T2: 3.69 l (3.00|4.03), p < 0.01). Moreover, there was a significant decrease in the biomarker human epididymis protein 4 (T1: 6.2 ng/ml (4.6|6.3) vs. T2: 3.0 ng/ml (2.2|3.7), p < 0.01) and a small but significant decrease in matrix metallopeptidase 9 (T1: 45.5 ng/ml (32.5|140.1) vs. T2: 28.2 ng/ml (18.2|33.6), p < 0.05). Neutrophil phenotype  and function  remained largely unaffected by ETI treatment. Likewise, monocyte phenotype and markers of platelet activation were similar at T1 and T2. In summary, the present study confirmed a positive impact on patients with CF after ETI treatment. However, neither beneficial nor harmful effects of ETI treatment on cellular innate immunity could be detected, possibly due to the study population consisting of patients with well-controlled CF.Cystic fibrosis (CF) is a monogenetic disease caused by an impairment of the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organs and is associated with acute and chronic inflammation. In 2020, Elexacaftor–Tezacaftor–Ivacaftor (ETI) was approved to enhance and restore the remaining CFTR functionality. This study investigates cellular innate immunity, with a focus on neutrophil activation and phenotype, comparing healthy volunteers with patients with CF before  and after six months  of ETI treatment. ETI treatment reduced sweat chloride (T1: 95 mmol/l (83|108) vs. T2: 32 mmol/l (25|62), p < 0.01, median, first|third quartile) and significantly improved pulmonal function (FEV CFTR isWhile the initial treatment focused on symptomatic intervention, for example, by assisting expectoration, nutritional supplementation, and antibiotic treatment of chronic and/or exacerbated infections, modern treatments also aim to directly restore CFTR function , 4. Depe1), pulmonary exacerbations, sweat chloride concentration, and body mass index (BMI = kg/m²)  was developed . The nex= kg/m²) , 9. ETI = kg/m²) . Current= kg/m²) .Sustained inflammation plays a critical role in CF lung disease, which is predominantly neutrophil driven but also promoted by monocytes and platelets , 13. RecNeutrophils, monocytes, and platelets can become activated by a variety of mediators of inflammation, for example, cytokines such as tumor necrosis factor (TNF), lipid-derived mediators such as platelet-activating factor (PAF), and microbe-associated molecular patterns  or lipopolysaccharide (LPS)), and others –23. UponIn summary, it remains a matter for debate whether dysregulation of innate immunity in CF is acquired or constitutive  and whet22.1All experiments were performed in accordance with the Helsinki declaration , after eBlood was drawn by peripheral venipuncture in adherence to the guidelines of the World Health Organization  and coll++  adjusted to pH 7.3. The total volume of blood and PBS including stimuli and staining reagents cumulated to 50 µL. Blood was stimulated with PBS as buffer control, 1 µM PAF , 100 ng/ml LPS from Escherichia coli , 1 µg/mL LPS from Pseudomonas aeruginosa , or a mixture of inflammatory mediators (hereafter referred to as the mixture of proinflammatory mediators or Cocktail in the figures) consisting of 1 µM PAF, 10 µM fMLF , and 2.3 µM TNF  as indicated in the figure captions. Subsequently, the cells were stained, chemically fixed, and measured as described below. PAF, LPS, fMLF, and TNF were chosen as commonly used and clinically relevant stimuli of cellular innate immunity , matrix metallopeptidase 9 , and human epididymal protein 4 , DuoSet® Human HE4/WFDC2 ELISA Kit, #DY6274-05, R&D Systems) was carried out using standard enzyme-linked immunosorbent assays (ELISAs) as indicated by the manufacturers.Citrate anticoagulated blood was centrifuged for 10 minutes at 400 × g. The sampled plasma was stored at −80°C until further use for the analysis of humoral markers of inflammation. Measurement of plasma levels of interleukin 6 , interleukin 8 , anti-CD11b , anti-CD62L , anti-CD66b , or corresponding isotype controls . In addition, the diluted whole blood was stimulated with either PBS++ , PAF, LPS or with the mixture of proinflammatory mediators described above.For the analysis of the neutrophil and monocyte phenotype as previously described , 22, 10 ++ adjusted to pH 7.3 (including prior added stimuli as listed above and fluorescent reagents as subsequently listed) and incubated for 30 minutes at 37°C in a light-protected water bath. Phagocytosis was analyzed using fluorescent microspheres . The microspheres were dissolved 1:10 in PBS++ followed by a washing procedure (3 × at 4000 × g for 5 minutes). Of this microsphere solution, 5 µl was added to the above-mentioned mixture resulting in a total volume of 50 µl. Radical oxygen species (ROS) generation was determined by adding 5 µM CellROX Deep Red . Following stimulation and staining of diluted whole blood as described above, erythrocytes were lysed and leukocytes fixed in a sample volume made up to 1 mL with 1 × BD FACS lysing solution  for 30 minutes and incubated at room temperature in the dark. Following centrifugation of the samples for 5 minutes at 340 × g, the pellet was resuspended in 100 µl PBS++ containing 0.1% bovine serum albumin (Sigma Aldrich) and stored at room temperature in the dark until further analysis.Similarly, to assess neutrophil activity, 10 µl heparin-anticoagulated blood were added to 40 µl PBSi) was monitored by using fluorescent dyes as described before  including anti-CD45 , 50 nM bis trimethine oxonol (DiBAC4(3), #D8189, Sigma Aldrich, for MP), and 2.4 µM SNARF 5-(and-6)-carboxy-SNARF-1 . After 10 min of incubation in the dark at room temperature, the diluted blood was mixed with 950 µl Hanks’ Balanced Salt Solution  adjusted to pH 7.3 including 50 nM DiBAC4(3) and transferred to a light-protected water bath at 37°C. After a resting period of 2 min, neutrophils were stimulated with either PBS++ (as buffer control), PAF or with the mixture of proinflammatory mediators described above. After exclusion of erythrocytes as CD45 negative cells, neutrophils were identified as described below. An increase in DiBAC4(3) indicates depolarization, and a decrease in PE/PerCP ratio in SNARF indicates alkalization, respectively  and intracellular pH  area versus the height. Neutrophils and monocytes were identified on the basis of their forward and side scatter (SSC) area properties. The spillover between the fluorescence channels was corrected by a compensation matrix. For all antigens, appropriate isotype controls and single staining controls were performed (data not shown). For all experiments, a minimum of 3000 neutrophils and 500 monocytes were recorded using a BD FACSLyric (BD Biosciences). The gating strategy for the analysis of neutrophils and monocytes is illustrated in 2.4++ adjusted to pH 7.3. Hereafter, 10 µl of this diluted whole blood was added to 40 µl PBS++ adjusted to pH 7.3 including prior added stimuli  and antibodies to CD61  and CD62P (FITC anti-human CD62P (P-Selectin), dilution 1:25, #304904, BioLegend). Following incubation for 10 minutes in a light-protected water bath at 37°C, 950 µl HBSS++ were added to the sample followed by immediate flow cytometry analysis. Platelets were identified by the properties of FSC, SSC, and CD61 expression. The gating strategy for the analysis of thrombocytes is summarized in For the brief analysis of platelet activation, 50 µl citrate-anticoagulated blood was diluted with 562.5 µl HBSS2.5++ adjusted to pH 7.3 and stimulated with either PBS++ as buffer control or 1 µM PAF. Blood smears were stained with the “Hemacolor Rapid staining of blood smear - staining set for microscopy” . For each sample, a minimum of 50 neutrophils per specimen were analyzed by two independent and blinded individuals. Each neutrophil with at least one thrombocyte in direct juxtaposition was counted as a PNC. Representative PNCs identified by light microscopy are shown in PNCs were analyzed by light microscopy and flow cytometry as previously described , 33, 37.2.66 cells/ml were suspended in HBSS++ adjusted to pH 7.3. Neutrophils were stained with the fluorescent dye BCECF  for 30 minutes at 37°C, subsequently centrifuged for 5 minutes (340 × g) and resuspended in HBSS++ + 0.1% BSA. A total of 33 µl chemoattractant PAF  or a mixture of PAF, fMLF, and TNF  was added to the wells of the lower plate. Subsequently, a silicone gasket and a framed filter with 3 µm pores (Neuro Probe) were placed on the lower wells. On top of the filter and the gasket, the upper plate was attached and the stained neutrophils were pipetted into the corresponding wells. During incubation for 30 minutes at 37°C, neutrophils migrated from the upper wells towards the lower wells containing the inflammatory stimuli, but became adherent to the filter, resulting in increased fluorescence. The fluorescence of the cells in the filter was determined at a wavelength of 485/538 nm using a Fluoroskan Ascent (Thermo Fisher Scientific) with Ascent Software Version 6.0.2.Polymorphonuclear granulocytes mainly consisting of neutrophils were isolated by Ficoll-Paque  density gradient centrifugation and subsequent dextran sedimentation followed by hypotonic lysis of the remaining erythrocytes, as previously described , 34, 36.2.7www.bflow.science, last accessed 28th February 2023). All data is presented as medians with bars indicating the interquartile range, for example, median (25th percentile|75th percentile). The flow cytometry data including of neutrophils, monocytes, and platelets were further analyzed using the custom-written, python-based flow cytometry analytics software “BFlow”  vs. T2: 26 U/L (23|32), p = 0.82), ALT (T1: 23 U/L (15|27) vs. T2: 31 U/L (23|43), p = 0.09), and creatinine values (T1: 63 U/L (54|74) vs. T2: 63 U/L (49|78), p = 0.75). 3.3i upon stimulation were similar comparing HV and patients with CF and remained unaffected by ETI treatment (Innate immunity was monitored by analyzing neutrophil cell physiology, phenotype, and function as well as monocyte phenotype. Neutrophil phenotype and cell physiology was largely similar comparing patients with CF at T1 and HV 5. Of noThe activity of platelets as reported by CD62P under resting conditions or after stimulation with PAF was similar when analyzing HV, T1, and T2 (4This study investigated the function and the phenotype of cellular innate immunity with a focus on neutrophils in patients with CF before and after with ETI treatment in comparison to healthy volunteers. In accordance with data from the original phase III clinical trial  and postIn accordance with a recent study , neutropIn contrast to the well-known causality between CFTR dysfunction and defective epithelial chloride transport, it remains a matter of debate whether CFTR directly affects cellular innate immune function. The CFTR protein was detected in the phagolysosome of human neutrophils . InteresThe role of CFTR modulators in innate immune cell function has been previously studied. IVA treatment improved bacterial killing in neutrophils and monocyte-derived macrophages , 46. MorThe present article has several strengths and limitations. Despite that there were some significant changes in cellular innate immunity, the authors interpreted the reported changes as likely to be without clinical consequences. The findings also indicated that ETI treatment had no negative effect on cellular innate immunity. Moreover, while the study population mimicked the typical characteristics of CF in general and the ETI treatment in particular, patients with CF were investigated during stable periods of disease without exacerbation in a monocentric prospective study. Therefore, we may have missed changes in neutrophil and/or monocyte phenotype and/or function, which potentially only become apparent during acute exacerbation . However5Patients with CF are affected by multiple severe organ function alterations, which in this study did not affect circulating innate immunity and only marginally humoral markers of inflammation. While this study confirmed previous beneficial effects of ETI on clinical data and markers of humoral inflammation, no major effects on innate immunity were detected, besides some alterations after additional stimulation in vitro with inflammatory mediators. Nevertheless, ETI treatment did not impair cellular innate immunity. The present study population consisted of patients with currently well-controlled CF. Further studies are needed to evaluate potential benefits of ETI treatment during acute exacerbation in patients with CF. In summary, in patients with CF without acute exacerbation, circulating innate immunity did not exhibit any alterations.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by Local Independent Ethics Committee of the University of Ulm. The patients/participants provided their written informed consent to participate in this study.Conceptualization: HS, MH-L, and DM. Data curation: HS, LH, LW, and DM. Formal analysis: HS, LH, LW, and DM. Funding acquisition: MH-L. and DM. Investigation: HS, LH, LW, CK, AM, LS, FM, AS, CB, PM, and DM. Methodology: HS, LH, LW, CK, and DM. Project administration: MH-L and DM. Resources: MH-L and DM. Visualization: LH and DM. Writing – original draft: HS, LH, and DM. Writing - review & editing: all authors. All authors contributed to the article and approved the submitted version.


F1-score, and AUC of 0.879, 0.879, 0.879, 0.875, and 0.971, respectively. Compared to the MLP classifier with the 3D CNN features selected by Lasso, our method based on the MLP classifier has improved the classification performance by 5.8% (accuracy), 5.3% (mean precision), 5.8% , 5.4% (mean F1-score), and 2.5% (AUC). Compared to the MLP classifier with lung radiomics features selected by Lasso, our method based on the MLP classifier has improved the classification performance by 5.0% (accuracy), 5.1% (mean precision), 5.0% , 5.1% (mean F1-score), and 2.1% (AUC). Therefore, it is concluded that our method is effective in improving the classification performance for COPD stage classification.Computed tomography (CT) has been regarded as the most effective modality for characterizing and quantifying chronic obstructive pulmonary disease (COPD). Therefore, chest CT images should provide more information for COPD diagnosis, such as COPD stage classification. This paper proposes a features combination strategy by concatenating three-dimension (3D) CNN features and lung radiomics features for COPD stage classification based on the multi-layer perceptron (MLP) classifier. First, 465 sets of chest HRCT images are automatically segmented by a trained ResU-Net, obtaining the lung images with the Hounsfield unit. Second, the 3D CNN features are extracted from the lung region images based on a truncated transfer learning strategy. Then, the lung radiomics features are extracted from the lung region images by PyRadiomics. Third, the MLP classifier with the best classification performance is determined by the 3D CNN features and the lung radiomics features. Finally, the proposed combined feature vector is used to improve the MLP classifier's performance. The results show that compared with CNN models and other ML classifiers, the MLP classifier with the best classification performance is determined. The MLP classifier with the proposed combined feature vector has achieved accuracy, mean precision, mean recall, mean  Chronic obstructive pulmonary disease (COPD) is a common and non-infectious lung disease characterized by persistent airflow limitation –3. BecauCompared with the GOLD criteria and other imaging equipment, computed tomography (CT) has been regarded as the most effective modality for characterizing and quantifying COPD . CompareRadiomics features in COPD develop slower than those in other lung diseases, such as lung cancer and pulmonary nodules. Until 2020, reference  points oCurrently, radiomics features also have been used in COPD for survival prediction , 21, COPThe participants are enrolled by the national clinical research center of respiratory diseases, China, from May 25, 2009, to January 11, 2011. Finally, 465 Chinese subjects participated in the study after being strictly selected by the inclusion and exclusion criteria . The 465First, 465 sets of chest HRCT images are automatically segmented by a trained ResU-Net , obtainiA truncated transfer learning strategy is proposed to extract the 3D CNN features based on the pretrained Med3d . Med3d, N) are cropped into the size 280 × 400 × N′, retaining the lung region. The non-lung images are also deleted, so N changes into N′ (N′ < N). Second, the cropped lung images are preprocessed by the method in reference , and j ∈ .Second, the combined feature vector is generated by concatenating the selected CNN feature vector and the selected radiomics feature vector. Finally, the combined feature vector is the size 1 × 166 per subject. Our proposed method uses the combined feature vector of 3D CNN features and lung radiomics features for COPD stage classification based on the MLP classifier. Our experiment includes five experiments in this section to verify the effectiveness of our proposed method.∗ (input size (2D/3D)), 50/50 (epoch (2D/3D)), and 0.5/0.2 (drop rate (2D/3D)) in experiment 1. The training parameters of 2D GoogleNet and 3D GoogleNet are set: 16/2 (batch size (2D/3D)), 512 × 512/512 × 512 × 20∗ (input size (2D/3D)), 50/50 (epoch (2D/3D)), and 0.2/0.2 (drop rate (2D/3D)) in experiment 1. ∗MIL: each case (a set of chest HRCT images) was equally divided into 20 segments, with one slice taken equidistantly to obtain 20 slices in each case. The training parameters of 2D DenseNet with MIL (2D DenseNet_MIL) and 3D DenseNet with MIL (3D DenseNet_MIL) are set: 16/2 (batch size (2D/3D)), 512 × 512∗∗/512 × 512 ×  × 512 × 16∗∗∗ (input size (2D/3D)), 50/50 (epoch (2D/3D)), and 0.5/0.2 (drop rate (2D/3D)) in experiment 2. The training parameters of 2D GoogleNet with MIL (2D GoogleNet_MIL) and 3D GoogleNet with MIL (3D GoogleNet_MIL) are set: 16/2 (batch size (2D/3D)), 512 × 512∗∗/512 × 512 × 16∗∗∗ (input size (2D/3D)), 50/50 (epoch (2D/3D)), and 0.2/0.2 (drop rate (2D/3D)) in experiment 2. ∗∗MIL: each case was equally divided into 10 bags, with one slice taken randomly to obtain 10 slices in each case. ∗∗∗MIL: each case was equally divided into 16 bags, with one slice taken equidistantly to obtain 16 slices in each case.Specifically, the training parameters of 2D DenseNet and 3D DenseNet are set: 20/2 (batch size (2D/3D)), 512 × 512/512 × 512 × 20Specifically, experiments 3–5 are designed to compare the classification performance of the six different classifiers based on the CNN feature vector 13824), radiomics feature vector (1316), their selected feature vector by Lasso, and the proposed combined feature vector (166), respectively. First, based on 3D ResNet10, we use six classic classifiers . The above standard evaluation metrics are defined as in equations . The. TheF1-sThis section reports the experimental results, including (1) the classification performance of the parenchyma images based on the DenseNet and GoogleNet; (2) the classification performance of the CNN feature vector and lung radiomics vector based on different classifiers; (3) the MLP classifier's performance with the combined feature vector; and (4) the MLP classifier's performance with combined feature vector based on different 3D ResNet.This section shows the classification performance of 2D/3D DenseNet, 2D/3D GoogleNet, 2D/3D DenseNet_MIL, and 2D/3D GoogleNet_MIL based on the parenchyma images, respectively.F1-score), and 0.809 (AUC). In addition, F1-score), and 0.787 (AUC).This section shows the classification performance of the CNN feature vector (13824), the CNN feature vector selected by Lasso (60), the lung radiomics vector (1316), and the lung radiomics vector selected by Lasso (106) based on different classifiers, respectively.F1-score), and 0.790 (AUC), respectively. F1-score), and 0.946 (AUC), respectively. F1-score), and 0.919 (AUC), respectively. F1-score), and 0.950 (AUC), respectively.4The best MLP classifier is determined with the CNN feature vector selected by Lasso (60) or the lung radiomics vector selected by Lasso (106) by F1-score), and 0.971 (AUC), respectively.The best MLP classifier is determined with the CNN feature vector selected by Lasso (60) or the lung radiomics vector selected by Lasso (106) by This paper proposes a features combination strategy by concatenating 3D CNN features and lung radiomics features for COPD stage classification based on the MLP classifier. Three sections are discussed in this section, and we also point out the limitations in this study and the future direction.First, 2D GoogleNet with parenchyma images performs the best in 2D CNN models. The main reason is that 2D GoogleNet is designed for 2D natural image classification (RGB images). Therefore, it achieves the best classification performance in 2D parenchyma images. Meanwhile, because of the ability to extract interlayer information, 3D DenseNet with parenchyma images performs the best classification in 3D CNN models. However, CNN models with parenchyma images fail to classify the COPD stage. One main reason is that the chest HRCT image cannot fully reflect COPD's characteristics for the CNN models. Specifically, the gold standard of COPD classification is characterized by airflow restriction with a slight difference in the chest HRCT image. The slight difference in COPD is mainly caused by small airway disease with an airway diameter<2 mm . BecauseSecond, Lasso can improve the classification performance of the MLP classifier with the 3D CNN features and the lung radiomics features. Lasso is often used with survival analysis models to determine variables and eliminate the collinearity problem between variables , 42. TheThird, the proposed feature combination strategy can further improve the classification performance of the MLP classifier. This paper does not improve the existing classic classifiers and starts with the classification features to enhance the classifier's performance. Many nonlinear classification features, the 3D CNN features, are obtained by a truncated transfer learning strategy. We concatenate the CNN feature vector and the radiomics feature vector for the COPD stage classification, which improves the MLP classifier's performance. The MLP classifier is good at handling complex nonlinear features by itself , 32. TheFinally, this study has some limitations, and we point out the future direction. First, from the materials used in this study, there are not enough cases at the COPD stages III and IV. Second, the existing classic classifiers are not improved. Third, the classification performance of the ML classifier with the 3D CNN features is also limited by the encoder backbone of the pretrained Med3d. In our future work, the recent networks, an auto-metric graph neural network , will beF1-score, and AUC of 0.879, 0.879, 0.879, 0.875, and 0.971, respectively. This shows that our method effectively improves the classification performance for COPD stage classification.This paper proposes a feature combination strategy by concatenating 3D CNN features and lung radiomics features for COPD stage classification based on the MLP classifier. First, the 3D CNN features are extracted from the lung region images based on a truncated transfer learning strategy. Then, the lung radiomics features are extracted from the lung region images by PyRadiomics. Compared with CNN models and other ML classifiers, the MLP classifier with the best classification performance is determined by the 3D CNN features and the lung radiomics features. Lasso plays a role in improving the classification performance of the MLP classifier with the CNN feature vector and the radiomics feature vector. The proposed combined feature vector also improves the MLP classifier's performance. The MLP classifier with the proposed combined feature vector has accuracy, mean precision, mean recall, mean


Integrated root phenotypes and transcriptome analysis have revealed key candidate genes responsible for maize root growth and development in potassium deficiency.+ uptake remains limited. To address this, we conducted hydroponic and field trials at different growth stages. K+ deficiency significantly inhibited maize root growth, with metrics like total root length, primary root length, width and maximum root number reduced by 50% to 80% during early seedling stages. In the field, RSA traits exhibited maximum values at the silking stage but continued to decline thereafter. Furthermore, K deprivation had a pronounced negative impact on root morphology and RSA growth and grain yield. RNA-Seq analysis identified 5972 differentially expressed genes (DEGs), including 17 associated with K+ signaling, transcription factors, and transporters. Weighted gene co-expression network analysis revealed 23 co-expressed modules, with enrichment of transcription factors at different developmental stages under K deficiency. Several DEGs and transcription factors were predicted as potential candidate genes responsible for maize root growth and development. Interestingly, some of these genes exhibited homology to well-known regulators of root architecture or development in Arabidopsis, such as Zm00001d014467 (AtRCI3), Zm00001d011237 (AtWRKY9), and Zm00001d030862 (AtAP2/ERF). Identifying these key genes helps to provide a deeper understanding of the molecular mechanisms governing maize root growth and development under nutrient deficient conditions offering potential benefits for enhancing maize production and improving stress resistance through targeted manipulation of RSA traits in modern breeding efforts.Potassium (K) is a vital macronutrient for plant growth, but our understanding of its regulatory mechanisms in maize root system architecture (RSA) and KThe online version contains supplementary material available at 10.1007/s00425-023-04260-7. K plays a fundamental role in plant growth and development by enhancing photosynthetic assimilation  How does the root system architecture of maize respond to K deprivation at different growth stages? (ii) What are the transcriptomic changes in maize roots under low K stress, and what are the hub genes and pathways involved in post-transcriptional modulation of the response to K stress? KTo answer these questions, we conducted a series of experiments to evaluate the effects of K deficiency on maize growth, biomass accumulation, root system architecture (RSA), and grain yield, as well as the changes in gene expression and regulation at five key developmental stages. We used a hydroponic pouch system to simulate K deprivation in early seedling stages and conducted field trials to assess the phenotypic traits and nutrient concentrations at different growth stages. We also performed transcriptomic analysis using RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) and co-expression modules in response to K stress and to identify the hub genes and pathways involved in post-transcriptional modulation of the response to K stress.Zea mays cv. Chengdan 30 (CD30), a high-yielding spring maize cultivar developed by the Sichuan Academy of Agricultural Sciences in 2004 and being currently dominant in the hilly region of central Sichuan 2·4H2O, 1 mM NH4NO3, 1 mM KCl, 0.85 mM MgSO4·7H2O, 0.25 mM NH4H2PO4, 77 μM Fe-EDTA, 25 μM H3BO3, 0.8 μM Na2MoO4·2H2O, 0.6 μM CuSO4·5H2O, 9 μM MnCl2·4H2O, 2 μM ZnSO4·7H2O. For a nutrient solution with low-K (LK) treatment, the potassium concentration was adjusted to 10 μM and 1000 μM as sufficient K conditions (SK), respectively. The pH of the solution was adjusted to 5.7 ± 0.1 with NaOH or HCl as required and was continuously aerated and replaced every 3 days. The seedlings were cultured in a growth chamber . Each treatment had six replicates.Maize seeds were surface-sterilized with 8% (v/v) sodium hypochlorite for 20 min. The sodium hypochlorite was removed by washing the seeds with distilled water three times. The seeds were then germinated in moistened filter paper. Four days after germination, seedlings with similar growth vigor were transferred to 100 L polypropylene tubs and grown hydroponically using a specially designed and constructed plant growth pouch system, which holds the seedling and root system on the surface of filter paper, as described by Gladman et al. . The fulhttp://www.plantmineralnutrition.net/software/prida/index.html). The images were extracted as TIFF files for further processing and computation of root traits. WinRHIZO software  was used to quantify root growth and topology traits, while GiA Roots , primary root length (cm), root width (cm), average root diameter (cm), convex area (cm2), maximum root number, and root volume (cm3). After imaging, the whole maize seedlings were harvested, and the shoots and roots were dissected. The samples were heated at 105 ℃ for 30 min and then oven-dried at 65 ℃ for 72 h. The shoot dry weight, root dry weight, and root-to-shoot ratio (R/S) were determined by weighing the samples.After 14 days of growth in hydroponics, individual pouches with one maize plant were removed from the hydroponic tub. The plant on the pouch system was carefully placed horizontally in a glass tray containing water, and the root system was submerged in the water to collect root system images using Plant Root Imaging and Data Acquisition (PRIDA), a Python-based image acquisition and data management software (−1 (SK) and no K supplied (LK). No K fertilizer has been applied since 2012. The plots were fertilized with 300 kg ha−1 N and 90 kg ha−1 P2O5. Phosphorus and K fertilizers were applied before sowing, 50% of N fertilizer was applied as a base dressing, and the remaining 50% was applied at the stem jointing stage. Soil physical and chemical characteristics were evaluated at the beginning of the experiment for SK and LK treatments by analyzing three soil samples, respectively  5.9 mg kg−1, ammonium acetate extractable potassium 133 mg kg−1 and pH 8.60 (1:1.25 g/v). In LK treatment, chemical characteristics were organic matter 9.9 g kg−1, total N 0.76 g kg−1, alkali-hydrolyzable N 55 mg kg−1, available phosphorus (Olsen-P) 5.7 mg kg−1, ammonium acetate extractable potassium 93 mg kg−1 and pH 8.61. The subplot area was 20 m2 (5-m-long × 4-m-width). Maize was seeded on 1st April and harvested on 11th August 2021.A field experiment was conducted at Sichuan Agricultural Research Institute Modern Experimental Station, Deyang City, Sichuan Province. The experimental design was a spit-plot with potassium (K) fertilizer treatments in the main plots and the cultivar in the subplots. The variable between two application rates of K fertilizer, namely 150 kg K haDuring specific developmental stages, namely the 10th leaf stage (V10), tasseling stage (VT), silking stage (R1), milk-ripe stage (R3), and physiological maturity (R6), root samples were obtained for RNA sequencing. We selected three uniform phenotype plants for each stage, carefully extracting two layers of roots from beneath the soil surface. These root samples were subsequently cleaned using running water and combined into a single representative sample. To ensure robust analysis, we collected three replicates for each developmental stage, promptly freezing them in liquid nitrogen, and then storing them at − 80 °C for subsequent RNA isolation.Total RNA was isolated from root tissues using a Trizol reagent  following the manufacturer’s instructions. RNA integrity was confirmed using the 2100 Bioanalyzer system . Reverse transcription was performed to synthesize first-strand cDNAs as templates for preparing mRNA-seq libraries. All RNA-seq libraries were constructed using a Ribo-Zero rRNA Removal kit  followed by a TruSeq Stranded Total RNA Library Prep Plant and then sequenced on an Illumina HiSeq2500 Platform .Root system architecture (RSA) was evaluated at each developmental stage. Three uniform phenotype plants were selected from the central rows of each subplot, then the soil volume with 33 cm in width, 50 cm in length and 30 cm in depth surrounding the plant was excavated, and the whole seedlings were dissected into shoot and root. The roots were cleaned with running water. Root segments were floated in water in an acrylic tray on the scanner (ESPON Perfection V850). The scanned images were analyzed with WinRHIZO (Regents Instruments Inc.) commercial software to obtain total root length, surface area, volume and average root diameter. After imaging, the shoot and root were heated at 105 ℃ for 30 min and then oven dried at 65 °C for 72 h to obtain shoot and root dry weight. The grain yield was determined and then standardized to 14% moisture. Total N concentration in grain and plant shoot was determined by the semi-micro Kjeldahl procedure Bremner , total Phttp://ensemblgramene.org/Zea_mays/Info/Index) using Hisat2  with singular enrichment analysis  terms at a Bonferroni-corrected The FPKM value of each gene from the five comparisons  was used for co-expression analysis using the WGCNA  deficit Figs.  and 2. IP < 0.05) decrease in grain yield, a 2.2% (P < 0.05) decrease in seed K concentration, and a 6.8% (P < 0.01) in seed P concentration, respectively, but not altered seed N concentration  were covered, and an average of 4,811 new genes were detected among the five developmental stages. The detected known gene number of each stage were 31,276 (LK) and 31,063 (SK) for V10, 30,673 (LK) and 31,137 (SK) for VT, 30,783 (LK) and 30,221 (SK) for R1, 30,071 (LK) and 30,056 (SK) for R3, 30,124 (LK) and 30,294 (SK) for R6 (Suppl. Table S4) The Pearson correlation analysis plots showed strong correlation coefficient among the replicates under SK and LK conditions, respectively, indicating the reliability of our RNA-seq data . To mitigate background influences, the roots from three plants were pooled as one sample. Thirty RNA-seq libraries were constructed and sequenced from maize roots at different developmental stages with three biological replicated under SK and LK conditions. Totally, 142 million raw reads were generated for all the samples, and 138 million clean reads (97.27%) were obtained after performing quality control with a Q20 value of 97.68%. The average GC content is 54.83% , 740 (VT), 1,090 (R1), 1,306 (R3), and 520 (R6), respectively. Of those DEGs, 4,315 specific genes were identified at all stages, 678 genes were shared by two development stages, 93 genes by three, two genes by four, and one gene by five stages, respectively , was a component of the low K signal transduction pathway in Arabidopsis roots. It was reported that RCI3 was induced upon K deprivation and participated in the induction of the high-affinity K+ uptake transporter HAK5, which ultimately facilitated K acquisition under K starvation , CALCINEURIN B-LIKE INTERACTING PROTEIN KINASE 23 (Zm00001d018799), CALCINEURIN B-LIKE PROTEIN 10 (Zm00001d044285) were also observed up-regulated in specific developmental stage under LK conditions. Moreover, important transcription factors (TFs) related to K metabolism were found on the list. Maize WRKY TRANSCRIPTION FACTOR 9  was significantly induced by LK in VT, R3, and R6 stages  was notably induced in response to K starvation at V10, VT, and R1 stages, respectively , green (P < 0.01), and red (P < 0.001) CMs with shoot dry weight and a positive correlation of the pink (P < 0.001) module with shoot K concentration. A negative correlation with shoot dry weight was observed in light yellow, midnight blue, and pink CMs.A gene regulation network was built using the WGCNA method to obtain a comprehensive understanding of the transcriptional network in the successive developmental stages and to clarify that it controls the adaptation of LK condition in maize roots. Totally, 24,043 expressed genes identified in 30 samples were divided into 23 co-expressed modules (CMs), color-coded for reference, with the CM grey set containing the genes that could not be assigned to other CMs Fig. a. The 23We further analyzed the magenta, green, red, light green, light yellow, midnight blue, and pink modules among all K deficit responding modules. Gene ontology enrichment analysis of each selected module highlighted key biological processes by a set of co-expressed genes , spliceosome (zma03040), RNA transport (zma03013), purine metabolism (zma00230), ubiquitin-mediated proteolysis (zma04120), and aminoacyl-tRNA biosynthesis (zma00970). In CM light green, a noteworthy KEGG pathway enriched was zeatin biosynthesis (zma00908). In contrast, two significant KEGG pathways, glycerophospholipid metabolism zma00564) and glycerolipid metabolism (zma00565), were observed in the CM light yellow. In the CM midnight blue, the DEGs were notably enriched in four KEGG pathways: Galactose metabolism (zma00052), plant hormone signal transduction (zma04075), MAPK signaling pathway—plant (zma04016), and carotenoid biosynthesis (zma00906). In the case of CM magenta and red, the DEGs were significantly enriched in the RNA polymerase (zma03020) pathway  score  reduction in grain yield  are important regulatory nodes in the regulatory network of plant K deficiency stress response. ARF2 (Auxin Response Factor 2) is well-characterized TF responsible for the negative regulation of AtHAK5 expression. Under KAtbHLH121, AtMYB59, and OsMYBc, have been demonstrated to bind to the promoters of high-affinity K+ transporters and activate gene expression under K+ limiting conditions  was identified as one of the hub genes, exhibiting significant downregulation across all developmental stages. Interestingly, the maize homolog of RAP2.11, Zm00001d030862, displayed downregulation in the root tissue during the early developmental stage -CIPK module . The phosphorylation of AKT1 enhances its K+ uptake activity , was identified as a component of the low K signal transduction pathway in Arabidopsis roots. RCI3 is known to be induced in response to K deprivation and is involved in activating of the high-affinity KPhosphorylation and dephosphorylation may be important regulatory mechanisms in the plant responses to K deficiency. In the present study, several kinase genes were transcriptionally regulated by K deficiency. Among them, an important serine/threonine-protein kinase TOR (Target of rapamycin) was in the hub gene list Fig. . TOR proAtRCI3), Zm00001d011237 (AtWRKY9), and Zm00001d030862 (AtAP2/ERF). These findings provide a valuable resource for future functional experiments, enriching our comprehension of how root system architecture influences nutrient uptake efficiency and contributes to the continued enhancement of maize grain yield under limiting nutrient availability environments.In summary, our comprehensive analysis of root phenotypes and transcriptome data from five key developmental stages has successfully identified important root system architectures and gene regulatory networks that are implicated in both root development and the response to potassium deficiency in maize. Additionally, we have identified a diverse set of TFs and potential hub genes that are closely associated with the response of roots to potassium deficiency. Interestingly, some of these genes exhibited homology to well-known regulators of root architecture or development in Arabidopsis, such as Zm00001d014467 (Supplementary file1 (TIF 47994 KB)Supplementary file2 (XLSX 64 KB)Below is the link to the electronic supplementary material.


Overall, the developed approach circumvents uncertainty in choosing the relevant compositional model (close-to-equilibrium or kinetic), eliminates unknown parameters in the vapor–solid distribution of vapor–liquid–solid nanowires based on group-III intermix, and should be useful for the precise compositional tuning of such nanowires.Compositional control in III–V ternary nanowires grown by the vapor–liquid–solid method is essential for bandgap engineering and the design of functional nanowire nano-heterostructures. Herein, we present rather general theoretical considerations and derive explicit forms of the stationary vapor–solid and liquid–solid distributions of vapor–liquid–solid III–V ternary nanowires based on group-III intermix. It is shown that the vapor–solid distribution of such nanowires is kinetically controlled, while the liquid–solid distribution is in equilibrium or nucleation-limited. For a more technologically important vapor-solid distribution connecting nanowire composition with vapor composition, the kinetic suppression of miscibility gaps at a growth temperature is possible, while miscibility gaps  always remain in the equilibrium liquid–solid distribution. We analyze the available experimental data on the compositions of the vapor–liquid–solid Al It is known that III–V ternary nanomaterials with widely tunable compositions are paramount for the fundamental research of semiconductor properties at the nanoscale, bandgap engineering, and fabrication of functional heterostructures for different device applications in nanoelectronics and optoelectronics . FurtherxGa1−xAs [xGa1−xP [xGa1−xAs [xGa1−xN [xB1−xC, pseudo-binary (AC)x(BC)1−x NW xB1−xC ternary NW based on group-III intermix (with A and B belonging to group III and C belonging to group V) is close to equilibrium [xGa1−xAs, InxGa1−xAs, InxGa1−xP, and InxGa1−xN nanowires, which are very well described within the model. In particular, we demonstrate that the miscibility gaps in the material systems with strong interactions between dissimilar III–V pairs in solids, such as highly mismatched InGaAs, InGaP, and InGaN ternaries, can be fully circumvented by fast VLS growth kinetics.Compositions of VLS III–V ternary NWs based on group-III intermix, including InxGa1−xAs ,14,15,16[xGa1−xP , AlxGa1−xGa1−xAs ,21,22,23[xGa1−xN  material[xGa1−xN ,26,27 foliquid y ,32,33,34 vapor z ,35,36. A vapor z , the liqilibrium ,28, whicilibrium ) derivedilibrium ,30,31. Tilibrium ,38 for tilibrium . On the  in Ref.  and InGa in Ref.  NWs. The in Ref. . Herein,xB1−xC NW based on group-III intermix from atomic vapor fluxes The VLS growth of a ternary Aof Refs. ,35,36,37of Refs. ,29,31,32of Refs. , or InGaof Refs.  NWs are of Refs. ,29,31. HThe condition time ago  but not xB1−xC NW presents the solid composition A over the total concentration of A and B atoms in liquid, and is considered as a governing parameter for NW composition. As discussed in detail in Ref. [The liquid–solid distribution of a VLS III–V A in Ref.  and Ref. in Ref.  specific in Ref. ,27,28 ,35,36,37given by ,35,36,37material ,27. The the form (5)y=11+Here, As discussed in detail in Refs. ,28,37, t of Ref.  requires unknown ,37.One difficulty in joining the liquid–solid and vapor–solid distributions of VLS III–V ternary NWs is that the liquid–solid distributions are obtained by treating the nucleation step ,30,31 orectively ,29,37. UThe arrival rates of group-III atoms are given byHere,y (HVPE) ,40,41. MA and λB ,40,41,42A and λB .The outgoing fluxes of group-III atoms A and B depend on the chemical potentials of these atoms in liquid, A and μB ,40. In oA and μB (9)2μC=μC2 dimers at a growth temperature Here,  in Ref. .Assuming that group-III atoms do not desorb directly from the droplet surface but rather leave the droplet by “negative” diffusion onto the NW sidewalls and then desorb from there ,45, the Here, droplet ,37. UsinCalculating the adatom diffusion fluxesUsing Equations (8) and (15) in Equation (7), and the definition for the fraction of A atoms in vapor, of Ref. , which w of Ref.  to accou of Ref. ,32,33,34 of Ref. , the VLSThe equilibrium vapor–solid distribution, which sets a limit for the solid composition under no-growth conditions, is obtained fromThis equilibrium vapor–solid distribution has the same form as the equilibrium liquid–solid distribution given by Equation (5), but with modified parameters which correspond to the vapor rather than the liquid phase.xGa1−xAs [xGa1−xP [xGa1−xN [xGa1−xAs material system [According to Equations (5) and (19), the equilibrium liquid–solid and vapor–solid distributions of VLS III–V ternary NWs based on group-III intermix are determined by the two control parameters, the pseudo-binary interaction parameter xGa1−xAs ,30,31,35[xGa1−xN  materiall system ,30,31 foThe kinetic vapor–solid distribution given by Equation (17) is very different from the equilibrium distributions. First, this distribution is five-parametric and is reduced to the four-parametric function of Ref.  at larger fluxes ). In parg to Γ→0 ,35,36,37xGa1−xAs NWs grown by MBE often feature the spontaneous formation of core–shell radial structures, where cylindrical cores have lower AlAs fractions compared to the tapered shells [xGa1−xAs NWs grown by MBE on Si(111) substrates at 510 °C, where different vapor compositions xGa1−xAs NWs [Au-catalyzed Ald shells ,46. The d shells . The lowtio of 3 ,35,37. A−xAs NWs ,29. Henc−xAs NWs , where txGa1-xAs NWs, obtained using in situ growth and compositional monitoring of NW growth inside an environmental transmission electron microscope [Let us now discuss the data of a highly mismatched InGaAs material system, where the miscibility gap closes for temperatures above the critical temperature of 543 °C . Figure croscope . These I in Ref. , where a ω=2.724  and βl=0xGa1−xAs NWs grown by MOVPE at 450 °C and 470 °C on InAs(111)B substrates [xGa1−xAs NWs grown by MOVPE at 570 °C on graphene [xGa1−xAs at 420 °C and 450 °C. Overall, the measured vapor–solid distributions are centered around the simplest curve, The measured vapor–solid distributions of Au-catalyzed Inbstrates , and at bstrates , are verbstrates , but quigraphene . All thef 420 °C ), positiKinetically driven compositional trends in InGaAs NWs are further demonstrated in xGa1−xP NWs of Ref. [xGa1−xP ternary, obtained from Equation (19) at  of Ref. . These Nnd 2.438 , yieldinxGa1−xN NWs grown by HVPE on Si substrates at different temperatures from 630 °C to 680 °C, at a fixed In content in vapor of 0.6 [Highly mismatched InGaN is one of the most difficult ternary systems based on group-III intermix for obtaining high InN fractions without material segregation and compositional inhomogeneities ,49. The r of 0.6 . All theeratures . TherefoTo summarize, the developed approach for obtaining the analytical vapor–solid distribution of VLS III–V ternary NWs based on group-III intermix uses close-to-equilibrium conditions for group-III atoms at the liquid–solid interface of a growing ternary island and close-to-equilibrium conditions for highly volatile group-V atoms at a droplet surface. Whenever the liquid–solid distribution is close to equilibrium, or nucleation-limited, as given by Equation (5), and the chemical potential of group-V atoms in the droplet is close to equilibrium with vapor, as given by Equation (9), the vapor–solid distribution has the kinetic form given by Equation (17). This kinetic distribution contains no characteristics of liquid and circumvents uncertainty in the unknown group-V concentration in a catalyst droplet and other parameters of the chemical potentials of different atoms in liquid, which is an important step with respect to the previously obtained results ,35,37. T


Chiral sulfoxidesare valuable in the fields of medicinal chemistryand organic synthesis. A recycle photoreactor utilizing the conceptof deracemization, where a racemate is converted into a pure enantiomer,is developed and successfully applied in the syntheses of chiral alkylaryl sulfoxides. The recycling system consists of rapid photoracemizationusing an immobilized photosensitizer and separation of the enantiomersvia chiral high-performance liquid chromatography, and the desiredpure chiral sulfoxides are obtained after 4–6 cycles. The keyto the success of the system is the photoreactor site, wherein thephotosensitizer 2,4,6-triphenylpyrylium is immobilized on the resinand irradiated (405 nm) to enable the rapid photoracemizations ofthe sulfoxides. As the green recycle photoreactor requires no chiralcomponents, it should be a useful alternative system for applicationin producing chiral compounds. From this perspective, we focused on the conversion ofa racemate to a single enantiomer using chiral HPLC,2f which may be summarized by the following process: (1) isolate eachenantiomer, (2) racemize the undesired enantiomer, and (3) recyclethe racemic mixture by subjecting it to the resolution conditions.Using this approach, only five cycles are theoretically required toproduce the enantiomerically pure compound in a yield of >96%.Mucheffort has been focused on the enantiomeric enrichment of stereolabilechiral compounds using HPLC methods. However, even if on-column stopped-flowbidimensional recycling HPLC is employed,2d the procedure is so complicated that the potential of the deracemization-recyclingapproach has not been realized. A key solution to this problem maybe the use of an on-column rapid photoracemization reaction. Therefore,we developed a recycling HPLC system equipped with an immobilizedphotoracemization site to realize a rapid reaction, which should representa powerful tool in generating a pure enantiomer. Compared to thermalreactions, photoreactions3d are more practicalin terms of green,sustainable chemistry. In this regard, Kappe et al. reported efficientphotochemical deracemization using a flow system.3e In contrast, rapid, reversible photoracemization withoutheating was integrated with the flow system of the recycling HPLC.Among numerous deracemization approaches,4l the photoracemizations of chiral sulfoxides5e are the most promising reactions for applicationin our system. Chiral sulfoxides are critical bioactive compoundsand intermediates in chemical reactions.6e Chiral sulfoxides, e.g., esomeprazole7b and dexlansoprazole,7c also attract attention as antiulcerdrugs (Recycling high-performance liquid chromatography(HPLC) enablesthe recycling of samples to increase the efficiency of separation.It is widely used to isolate and purify enantiomers, diastereomers,and various structurally related or unrelated compounds.erdrugs 1.8b In contrast, the racemizationsof chiral sulfoxides are facilitated by light irradiation.9bThe characteristic properties of chiral sulfoxides include theirthermal stabilities. The pyramidal inversion of the S center requires159.1–171.7 kJ/mol, which entails extreme temperatures (approximately200 °C).9a the pyramidal inversions of alkyl aryl sulfoxides in the presenceof photosensitizers have been investigated,10 and S inversion may occur in an exciplex comprising the photosensitizerand sulfoxide.11b Recently, we reported that theuse of 1 mol % of the photosensitizer 2,4,6-triphenylpyrylium tetrafluoroborate(TPT+) enables the rapid photoracemizations of chiral alkylaryl sulfoxides 1. Under irradiation, no photoracemizationoccurred without a photocatalyst. Various substitution patterns aretolerated, and the racemizations proceed extremely rapidly  isolation of the desired enantiomer and(2) rapid racemization of the other enantiomer to produce an enantiomericallypure sulfoxide in a good yield. The rapid photoracemization on thesolid phase is critical.The rapid photoracemizations of chiral sulfoxides should be applicablein deracemization. Although the one-pot deracemization of chiral sulfoxidesusing chemoenzymatic methods has been reported,+ should be immobilizedon the solid phase. We analyzed commercially available supportingmaterials, and the cation-exchange resin was the most suitable becausecationic TPT+ could be ionically bonded to the sulfonicacid anion (RSO3–) of the resin.14 Initially, TPT+ was immobilized oncommercial cation-exchange resin via ion exchange in H2O. However, the removal of BF4– fromthe prepared immobilized catalysts was challenging, and multiple cumbersomepost-processing steps, such as washing with various solvents, werenecessary. Furthermore, the BF4– contaminanthindered the estimation of the loading ratio of TPT+, andthus, an alternative procedure was developed. 1,3,5-Triphenyl-2-pentene-1,5-dione(2),15 which is prepared usingTPT+ via hydrolysis with sodium acetate (NaOAc), is treatedwith several cation-exchange resins containing sulfonic acid to generateionically bonded immobilized catalysts without counter ions -1a, >98% ee} in CH3CN served as a model reaction (3CNas the mobile phase may separate the racemate 1a(kobs) of A–C, we irradiated (425 nm) a 10 mM solutionof (+)-1a in the presence of 5 mg of each immobilizedcatalyst . After the racemization was completed, the solid catalyst was removedby filtration. The filtrate was evaporated, and the residue was analyzedusing HPLC and 1H nuclear magnetic resonance (NMR) spectroscopyto confirm that no other products were formed.Using this procedure, we maycalculate the loading ratio of f free 2 1, and imreaction 1. We scrcemate 1a into theB displays a somewhat low activity, A (kobs = 2.59 × 10–2 M–1 s–1) and C (kobs = 1.99 × 10–2 M–1 s–1) yield desirable results. ImmobilizedTP+ exhibits a comparable catalytic activity12 to that of soluble TPT+.Although + from A–C were then examined following the modelreaction. After irradiation (425 nm) for 2 min, the solid catalystwas separated by filtration, and the possibility of photoracemizationin the filtrate due to catalyst leaching was examined. Photoracemizationscaused by catalyst leaching are observed in the filtrates of A and B. However, no photoracemization is observedin the filtrate of C, which indicates that no leachingof TP+ into the solvent occurs . Given the good resultsobtained using C, we then explored catalyst recyclability,and recycling studies of C were conducted using the modelreaction. The reaction conditions were the same as those describedabove, and the progress of each run was determined by the enantiomericexcess of the recovered 1a. After completion of eachrun (10 min), the solid catalyst was separated by filtration, washedsuccessively with CH3CN and CH2Cl2, dried, and then reused in the subsequent run. C wasreused up to 10 times, with the reaction rate remaining almost unchanged. Consideringthat Dowex 50WX8 produced the best results, the highest cross-linkage(8%) of styrene divinylbenzene in Dowex 50WX8 might be the key totightly bond TP+ and resin.Thepossibilities of the leaching of TPC is packed into a glass tube , which is covered with a device that irradiateslight. In this device, a light-emitting diode (LED) tape (λ= 405 nm) is attached to an alumina block that maintains the constanttemperature of the photoreaction. The photoreactor incorporated intothe recycling HPLC system should racemize the chiral sulfoxide rapidlyunder light irradiation . However, C adjacent to the inner wall of the glass tube darkens incolor due to irradiation. The light absorption of TP+ immobilizedon the resin adjacent to the inner wall may be too intensive, leadingto self-destructive reactions, whereas most C in thecentral region of the glass tube remains under poor lighting conditions.Increasing light transmission to the central region of the glass tubeshould be key to success, and thus, light-permeable glass beads, whichare almost identical in radius to C, are employed toyield a more efficient light distribution. A total of 6 g of a mixtureof the glass beads and C is uniformly mixed and packedinto the glass tube, and the efficiencies of the photoreactions withdifferent ratios of glass beads and C were evaluatedvia the photoracemization of 2-methoxyethyl phenyl sulfoxide (+)-1b.12 The injected (+)-1b flows at 2.0 mL/min through the packed glass tube under opticalirradiation (405 nm). The progress of the reaction was evaluated viain-line HPLC using a chiral column (CHIRALPAK ID).Initially, the glass tube containing C leads to self-destructivereactions,and the absorption of more light by the resin adjacent to the innerwall of the glass tube compared to that absorbed by the central regionappears inevitable.The reactionrate increases as the proportion of glass beads increases,indicating the significance of light permeability in the photoreactor.However, using >3 wt % C decreases the efficiencyof racemization -1c12 and methoxyphenyl methyl sulfoxide(+)-1d12 were examined, andrapid racemizations are observed in both cases . Although complete racemizationsare not observed in the first runs  was injected tothe recycling HPLC equipped with the photoreactor and separated bythe HPLC component -1b fraction was recycled and flowedat 2.0 mL/min through the photoreactor  under optical irradiation (405 nm). During the passing of (+)-1b through the photoreactor, rapid photoracemization occurredand the isomerized 1b was again separated via HPLC. Theratio of (+)-1b/(−)-1b is 56:44 (1b was collected and (+)-1b was simultaneouslyapplied in the next cycle. After the 3rd and 4th runs, the obtainedratios of (+)-1b/(−)-1b are 51:49and 52:48, respectively.To generate chiral alkyl arylsulfoxides, the following closed-looprecycling runs were conducted 3. Racema1b/(−)-1b is53:47. The desired (−)-1b is accumulatedover five cycles in a yield of 91% with a 98% ee . Similarly, (−)-1c and (−)-1d were deracemized using the recyclephotoreactor . (−)-1c is accumulatedover five cycles in a yield of 85% with a 98% ee -1c, and (−)-1d, which are eluted after their antipodes,are collected. (+)-1c, which is eluted before its antipode,is collected and accumulated over four cycles in a yield of 87% witha 99% ee . The order of elution of the enantiomers did not affectthe yield or enantiomeric purity -3CN as the mobile phase was suitable for the resolution of enantiomersand photoracemization. These limitations should be addressed in futurework. Nevertheless, a continuous deracemization process was accomplishedusing this recycling HPLC system.Gradual broadening of the fraction peaks during later cycle runscomplicated the separation process. Therefore, accumulated yieldsafter several cycles were lower than expected. Careful selection ofHPLC conditions is required, as the solvent used in HPLC is determinedby the solvent used in photoracemization. In this case, CH1. As chiral sulfoxidesoccur within numerous bioactive compounds, developing an efficientmethod of producing the desired enantiomer should be very attractivein pharmaceutical applications. Notably, this green photoreactionsystem did not require any chiral components to provide enantiopurechiral sulfoxides.16 Our procedure should also beapplicable in the enantiospecific preparation of other racemic compounds.There are several issues to resolve, such as avoiding the gradualbroadening of the fraction peaks during later cycle runs and preparingsuitable separation conditions on the chiral column. If such problemsare resolved, the scale-up and automatization of this system may yielda powerful tool to generate a pure enantiomer using racemic compoundswithout a cumbersome stereoselective synthesis or high-cost enantiopurificationprocesses. Rapid photoracemization on the solid phase was criticalto success, and improvements to the photoreactor system shall be reportedin due course.Arecycle photoreactor was developed and applied in the resolutionof chiral alkyl aryl sulfoxides 2SO4. Solvents wereevaporated under reduced pressure, and 1H and 13C NMR spectroscopy was performed at 296 K at 600 and 150 MHz, respectively,unless otherwise stated. Tetramethylsilane  orresidual internal CHCl3 (1H NMR: δ 7.26and 13C NMR: δ 77.16) was used as an internal referencein 1H and 13C NMR spectroscopy of samples inCDCl3. Coupling constants (J) are reportedin Hertz (Hz), and splitting patterns are abbreviated as follows:singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m);and broad (br). High-resolution mass spectrometry was conducted usingan electrospray ionization time-of-flight (TOF), atmospheric pressurechemical ionization-TOF, or electron impact mass spectrometer. Infrared(IR) spectroscopy was performed using a Fourier transform IR spectrometerequipped with attenuated total reflectance (diamond). Melting points(mps), which are uncorrected, were recorded using a melting pointapparatus. Room temperature means 23–25 °C. An opticalirradiation device (Evoluchem PhotoRedOx Box) and chemistry screeningkits  were used for LED irradiation. Inthe photoreactor, the chiral sulfoxides were irradiated with a blueLED light tape (405 nm) .All reagents were purchased fromcommercial suppliers and used as received. Reaction mixtures werestirred magnetically, and the reactions were monitored using thin-layerchromatography on precoated silica gel plates. An oil bath was usedfor the reactions that require heating. Column chromatography wasperformed using silica gel (45–60 μm) and extracted solutionswere dried over anhydrous Na1a–d are described in our previous study.12All resins were purchasedfrom commercial suppliers and used as received, and the characterizationsof +  in 25 mL of CHCl3:H2O (1:1 v/v). After stirring at 50 °C overnight,the mixturewas poured into H2O and extracted using CH2Cl2. The resulting extract was washed with brine, dried, andconcentrated. To remove counter ions, the residue was purified viacolumn chromatography  toafford 2  as a colorless amorphoussolid. mp: 114–116 °C.NaOAc  was added to a stirredsolution of TPT2  in CH3CN (25 mL). After the mixture wasstirred at 60 °C for 3 h, the solid catalyst was removed by filtrationand stirred in CH3CN (200 mL) at room temperature for 3h. After filtration, the solid catalyst was then stirred in CH2Cl2 (200 mL) at room temperature for 3 h. Afterfiltration, the solid catalyst was dried in vacuo overnight to afford A. The washes were collected and evaporated to provide 2.2mg of unreacted 2, indicating that 97.3% of 2 was loaded on A. The catalyst loading was 0.973 mmol/g.Dowex 50WX2 200–400 mesh  was added to a stirred solution of B was prepared according to the procedure describedabove for the preparation of A. B was preparedusing 2  and Dowex 50WX4 200–400mesh , and 1.6 mg of 2 was recovered, indicating that 98.1% of 2 was loadedon B. The catalyst loading was 0.980 mmol/g.C was prepared according to the procedure describedabove for the preparation of A. C was preparedusing 2  and Dowex 50WX8 200–400mesh , and 2.0 mg of 2 was recovered, indicating that 97.5% of 2 was loadedon C. The catalyst loading was 0.976 mmol/g.1a  and A/B/C (5 mg) in CH3CN (2 mL) was stirred ina photoreactor equipped with blue LEDs  using PhotoRedOxBox EvoluChem  at room temperature.The extent of racemization was determined via HPLC using a CHIRALART Amylose-SA  column and 100% CH3CN as the mobile phase .A solution containing(+)-1b: CHIRALPAK ID (1.0 × 50cm), eluent: CH3CN, flow rate: 2.0 mL/min, temperature:23 °C, detection:254 nm, initial peak retention time = 23 min, and latter peak retentiontime = 29 min.1c: CHIRALPAK IH (1.0 × 25cm), eluent: CH3CN, flow rate: 2.0 mL/min, temperature:23 °C, detection: 254 nm, initial peak retention time = 15 min,latter peak retention time = 22 min.1d: CHIRALPAKIH (1.0 × 25 cm), eluent: CH3CN, flow rate: 2.0 mL/min,temperature: 23 °C, detection:254 nm, initial peak retention time = 20 min, and latter peak retentiontime = 28 min.1b  was injected into therecycling HPLC equippedwith the photoreactor. The fractions of the desired enantiomer wereaccumulated after five cycles to yield (−)-1b asa colorless oil .Racemate 1c was prepared according to the procedure described above for thepreparation of (−)-1b. Using racemate 1c as a colorless oil  yielded (−)-1c  after five cycles.(−)-1d was prepared according to the proceduredescribed above for the preparation of (−)-1b.Using racemate 1d as a colorless oil yielded (−)-1d  after sixcycles.(−)-1c was prepared according to the proceduredescribedabove for the preparation of (−)-1b. Using racemate 1c  yielded (+)-1c  after four cycles.(+)-


We previously demonstrated that neural stem/progenitor cells (NSPCs) were induced within and around the ischemic areas in a mouse model of ischemic stroke. These injury/ischemia-induced NSPCs (iNSPCs) differentiated to electrophysiologically functional neurons in vitro, indicating the presence of a self-repair system following injury. However, during the healing process after stroke, ischemic areas were gradually occupied by inflammatory cells, mainly microglial cells/macrophages (MGs/MΦs), and neurogenesis rarely occurred within and around the ischemic areas. Therefore, to achieve neural regeneration by utilizing endogenous iNSPCs, regulation of MGs/MΦs after an ischemic stroke might be necessary. To test this hypothesis, we used iNSPCs isolated from the ischemic areas after a stroke in our mouse model to investigate the role of MGs/MΦs in iNSPC regulation. In coculture experiments, we show that the presence of MGs/MΦs significantly reduces not only the proliferation but also the differentiation of iNSPCs toward neuronal cells, thereby preventing neurogenesis. These effects, however, are mitigated by MG/MΦ depletion using clodronate encapsulated in liposomes. Additionally, gene ontology analysis reveals that proliferation and neuronal differentiation are negatively regulated in iNSPCs cocultured with MGs/MΦs. These results indicate that MGs/MΦs negatively impact neurogenesis via iNSPCs, suggesting that the regulation of MGs/MΦs is essential to achieve iNSPC-based neural regeneration following an ischemic stroke. The brain comprises various cells, including those of neural lineage  and vascular lineage . Neural lineage cells are particularly vulnerable to ischemia/hypoxia and rapidly undergo cell death after an ischemic stroke . HoweverIn addition to iNSPC induction, inflammatory cells such as microglial cells (MGs) and macrophages (MΦs), which play an important role in tissue repair following various types of brain injury ,5, emergIn the present study, we utilized iNSPCs isolated from the site of injury in a mouse model of ischemic stroke to investigate whether brain-derived MGs/MΦs impacted the fate of iNSPCs in coculture experiments. We found that the presence of MGs/MΦs suppressed iNSPC proliferation and reduced neuronal differentiation of iNSPCs, which were not observed in iNSPCs coincubated with other cell types including astrocytes (ACs), endothelial cells (ECs), and pericytes (PCs). Additionally, MG/MΦ depletion using clodronate liposomes attenuated the MG/MΦ-mediated effects on iNSPCs. These results show that the regulation of MGs/MΦs can be a potential strategy to accomplish iNSPC-based neural regeneration following an ischemic stroke.Experimental procedures were approved by the Animal Care Committee of Hyogo Medical University . Adult (6–10 weeks-old) male CB-17 wild-type mice  were used to establish the mouse model of permanent focal cerebral ischemia by ligating and interrupting the distal portion of the left middle cerebral artery (MCA) as previously described [At the time of tissue harvest, mice were intraperitoneally administered a mixture containing medetomidine 0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) ,6, follo mg/kg, mUsing previously described immunohistochemistry protocols ,2,3,10, Positive areas for nestin and Iba-1 were evaluated in ischemic and peri-ischemic areas in coronal brain sections obtained from the same region across all animals. A total of 27 data points  using ImageJ, as previously described . IschemiRegionally-derived endogenous iNSPCs were isolated from ischemic areas of mice after MCAO and maintained in Dulbecco’s modified Eagle’s medium/F12  containing 20 ng/mL basic fibroblast growth factor , 20 ng/mL epidermal growth factor , 1% N2 (Thermo Fisher Scientific), and 2% fetal bovine serum (FBS), as previously described . Commerc+ iNSPCs (nestin+/mCherry− cells) or Sox2+ iNSPCs (Sox2+/mCherry− cells) was compared between iNSPC monocultures (control) and iNSPCs cocultured with mCherry+ MGs/MΦs using 12 data points , as described [+ iNSPCs (Ki67+/mCherry− cells) to all iNSPCs (DAPI+/mCherry−) cells was compared between iNSPC monocultures and iNSPCs cocultured with mCherry+ MGs/MΦs using 12 data points , as described [iNSPCs were cocultured with MGs/MΦs to investigate the effect of MGs/MΦs on iNSPC proliferation. Briefly, iNSPCs alone or in coculture with MGs/MΦs were plated on poly-D-lysine-coated dishes and incubated in Dulbecco’s modified Eagle’s medium/F12 containing bFGF, EGF, N2, and 2% FBS. The monocultures and cocultures were fixed and immunostained with antibodies against nestin , Sox2 , Ki67 , and mCherry , followed by incubation with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies . Nuclei were counterstained with DAPI (1:500), and fluorescence images were captured using a laser scanning microscope (LSM780). The number of nestinescribed . Additioescribed .+ iNSPCs were cultured alone or in combination with mCherry+ MGs/MΦs. Briefly, approximately 2–5 GFP+ neurospheres/well were plated onto mCherry+ MGs/MΦs in poly-L-lysine-coated dishes and incubated in neurobasal medium (Thermo Fisher Scientific) supplemented with bFGF, B-27 supplement (Thermo Fisher Scientific), and 2% FBS as described [+ neurospheres were plated onto mCherry+ ACs, ECs, or PCs, which were incubated under the same conditions. After fixation, the cocultures were immunostained with antibodies against GFP  and mCherry . The areas of GFP+ neurites were evaluated per cell type in total 3 wells.To investigate the effect of MGs/MΦs on iNSPC differentiation, neurospheres derived from green fluorescent protein (GFP)escribed . As a co+ MGs/MΦs. Briefly, approximately 5–10 neurospheres/well were plated onto mCherry+ MGs/MΦs and incubated in a neurobasal medium with bFGF, B-27 supplement, and 2% FBS. After fixation, the cocultures were immunostained with antibodies against Tuj1 , microtubule-associated protein 2 , myelin basic protein , and glial fibrillary acidic protein . The population of neurospheres that produced neurons (Tuj1+ cells and MAP2+ cells), astrocytes (GFAP+ cells), and oligodendrocytes (MBP+ cells) to all neurospheres were evaluated per well .To further investigate the effect of MGs/MΦs on iNSPC differentiation, neurospheres derived from iNSPCs were cultured alone or in combination with mCherryFor in vitro MG/MΦ depletion, clodronate encapsulated in liposomes, i.e., CD (+) liposomes , was added to the culture medium. Liposomes without clodronate, i.e., CD (−) liposomes, were used as control.+ MGs/MΦs were collected by FACS. Additionally, GFP+ iNSPCs collected by FACS were processed for microarray analysis.Analysis of MGs/MΦs with fluorescence-activated cell sorting (FACS) was performed using PE-conjugated antibodies against CD11b, CD86, and CD206, all obtained from Thermo Fisher Scientific with BD LSRFortessa™X-20 (BD Pharmingen), as described [+ iNSPC monocultures and GFP+ iNSPCs cocultured with MGs/MΦs were collected by FACS. Next, total RNA isolated from cells with the RNeasy Micro Kit  was used in microarray analysis, as described [Control GFPescribed ,6,13. Thescribed ,6,13 andescribed , as descescribed .p value of <0.05 was considered statistically significant.Data were presented as means ± standard deviation. Comparisons among three or more groups were performed using a one-way analysis of variance, followed by Bonferroni post hoc tests. Comparisons between the two groups were performed using Student’s t-test. In all analyses, a + cells were present in both the ischemic and peri-ischemic regions on poststroke day 1   A or in cntration B in 6-wentration  showed tntration  but alsontration , gradualntration  and the ntration . Howeverntration  comparedntration .+ iNSPCs (nestin+/mCherry− cells) was significantly lower in cocultures containing MGs/MΦs (+ iNSPCs (Sox2+/mCherry− cells) was significantly reduced by the presence of MGs/MΦs  was lower in iNSPCs cocultured with MGs/MΦs (+ iNSPCs (Ki67+/mCherry− cells) among all iNSPCs (DAPI+/mCherry− cells) was significantly lower in iNSPCs cocultured with MGs/MΦs than in iNSPC monocultures  in 12-well dishes, transwell inserts alone  were cultured in 6-well dishes in the presence of various numbers of MGs/MΦs  with direct cell–cell contact for 3 days. Compared to the control iNSPC monocultures  following IL4 treatment, i.e., IL4(+) MGs/MΦs, and used CD206+ MGs/MΦs from cultures not treated with IL4, i.e., IL4(−) MGs/MΦs, as controls to coculture with iNSPCs. Although the number of nestin+ iNSPCs was significantly decreased in both coculture groups, the number of nestin+ iNSPCs was significantly higher in iNSPCs cocultured with IL4(+) MGs/MΦs than in those cocultured with IL4(−) MGs/MΦs  in 24-well dishes, CD (+) or CD (−) liposomes (1 mM each) were added to the cultures and the number of MGs/MΦs was counted on post-treatment day 3. As shown in 4 cells/well) were plated in 24-well dishes, followed by the addition of CD (+) or CD (−) liposomes (1 mM each) 24 h later. The evaluation of cultures on post-treatment day 3 revealed that the number of iNSPCs was not significantly different between the CD (−) and CD (+) liposome groups  liposomes could prevent the negative effect of MGs/MΦs on iNSPCs. First, 24 h after plating MGs/MΦs  liposomes  (4 cells/well) were plated in 24-well dishes  A. In parl dishes B, to whir 8 days B.+ neurospheres, the cells differentiated well and generated many neurites  of mCherry+ ACs  were placed in the dishes    or the Gr 8 days . As show3 cells/well) were plated in 24-well dishes. One day later, GFP+ neurospheres were added to the same dishes and incubated for seven days. Additionally, CD (−) or CD (+) liposomes were added to the cultures 1 and 4 days after the plating of MGs/MΦs (+ neurite area was significantly larger in cultures treated with CD (+) liposomes than in those treated with CD (−) liposomes, indicating that MG/MΦ depletion with CD (+) liposomes attenuated the negative effect of MGs/MΦs on iNSPC differentiation.We further investigated whether the observed suppressive effect of MGs/MΦs on iNSPC differentiation was attenuated by CD (+) liposomes. MGs/MΦs , astrocytic (GFAP), and oligodendrocyte (MBP) markers, and the number of neurospheres with neurites that were positive for Tuj1, MAP2, GFAP, or MBP were counted. Compared with the control cultures, neuronal differentiation determined based on the population of neurospheres with Tuj1+ D–G and M exhibited a significant upregulation of more than 2 fold in iNSPCs cocultured with MGs/MΦs compared to the iNSPCs monocultures, 10 cell cycle-related genes  exhibited a significant downregulation of more than 2 fold in iNSPCs cocultured with MGs/MΦs compared to the iNSPCs monocultures  were significantly downregulated for more than 2 fold in iNSPCs cocultured with MGs/MΦs compared to the iNSPCs monocultures, a total of 22 apoptosis-related genes  were significantly upregulated for more than 2 fold in iNSPCs cocultured with MGs/MΦs compared to the iNSPCs monocultures    migrate toward the site of injury following an ischemic stroke . HoweverAlthough the precise origins and traits of iNSPCs remain unclear, evidence that iNSPCs are present in ischemic areas indicates the potential of iNSPCs to regenerate damaged tissue following an injury such as an ischemic stroke. However, we previously demonstrated that iNSPCs at the site of injury gradually declined after an ischemic stroke ,3. Consi+ T lymphocytes were involved in the death of iNSPCs [In support of this hypothesis, we previously demonstrated that certain subsets of CD4f iNSPCs . Under if iNSPCs . Howeverf iNSPCs . Additiof iNSPCs ,6. Thus,Although the precise mechanism underlying the negative impact of MGs/MΦs on iNSPCs remains unclear, previous studies on embryonic and fetal brains during early developmental stages demonstrated that MGs/MΦs negatively regulated the number of NSPCs to inhibit excessive neurogenesis through several mechanisms including phagocytosis, inhibition of cell proliferation, and promotion of cell death ,8,9. SimIn the present study, MG/MΦ depletion by CD (+) liposomes attenuated the negative effect of MGs/MΦs on iNSPCs in vitro. However, whether MG/MΦ depletion is indeed beneficial for iNSPCs, such as the proliferation, survival, and neuronal differentiation of iNSPCs, requires further investigation in in vivo studies. Although the precise roles of MGs/MΦs in pathologic conditions are not fully elucidated, previous studies using animal models of stroke revealed that MG/MΦ depletion aggravated brain injury and inflammation after ischemia ,32,33. I++ MGs/MΦs. Although both M1 and M2 MGs/MΦs accumulate in the ischemic area, a previous study using mouse brains showed that the expression of M1 MGs/MΦs (CD16/32) peaked around poststroke day 14, while M2 MGs/MΦs (CD206) peaked around poststroke day 7 [+/CD206+) [Although the cause for this discrepancy between our findings and previous reports remains unclear, the use of different pharmacologic agents  liposomes, PLX3397, and PLX5622) in different studies might be a contributor. For example, both PLX3397 and PLX5622 inhibit colony-stimulating factor-1 receptors, although PLX3397 also inhibits other receptors, such as c-kit . Given tke day 7  that do ke day 7 . Additio/CD206+) ,43, as o/CD206+) ,45. TherThe present study has several limitations. For example, we used MG/MΦ cell lines because of the difficulty of isolating primary cultured MGs/MΦs from mouse brains. However, it is possible that the features of MGs/MΦs differ between these cells. Thus, further studies are warranted to determine the precise roles of MGs/MΦs in relation to iNSPCs using primary cultured MGs/MΦs isolated from ischemic and non-ischemic regions.In conclusion, we demonstrated that MGs/MΦs negatively regulated the proliferation and neuronal differentiation of iNSPCs isolated from the brains of mice after stoke induced by MCAO. Although the precise impact of MGs/MΦs on iNSPCs should be elucidated in future in vivo studies, the present study findings indicate that the regulation of MGs/MΦs should be considered as a therapeutic strategy to achieve iNSPC-based neural repair after an ischemic stroke.


When used as photocatalysts for Rhodamine B (RhB), hollow CuS microspheres can degrade more than 99% of the initial RhB within 21 min. These excellent Li-ion storage properties and photocatalytical performances are attributed to their unique hierarchical hollow structure.Herein, well-defined hollow CuS microspheres assembled from nanosheets were successfully synthesized through a facile solvothermal method. Hollow CuS microspheres have an average diameter of 1.5 μm; moreover, the primary CuS nanosheets have an ultrathin thickness of about 10 nm and are bound by {0001} polar facets. When used as anodes for lithium-ion batteries (LIBs), hollow CuS microspheres exhibit excellent electrochemical properties, including a large discharge capacity (610.1 mAh g Recently, with energy and environment issues becoming more and more prominent, developing high-performance electrochemical energy storage (EES) devices and high-efficiency pollution control technologies has attracted tremendous research attention ,2. For t2O3, TiO2, Nb2O5, and so on, have not only served as anodes for LIBs, but have also acted as photocatalysts for wastewater treatment 2+ complex ions (Cu2+ + 2TAA → [Cu(TAA)2]2+) and then these [Cu(TAA)2]2+ ions are converted into CuS nuclei and H2S gas ([Cu(TAA)2]2+ + 4H2O → CuS↓ + 2H+ + 2CH3COONH4 + H2S↑). It is known that the growth orientations of crystals are closely related to their intrinsic crystal structures  and [Cu3S]) are alternatively arranged along the [001] direction. Specifically, in the [CuS3] layer, each Cu atom is coordinated by four S atoms to form a tetrahedron, whereas each Cu atom in the [Cu3S] layer is coordinated by three S atoms in a triangle [ab planes rather than along the c-axis, resulting in the generation of hexagonal sheet-like building blocks [2S gas bubbles [During the synthesis, the Cuructures . Accorditriangle . Owing tg blocks . During  bubbles . FinallyIn the synthetic system, it is deduced that glycerol content plays an important role in the formation of S-1. Glycerol has a considerably higher viscosity than water (243 vs. 3 mPa s), so the primary CuS nanosheets have sufficient time to search for the low-energy configuration interface and assemble into a well-defined hollow microstructure in the deionized water–glycerol solvent . Control−1 over a voltage range of 0.01–3.0 V (vs. Li+/Li), and the result of this is shown in xCuS (CuS + xLi+ + xe− → LixCuS) and the oxidation peaks in the charge process are associated with the formation of CuS. The Li-ion storage properties of S-1 were researched via the galvanostatic discharge–charge method. +/Li) at different current densities. As the current densities are 0.5, 1, 2, and 5 C, the first discharge capacities are 610.1, 230.7, 194.9, and 158.9 mAh g−1, respectively. The rate capability of S-1 was studied using a progressive discharge and charge from 0.5, 1 to 2, and 5 C for 10 cycles at each current density. As shown in −1 at 0.5, 1, 2, and 5 C, respectively. Furthermore, the discharge capacity returns to 192.3 mAh g−1 as the current density is reduced to 1 C. These results indicate that the anode has an excellent rate performance and good structural stability at a high current density. The cyclic stability of S-1 was also investigated. The anode was first activated at a low current rate of 0.5 C for 10 cycles, and then the anode was repeatedly charged and discharged at 1 C. As shown in −1 after 500 cycles, keeping a capacity retention of 88.2% compared to the first cycle. Additionally, compared to other copper-sulfide-based anodes [The cyclic voltammogram (CV) curve of S-1 was tested at a scan rate of 0.1 mV sd anodes ,42,43,44+ storage properties, which may be attributed to its unique structure. At first, its hierarchical hollow structure is beneficial for the penetration of electrolyte into electrode and it can supply extra active sites for the storage of Li+ ions. Thus, S-1 has a high discharge capacity [+/e− diffusion in the electrode is proportional to the diffusion length. The primary CuS nanosheets have a thin thickness (ca. 10 nm), so the transfer rate of Li+ and e− can be effectively enhanced, resulting in a good rate capacity [Such results indicate that S-1 possesses excellent Licapacity ,45. Secocapacity ,46,47. T−) and holes (h+) (CuS + hυ → e− + h+). Then, the as-formed electrons leap into the bottom of the conduction band (CB) and react with O2 on the surface of the photocatalyst to form a superoxide radical (e− + O2 → ·O2−). In addition, the holes stay at the top of the valence band (VB) and react with H2O to generate hydroxyl radicals (h+ + H2O → ·OH + H+). Finally, the active substances (O2− and ·OH−) can oxidize RhB into smaller molecules .S-1 can be used as a photocatalyst for the degradation of organic dyes  due to its unique hierarchical hollow structure. The photocatalytic performance of S-1 was tested by the photocatalytic degradation of RhB under visible light irradiation. As a comparison, S-2 was also tested under the same conditions. In the photocatalytic process ,48, unde2− can be oxidized by h+ into S° or SO42−, thus decreasing the photocatalytic activity [activity ,55. Acco−/h+ recombination problem significantly affects the photocatalytic performance of photocatalysts. As for S-1, the ultrathin thickness of the primary nanosheets provides a short distance for the e− transfer from the interior to the surface; moreover, the charge transfer is facilitated by the high crystallinity of the nanosheets. Hence, the h+/e− recombination in the bulk is suppressed and the photocatalytic performance of S-1 is enhanced accordingly [+ and e− is effectively prevented, endowing S-1 with a good photocatalytic activity and stability for organic pollutant degradation.It is deduced that the outstanding degradation efficiency of S-1 stems from the following reasons. Firstly, S-1 has a large specific surface area, so it conveniently absorbs more photons and produces plenty of electron–hole pairs . Secondlordingly ,59. Fourordingly ,61. Acco−1 and they can retain a high capacity of 143.4 mAh g−1 even at 5 C. In addition, after 500 cycles at 1 C, they exhibit a high discharge capacity of 196.3 mAh g−1. On the other hand, hollow CuS microspheres degrade more than 99% of RhB within 21 min. Hence, hollow CuS microspheres show potential applications as anodic material for LIBs and photocatalysts for the treatment of organic dyes.In summary, we report the solvothermal syntheses, formation mechanisms, Li-ion storage properties, and photocatalytic performances of hollow CuS microspheres. Owing to the synergistic effects of their hollow structures, ultrathin nanosheets, special exposed facets, and hierarchical superstructures, hollow CuS microspheres have improved electrochemical and photocatalytic properties. At 0.5 C, hollow CuS microspheres exhibit a high discharge capacity of 610.1 mAh g


It is now widely accepted that radiotherapy (RT) can provoke a systemic immune response, which gives a strong rationale for the combination of RT and immune checkpoint inhibitors (ICIs). However, RT is a double-edged sword that not only enhances systemic antitumor immune response, but also promotes immunosuppression to some extent. Nevertheless, many aspects regarding the efficacy and safety of this combination therapy remain unknown. Therefore, a systematic review and meta-analysis was performed in order to assess the safety and efficacy of RT/chemoradiotherapy (CRT) and ICI combination therapy for non-small cell lung cancer (NSCLC) patients.th of February 2022.PubMed and several other databases were searched (according to specific criteria) to find relevant studies published prior to the 282: 96.7%) and 2.03% , respectively. Fatigue (50.97%), dyspnea (46.06%), dysphagia (10%-82.5%), leucopenia (47.6%), anaemia (5%-47.6%), cough (40.09%), esophagitis (38.51%), fever (32.5%-38.1%), neutropenia (12.5%-38.1%), alopecia (35%), nausea (30.51%) and pneumonitis (28.53%) were the most common adverse events for the combined treatment. The incidence of cardiotoxicity (0%-5.00%) was low, but it was associated with a high mortality rate (0%-2.56%). Furthermore, the incidence of pneumonitis was 28.53% , grade ≥ 3 pneumonitis was 5.82%  and grade 5 was 0%-4.76%.3,652 articles were identified for screening and 25 trials containing 1,645 NSCLC patients were identified. For stage II-III NSCLC, the one- and two-year overall survival (OS) was 83.25% : 79.42%-86.75%) and 66.16% (95% CI: 62.3%-69.92%), respectively. For stage IV NSCLC, the one- and two-year OS was 50% and 25%. In our study, the pooled rate of grade 3-5 adverse events (AEs) and grade 5 AEs was 30.18% (95% CI: 10.04%-50.33%, IThis study suggests that the addition of ICIs to RT/CRT for NSCLC patients may be both safe and feasible. We also summarize details of different RT combinations with ICIs to treat NSCLC. These findings may help guide the design of future trials, the testing of concurrent or sequential combinations for ICIs and RT/CRT could be particularly useful to guide the treatment of NSCLC patients. Immune via T cell activation  (P=<0.001). Additionally, radiation recall pneumonitis localized within a previously irradiated area may occur when using ICIs  . BotticeP=0.046)  also obsing ICIs . Hence, The efficacy and safety of RT/chemoradiotherapy (CRT) when combined with ICIs remains controversial, with the selection of ICIs needing further exploration and optimization. This is also true for aspects regarding the accompanying RT, including: ICI agent selection, treatment sequencing, RT dose, fractionation and irradiated site applicability. Therefore, a systematic review and meta-analysis to elucidate RT&ICI combination therapy efficacy and toxicity for NSCLC was performed and described herein.22.1This systematic review and meta-analysis was conducted using the Preferred Reporting Items for Systematic Review and Meta-analysis statement . Ethicalth of February 2022, without a lower date boundary. The following search terms were used: 1) “non-small cell lung neoplasm (s)/cancer (s)/carcinoma (s)” or “NSCLC”. 2) “radiotherapy” or “radiation therapy” or “radiation treatment” or “irradiation” or “SABR” or “stereotactic ablative radiotherapy” or “SBRT” or “stereotactic body radiation therapy” or “SRS” or “stereotactic radiosurgery” or “SRT” or “stereotactic radiotherapy” or “radio-chemotherapy” or “chemoradiotherapy”. 3) “immunotherapy” or “immune checkpoint inhibitors” or “checkpoint inhibitor “ or “checkpoint blockade” or “programmed cell death 1 receptor” or “programmed cell death 1 ligand 1” or “programmed death-1” or “PD-1” or “programmed death ligand-1” or “PD-L1” or “cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) antigen” or “anti-CTLA-4” or “anti-PD-1” or “anti-PD-L1” or “CTLA-4” or “Durvalumab” or “Imfinzi” or “ MEDI-4736” or “Atezolizumab” or “ MPDL3280A” or “Tecentriq” or “RO5541267” or “RG7446” or “Pembrolizumab” or “Lambrolizumab” or “Keytruda” or “SCH 900475” or “MK-3475” or “Nivolumab” or “Opdivo” or “Ono-4538” or “MDX-1106” or “BMS-936558” or “Nivo” or “Avelumab” or “Barvencik” or “MSB0010718C” or “Toripalima” or “Tislelizumab” or “Camrelizumab” or “Sintilimab” or “Tremelimumab” or “Ipilimumab” or “Cemiplimab” or “Libtayo” or “PDCD1” or “CD274”.The comprehensive search string is available as PubMed, Web of Science and the Cochrane Library were searched to identify literature in English language journals published prior to the 28Studies that met the following criteria were included in the meta-analysis: 1) describe participants with histologically confirmed lung cancer; 2) original papers describing human clinical trials that reported the outcomes for CRT/RT combined with ICIs; 3) prospective or retrospective study; 4) outcomes included treatment safety and efficacy; 5) published in English.Conference abstracts, case reports, comments, reviews, animal studies, and mechanistic studies were excluded. Studies that did not have sufficient data  or unclear descriptions  were also excluded. When articles described the same study population, only the most recent or complete analysis was included. Disagreements related to article selection were resolved in a discussion with all the authors of this study.2.2The data was extracted by two independent investigators, including: first author name, time of publication, country, number of cases, pathological subgroups, type of study, treatment regimens, radiotherapy type and dose, treatment characteristics including timing of ICI therapy and rates of pneumonitis and treatment related deaths. Any discrepancies were resolved through group discussions until a consensus was reached. The revised Cochrane Risk of Bias tool for randomized trials  and Risk2.32 statistics were used to assess heterogeneity. 0%, 25%, 50% and 75% of I2 represented no, low, moderate and high heterogeneity, respectively. Meta-regression was considered inappropriate due to the insufficient number of studies (<10). Subgroup and sensitivity analyses were performed to explore study heterogeneity and to determine the impact of each individual study. Publication bias was estimated using the Begg’s and Egger’s test. P=<0.05 was used as the threshold for considering statistical significance.The random effects model was used for the meta-analysis with the “meta” package implemented in R . A 95% confidence interval (CI) was used, and Cochran’s Q and I3The search process Figure 13.1The trials included in this study had different primary efficacy variables, which prevented the performance of an efficacy based meta-analysis. The objective response rate (ORR) was 25.8%-73.4% for II-III stage NSCLC and 45%-45.9% for IV stage NSCLC. The one-year to five-year PFS and OS for II-III stage NSCLC and IV stage NSCLC were shown in 3.22: 96.7%) and 2.03%  for grade 5 adverse events (All adverse events are described in e events 3. The m3.32: 92.00%). For conventional RT, the incidence of grade 1-5 pneumonitis was 29.92% , and 12.79%  for SBRT . For conventional RT the occurrence of grade 3-5 pneumonitis was 5.10% , and 11.62%  for SBRT   Figure 6In our study, only one trial  explored3.3.22: 89.0%) which was higher than those targeting programmed cell death protein-1 (PD-1) . The rate of grade 3-5 pneumonitis for PD-L1 was 5.07%  and 5.45%  for PD-1 (The pooled rate of grade 1-5 pneumonitis for patients treated with ICIs programmed cell death-ligand 1 (PD-L1) was 35.09%  and 4.79% , respectively. The rate of grade 1-5 and 3-5 pneumonitis for patients administered ICIs for less than one year, was 0%-17.24% and 0%-6.90%, respectively.When ICIs were administered for more than one year, the rate of grade 1-5 and grade 3-5 pneumonitis was 26.57%  and grade 3-5   7. A sub3.4The Begg’s and Egger’s tests are enumerated in 4The study included herein provides an overview of published NSCLC trials focusing on the use of RT with ICIs. This review and meta-analysis systematically, quantitatively and comprehensively analyzed the efficacy and toxicity of RT when combined with ICIs for 1,645 NSCLC patients from 25 studies.The efficacy of RT when combined with ICIs  appeared to be superior for patients that had previously received treatment for metastatic NSCLC. The efficacy (ORR) for nivolumab or pembrolizumab against pretreated metastatic NSCLC ranged from 19 to 36% in the Checkmate 017, Checkmate 057 and Keynote 011 trials , 53. The2:39.4%), one-year OS was 83.25%  and two-year OS was 66.16% . When viewed in relation to other recent reports, the one-year PFS was 48% in both the 60 Gy group from RTOG 0617  and 2.03%  for grade 5 AEs, which was lower than the aforementioned study.Zhou et al. , found tCardiotoxicity is a rare but potentially fatal adverse effect of ICI therapy, for NSCLC patients, the overall incidence of cardiotoxicity is low (0%-5%); but the mortality associated with cardiotoxicity is high (0%-2.56%). We found that the most common form of lethal cardiotoxicity was myocardial infarction (0.21%-2.56%). In our study, the incidence of grade 1-5 myocarditis was 0.76%, which is within the range found by previous studies (0.05%-1.14%) , 58. RT&2: 92.00%), with a 5.82%  incidence of grade ≥ 3 pneumonitis and 0%-4.76% for grade 5. Thus, the incidence of pneumonitis following RT&ICIs is similar to RT alone, and likely safe for use in NSCLC patients.Overall, the most common grade 5 adverse event was pneumonitis (0%-4.76%). Previous meta-analyses have demonstrated that the incidence of radiation pneumonitis is 1.5%–40%, with a 1.1%-6.6% incidence of grade ≥ 3 pneumonitis and 0%-3.1% for grade 5 , 60. In There remain concerns among researchers in the field that the RT&ICI prospective studies may not reflect the real-world clinical application. For example, patients with a history of interstitial lung disease were excluded from prospective trials. This notion is supported by Suresh et al. , who fouOnly one trial  includedElective node irradiation is performed for NSCLC patients to treat lymph nodes that are not known to contain metastases, including the bilateral hilar, mediastinum, and even supraclavicular areas. Grills et al. , has sugIn addition to involved-field irradiation, clinical target volume (CTV) omission is another strategy that has been used in conjunction with immunotherapy. CTV is defined as the volume of tissue containing gross tumor volume and subclinical microscopic malignant lesions. A retrospective study of 105 stage III NSCLC patients treated with (n=50) or without (n=55) CTV did not experience a significantly different rate of local recurrence, distant metastasis, PFS or OS. Notably, the incidence of grade 3–4 radiation-pneumonitis was significantly lower in group that did not receive CTV (P=0.044) . KilburnWe found higher rates of grade 1-5 pneumonitis in studies where a total radiation dose of >=60 Gy (vs. < 60 Gy) was administered (33.70% vs. 15.98%). Prior to the advent ICIs, the RTOG 7301 trial established that the standard radiation dose for unresectable NSCLC was 60–63 Gy in 1.8–2 Gy daily fractions . SubsequP < 0.05) better ORR  than both non-ablative radiotherapy (18% ORR with 45Gy/15 fractions) or pembrolizumab (20% ORR) alone  alone . AdditioR) alone . SeveralR) alone , 80. LimR) alone  demonstrRadiotherapy dose distribution also plays an important role, according to results from the phase III RTOG 0617 clinical trial, a V20 dose of less than 35% can minimize the risk of pneumonitis . Shaikh Our study identified only one trial that investigated the effects on pneumonitis for different radiotherapy techniques in combination with ICIs . HoweverAn analysis of two prospective metastatic NSCLC trials reported that the PFS associated with SBRT when combined with anti-PD1 treatment was significantly better than anti-CTLA4; although the efficacy was not statistically different . Gu et a2: 89.1%) rate of grade 1-5 pneumonitis and a 4.79%  rate of grade 3-5 pneumonitis. When the ICI treatment lasted for less than one year, the rate of grade 1-5 and grade 3-5 pneumonitis was 0%-17.24% and 0%-6.90%, respectively. Sukari et al.  showed improved PFS and OS ; anotherThe PACIFIC trial  found thSome difficulties remain for the identification of patients that will gain the greatest survival benefit from combination therapy or those who are more likely to have adverse effects. PD-L1 and tumor mutation burden (TMB) were widely used in clinical stratification of ICI patients, but their predictive effects are still controversial. Many other markers based upon tumor tissue, peripheral blood, and radiological images have been investigated, including: cell surface markers, various immune cells, immune related gene and imaging biomarkers . HoweverIt is important to note that most of the studies included in this meta-analysis were single-arm clinical trials, which prevents the comparison of the advantages and disadvantages associated with CRT/RT + ICIs and CRT/RT based upon a balanced baseline. Whilst the results from these trials may contain a high amount of statistical heterogeneity, subgroup analyses were performed to identify sources of study heterogeneity. Finally, due to data availability for lung cancer patients in this field, we could not explore certain details regarding the efficacy and safety of RT/CRT plus ICIs, including: the influence of different radiotherapy doses/fractionations, irradiation target volumes, chemotherapy regimen.5In conclusion, this comprehensive meta-analysis of 1,645 non-small cell lung cancer patients (from 25 studies) systemically and quantitatively explored the clinical efficacy and safety of immune checkpoint inhibitors when combined with radiotherapy/chemoradiotherapy. Based upon the studies presented herein, the combination of (chemo)radiotherapy and immune checkpoint inhibitors appears to be a both safe and feasible mode of treatment. These findings may help clinicians in the design of future trials testing concurrent or sequential combinations of immune checkpoint inhibitors and radiotherapy/chemoradiotherapy for the treatment of patients with non-small cell lung cancer.The original contributions presented in the study are included in the article/YuZ and YaZ conceptualized the study. JW, RD, YL, CF and TN collected the data. JW, TN, QinZ, FT, QiZ and YX analyzed the data. JW and YZ wrote the manuscript. All authors contributed to the article and approved the submitted version.