O3 are displayed in Figs. 2O3 in Fig. S3 and Table S1 revealed the low content of ZnO and Al2O3. Overall, the FSA@ZnO@Al2O3 gradient structure is constituted of inner FSA core, middle ZnO shell, and outer Al2O3 shell, forming a distinctive coaxial multi-interface layer-by-layer structure.The FSA@ZnO@Alectively . Similar022) and 11 planesectively . Finally3 superlattice structure [2O3 and ZnO are not found, which might be due to their lower content and beyond the detection limit of XRD. Therefore, XPS measurements were used to identify the existence of ZnO and Al2O3 in FSA@Al2O3@ZnO hybrid structure. The high-resolution Zn 2p showed a single Zn 2p3/2 peak at 1021.3 eV, corresponding to the Zn–O bond of the stoichiometric ZnO, evident in the formation of ZnO on the surface of FSA alloy  structure, with peaks located at 31.4°), (44.9°), (53.3°), (55.9°), (65.5°), and (83.1°) are corresponding to bcc B2 phase (200), (220), (311), (222), (400), and (422) planes, respectively, according to the JCPDS No. 45–1206, presented in Figs. .4°,  might be due to smaller size [c values among different samples might be due to the decrease of shape anisotropy, appropriate particle/crystal size, and the enhancement of crystallinity of FeSiAl components induced by the thermal treatment process [r = ε′- iε′′) and permeability (μr = μ′- iμ′′) in the frequency range of 0.5–18 GHz and layer thickness of 0.5–5.0 mm based on the transmission line theory [Further, the M-H curves show an S-like shape with apparent coercivity and remanence, which proved well ferromagnetic behavior of hybrid . The Ms values of FSA, FSA-500, FSA@Altructure . The Hc ler size . In addi process . To evale theory .2O3 gradient structure as depicted in three-dimensional (3D) RL maps  by adjusting the thickness from 1.5–5.0 mm , displayed in Fig. 2O3 shells. The potentiodynamic polarization curves are tested, which directly provide the stability behavior of FSA-based absorbers exposed to the corrosive solution. Figure Ecorr)) and corrosion dynamic kinetics (corrosion current (Icorr)) [2O3, FSA@ZnO, and FSA@ZnO@Al2O3 showed Ecorr values of -0.1783, -0.0628, -0.0263, and 0.0191 V, respectively. It can be found that ZnO, Al2O3, and ZnO/Al2O3 dual-oxide shells brought more positive potential than single FSA, suggesting that surface protection helps in preventing corrosion, however, the best results are only possible when both oxides are applied in conjunction. Further, the anodic current density of metal dissolution/corrosion rate of FSA@Al2O3, FSA@ZnO, and FSA@ZnO@Al2O3 is at least 1.0 orders of magnitude lower than that of pure FSA, indicating the FSA core is well protected by the oxide shells. Figure 0.01 Hz value is usually considered a semi-quantitative indicator to evaluate the corrosion protection performance of shells [0.01 Hz values of FSA@ZnO@Al2O3 are the highest (double the bare FSA) values among the four samples, indicating the good barrier capability of the dual-oxide shell. Furthermore, the time constant in Bode plots is also a significant parameter to analyze the anti-corrosion properties, which is related to different electrochemical processes. It is generally accepted that the high-frequency time constant is regarded as the charge transfer process at the interface between shell and electrolyte, the medium constant is considered to be the hydroxide layer/metal electrode interface, and the low-frequency time constant represents metal electrode/electrolyte interface [2O3 dual-oxide-shell in 5.0 wt% of NaCl solution.The electrochemical measurements  are carried out to investigate the corrosion protection role of the ZnO or Al(Icorr)) . The FSAf shells , 55. Thenterface , 57. Forr and μr. As can be observed in Fig. 2O3) > ε′ (FSA@ZnO) > ε′ (FSA@Al2O3) > ε′ (FSA). Meanwhile, the ε′′ also demonstrates a similar trend with that of ε′-f curves shown in Fig. 2O3 gradient structure, which might result from interfacial polarization related to the existence of ZnO, Al2O3, and FSA alloy in the hybrid [E value (tanδE = ε′′/ε′) of FSA@ZnO@Al2O3 gradient structure is the highest among the four samples in 0.5–18 GHz, as shown in Fig. 3 phase under heat treatment. Thus, the well-ordered structure DO3 possesses higher saturation magnetization than that of A2 and B2 phases. These findings are well-supported by the XRD results, where the as-prepared FSA@Al2O3, FSA@ZnO, and FSA@ZnO@Al2O3 present an obvious diffraction peak of DO3 (111) compared to FSA, indicating the presence of a higher magnetic ordered phase of FSA-based hybrids. On the other hand, the highly ordered DO3 phase is contributed to acquiring stronger magnetic behavior [M) of the complex permeability for FSA and FSA-based absorbers, the tan δM values of FSA@Al2O3 and FSA@ZnO are close to that of FSA. While the tan δM values of FSA@ZnO@Al2O3 gradient structure are smaller than that of FSA due to a certain amount of non-magnetic substance in the hybrid structure.To understand the microwave absorption mechanism, electromagnetic parameters are important evidence to reflect the intrinsic properties of absorbers, including εe hybrid , 58. Meae hybrid . In addie hybrid , 60. Thebehavior . Figure 2O3 also shows similar results with bare FSA, displayed in Fig. 2O3 are improved in L and S bands  means better impedance matching characteristics. As shown in Fig.  means be2O3 dual-oxide-shell could improve the attenuation capability. To further study the attenuation mechanism of microwave absorption property, dielectric and magnetic loss are also investigated in detail. Generally, the magnetic loss mechanism is attributed to the eddy current effect, exchange resonance and natural resonance [0 = μ"(μ')−2f−1≈2πμ0δd2/3, where δ is electric conductivity, d is the matching thickness [−2f−1 vs frequency should be kept constant in a specific frequency range or the whole frequency range of 0.5–18 GHz. Figure −2f−1 for FSA@ZnO@Al2O3 keep constant in 6.6–8.8 GHz and 10.1–12.8 GHz, demonstrating that the eddy current loss mechanism makes a greater contribution in the gradient structure. Besides, the peaks located at 13.0 GHz are related to exchange resonance, which usually occurs at a high-frequency range [2O3 gradient structure in low frequency covering L and S bands.The attenuation characteristic is also a key factor to evaluate microwave absorption performance, making sure that the electromagnetic wave entering the absorber is rapidly attenuated. The equation of attenuation constant α is given in Note S1 and the results are presented in Fig. esonance . The eddhickness . Accordicy range . Combine2O3/wax composites. Moreover, after the deposition of ZnO and ZnO/Al2O3, as shown in Fig. 2O3/wax systems, such as electron polarization, interfacial polarization and Debye relaxation [2O3, and FSA@ZnO@Al2O3-wax hybrids result in Maxwell–Wagner effect or interfacial polarization [2O3 shells. Similarly, electron polarization also contributes to the synergistic effect. Therefore, based on the free-electron theory 2O3 shells with good crystal structure result in a large dielectric loss, as shown in Fig. Dielectric loss property is another significant mechanism for microwave dissipation, which is usually investigated by Debye dipolar relaxation theory , 64. Baslaxation , 65. Therization , 61, 66. S bands .min value usually takes place when the thickness (dm) and the matching frequency (fm) obey the equation of m results are in good agreement with the simulated peak frequency using the quarter-wavelength (λ/4) cancellation model, demonstrating that the quarter-wavelength cancellation is also contributing to achieving excellent microwave absorption performance.Besides dielectric and magnetic loss mechanisms, the quarter-wavelength matching model should not be ignored to explain the microwave absorption properties. According to the quarter-wavelength cancellation model, the RL1,3,5,…) . The ref2O3, FSA@ZnO@Al2O3 are -0.01, 0.05, 0.03, and 0.12 V, respectively; a clear shift toward the direction of positive potential for FSA@ZnO@Al2O3 gradient structure. These results suggested that FSA-based absorbers are more inert than bare FSA in the NaCl solution, i.e., oxide shells form a very good anti-corrosion resistant barrier on FSA particles, especially ZnO/Al2O3 dual-oxide shell. The Icorr values for pure FSA, FSA@Al2O3, FSA@ZnO@Al2O3 are 8.196, 1.653, 0.8275, and 0.8351 μA cm−2, respectively, which demonstrate that the corrosion rate decreases clearly, displayed in Fig. Rct shows an increasing trend, indicating the enhanced protection for the dual-oxide shell, as shown in Fig. −1 of the formula weight, n is 2 of the chemical valency for Fe element, ρ is 7.86 g cm−3 of the density of Fe, F is 26.8 Ah of the Faraday constant. Figure 2O3 shows an obvious decline compared to pure FSA, revealing that its long-term corrosion protection. Generally, the porosity and the shielding-increase coefficient (β) are the significant parameters to evaluate the barrier effect. The porosity =G0.01 Hz/|Z|O0.01 Hz . The bigger the β values, the better the barrier effect of ceramic oxide [2O3 gradient structure is the highest among samples, suggesting that it possessed an excellent barrier effect to prevent FSA core from corrosion. Therefore, the high density and low porosity of ZnO/Al2O3 dual-oxide-shell could provide a good physical shielding effect to protect FSA alloy from corrosive media, such as Cl−, H2O, and O2, as shown in Fig. Additionally, to balance the rate of anodic and cathodic polarization, the OCP curves were measured in 5.0 wt% NaCl solution for ~ 25–26 min, as shown in Fig. S10. It is found that the OCP values after 25 min for pure FSA, FSA@AlρF×87600 . Where M2O3 gradient structure in a layer-by-layer fashion with atom-scale precision as an outstanding anti-corrosive microwave absorber through ALD technology. The as-synthesized gradient structure possesses a much lower RLmin and wider effective absorption bands than pristine FSA in low frequencies covering L and S bands. The RLmin of FSA@ZnO@Al2O3 gradient structure is as low as -50.6 dB, which is ~ 4 times lower than that of pristine FSA, and the effective absorption bands are 3 times wider than that of pristine FSA. The enhanced microwave absorption is attributed to dielectric loss from ZnO/Al2O3 dual-oxide-shells, interface relaxation, and Debye relaxation, magnetic loss from the inner FSA with DO3 superlattice structure, and the excellent impedance matching along with high attenuation characteristic. Moreover, the corrosion potential moves to 0.0191 V, the anodic current density is at least 1.0 orders of magnitude lower than that of pristine FSA and the corrosion rate decrease by about 9.0 times, confirming that the dual-oxide-shells are contributed to blocking the penetration of H2O, O2, and Cl− into the FSA/ceramic layers interface. It is believed that the FSA@ZnO@Al2O3 gradient structure would be a promising material for the next-generation microwave absorbers with enhanced anti-corrosion properties.Here, we have designed a FSA@ZnO@AlSupplementary file1 (DOCX 4041 kb)Below is the link to the electronic supplementary material.


Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. (2) Methods: To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed that combines metabolic labeling  with crosswise N-terminal dimethylation . (3) Results: The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion scores. (4) Conclusions: The quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL. Liquid chromatography coupled to mass spectrometry (LC–MS) is the common tool for the analysis of proteins in complex mixtures due to major improvements in LC–MS during the last few decades . MS-base2H, 13C, or 15N-enriched amino acids early in the experimental workflow during cell culture, which results in a robust and precise method enabling whole-proteome labeling and thus the quantification of living cells ) and Arg-d4 (L-arginine [2H4]) . Cells were harvested after reaching a density of 1 × 106 cells/mL, resuspended in 1 mL PBS, and centrifuged at 200 g. Cell pellets were frozen at −80 °C.If not stated differently, all chemicals were purchased from Merck Life Science AS/Sigma Aldrich, Oslo, Norway AS. The Jurkat T-cell line E6 was maintained in RPMI tissue culture medium  supplemented with 10% fetal calf serum and 200 mg/L proline and maintained in a humid incubator at 37 °C and 5% COg for 20 min at 4 °C in a Heraeus Biofuge pico  and the supernatant was divided in 20 μL aliquots. An aliquot was precipitated with 80 µL 100% ice-cold acetone over night at −20 °C. After centrifugation at 16,000× g for 20 min at 4 °C, the supernatant was discarded, and the precipitate was washed three times with acetone. The pellet was dissolved in 50 µL 6 M urea, and DTT was added to a final concentration of 10 mM, mixed thoroughly and incubated at 37 °C for 30 min using a Thermomixer . The reduction was followed by alkylation using iodoacetamide to a final concentration of 25 mM, incubated for 60 min in the dark at room temperature. Excess alkylation reagent was quenched by adding DTT to a final concentration of 30 mM and left for 30 min at 37 °C. For digestion, 240 µL 50 mM ammonium bicarbonate buffer and Trypsin/LysC  with an enzyme to protein ratio of 1:200 were added and incubated for 18 h at 37 °C. The digestion was stopped by adding formic acid to a final concentration of 1%. The tryptic peptides were purified by solid phase extraction using Strata C18-E SPE cartridge  and evaporated to dryness using a Speed Vac concentrator . The Strata C18-E SPE cartridges were activated using 500 µL acetonitrile and equilibrated with three times 500 µL 1% formic acid in water. The sample was loaded by passing through the column material three times. The column was washed using 500 µL water and the peptides were eluted with 500 µL 50% acetonitrile.Cell pellets were thawed and 800 µL cell lysis buffer  was added, followed by homogenization and mechanical breakage with a blue pestle. The samples were centrifuged at 16,000× The frozen, dried, and purified tryptic digest was thawed and dissolved in 40 µL 1% acetic acid, pH 2.7, and vortexed for about 5 min. A total of 2 µL 4% formaldehyde or formaldehyde-d2 in water and 2 µL sodium cyanoborohydride were added to a final concentration of 30 mM, mixed thoroughly, and left for incubation for 30 min. Subsequently, 8 µL of 1% ammonia hydroxide was added, vortexed, and incubated for 1 min. The reaction was stopped by adding 1 µL 5% formic acid. The tryptic peptides were purified with 10 µL OMIX-C18 Tips and dried using a Speed Vac concentrator. The OMIX-C18 Tips were activated using 50 µL acetonitrile and equilibrated with three times 50 µL 1% formic acid in water. The sample was loaded by passing through the column material three times. The column was washed using 50 µL of water and eluted with 10 µL 70% acetonitrile.m/z 400 to 2000) were acquired in the Orbitrap with a resolution of 70,000 at m/z 200 . Top ten most intense ions were isolated and fragmented in the HCD cell using high-energy collision dissociation (HCD) and a target value of 1 × 105 and an injection time of 100 ms. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS1 and MS2 acquisition. Target ions already selected for MS2 were dynamically excluded for 15 s. General mass spectrometry conditions were as follows: electrospray voltage, 2.0 kV; no sheath and auxiliary gas flow; heated capillary temperature of 275 °C; normalized HCD collision energy 28%. Ion selection threshold was set to 10,000 counts, and an isolation width of 2.0 Da was used.For LC–MS/MS analysis of the Jurkat T cell proteome, the dried peptides were dissolved in 10 µL 1% formic acid in water. For LFQ, 10 µL of the control was taken to be measured in eight replicates. For IPTL, 5 µL of control  and 5 µL of SILAC sample  were crosswise N-terminally dimethylated , and the isobaric peptides were mixed and measured in eight replicates. The samples were injected into a Dionex Ultimate 3000 nano-UHPLC system  connected to a quadrupole-Orbitrap (QExactive) mass spectrometer . For ionization, a nano electrospray ion source was used and the UHPLC was equipped with an Acclaim PepMap 100 column  . The flow rate was 0.3 µL/min with a solvent gradient of 4–35% B in 180 min. Solvent A was 0.1% formic acid and solvent B was 0.1% formic acid/90% acetonitrile. The mass spectrometer was operated in data-dependent acquisition and switched automatically between MS1 and MS2 acquisition. Survey full-scan MS1 spectra  files using ProteoWizard Analysis 3.0.331 applying peak picking: 2, filter: MS2 deisotope, filter: MS2 denoise 6, 100. For protein identifications, PEAKS Studio X+ and Mascot 2.4 were used. For relative quantification, LFQ samples were quantified using PEAKS Studio X+, whereas Mascot 2.4 in combination with IsobariQ version 2.0a was applied to IPTL data . General−5, charge: 2–5, peptide ID count per group ≥ 1 in at least 2 samples per group, significance ≥ 0, and significance method: ANOVA with at least 2 peptides. Ten internal standard proteins were used for normalization. For quantification of IPTL data using IsobariQ, the Mascot dat file was used with the following parameters: ignore peptide assignments with ion score below 20, peptide scoring standard for identification and minimal number of quantified peptides (PSMs) per protein: 4, require all labeling schemas, require bold red, and use unique and razor peptide for quantification. Normalization in IsobariQ was performed by division by median in utilizing the ratios of all peptides (reporter ions) or quant points.For quantification of LFQ data using PEAKS, the following parameters were applied: quality ≥ 4, average area ≥ 1 × 10