Development and External Validation of an Artificial Intelligence-Based Method for Scalable Chest Radiograph Diagnosis: A Multi-Country Cross-Sectional Study.

Problem: Chest radiography is a crucial tool for diagnosing thoracic disorders, but interpretation errors and a lack of qualified practitioners can cause delays in treatment. Aim: This study aimed to develop a reliable multi-classification artificial intelligence (AI) tool to improve the accuracy and efficiency of chest radiograph diagnosis. Methods: We developed a convolutional neural network (CNN) capable of distinguishing among 26 thoracic diagnoses. The model was trained and externally validated using 795,055 chest radiographs from 13 datasets across 4 countries. Results: The CNN model achieved an average area under the curve (AUC) of 0.961 across all 26 diagnoses in the testing set. COVID-19 detection achieved perfect accuracy (AUC 1.000, [95% confidence interval {CI}, 1.000 to 1.000]), while effusion or pleural effusion detection showed the lowest accuracy (AUC 0.8453, [95% CI, 0.8417 to 0.8489]). In external validation, the model demonstrated strong reproducibility and generalizability within the local dataset, achieving an AUC of 0.9634 for lung opacity detection (95% CI, 0.9423 to 0.9702). The CNN outperformed both radiologists and nonradiological physicians, particularly in trans-device image recognition. Even for diseases not specifically trained on, such as aortic dissection, the AI model showed considerable scalability and enhanced diagnostic accuracy for physicians of varying experience levels (all P < 0.05). Additionally, our model exhibited no gender bias (P > 0.05). Conclusion: The developed AI algorithm, now available as professional web-based software, substantively improves chest radiograph interpretation. This research advances medical imaging and offers substantial diagnostic support in clinical settings.

Alpha sensory stimulation modulates theta phase during speech-print associative learning.

Applying 10 Hz (α-rate) sensory stimulation, not 5 Hz (θ-rate), prior to introducing novel speech-print pairs can reset the phase of θ oscillations and enhance associative learning. This rapid gain indicates coordinated mechanisms to regulate attentional/cognitive resources (α oscillations) and facilitate memory storage (θ oscillations) early in learning. The present findings may inform educational practices for children with reading difficulties.

Association between glucose levels and all-cause mortality in cancer survivors: findings from NHANES 1999-2018.

BACKGROUND: Hyperglycemia is a rapidly increasing risk factor for cancer mortality worldwide. However, the dose‒response relationship between glucose levels and all-cause mortality in cancer survivors is still uncertain. METHODS: We enrolled 4,491 cancer survivors (weighted population 19,465,739) from the 1999-2019 National Health and Nutrition Examination Survey (NHANES). Cancer survivors were defined based on the question of whether they had ever been diagnosed with cancer by a doctor or a health professional. Hemoglobin A1c (HbA1c) was selected in this study as a stable marker of glucose level. Mortality was ascertained by linkage to National Death Index records until December 31, 2019. Cox proportional hazard, Kaplan‒Meier survival curves and Restricted cubic spline regression models were used to evaluate the associations between HbA1c and all-cause mortality risk in cancer survivors. RESULTS: In NHANES, after adjusting for confounders, HbA1c had an independent nonlinear association with increased all-cause mortality in cancer survivors (nonlinear P value < 0.05). The threshold value for HbA1c was 5.4%, and the HRs (95% CI) below and above the threshold value were 0.917 (0.856,0.983) and 1.026 (1.010,1.043), respectively. Similar associations were found between fasting glucose and all-cause mortality in cancer survivors, and the threshold value was 5.7 mmol/L. CONCLUSIONS: HbA1c was nonlinearly associated with all-cause mortality in cancer survivors, and the critical value of HbA1c in decreased mortality was 5.4%, suggesting optimal glucose management in cancer survivors may be a key to preventing premature death in cancer survivors.

A risk score based on polyamine metabolism and chemotherapy-related genes predicts prognosis and immune cells infiltration of lung adenocarcinoma.

We aimed to explore whether the genes associated with both platinum-based therapy and polyamine metabolism could predict the prognosis of LUAD. We searched for the differential expression genes (DEGs) associated with platinum-based therapy, then we interacted them with polyamine metabolism-related genes to obtain hub genes. Subsequently, we analysed the main immune cell populations in LUAD using the scRNA-seq data, and evaluated the activity of polyamine metabolism of different cell subpopulations. The DEGs between high and low activity groups were screened to identify key DEGs to establish prognostic risk score model. We further elucidated the landscape of immune cells, mutation and drug sensitivity analysis in different risk groups. Finally, we got 10 hub genes associated with both platinum-based chemotherapy and polyamine metabolism, and found that these hub genes mainly affected signalling transduction pathways. B cells and mast cells with highest polyamine metabolism activity, while NK cells were found with lowest polyamine metabolism activity based on scRNA-seq data. DEGs between high and low polyamine metabolism activity groups were identified, then 6 key genes were screened out to build risk score, which showed a good predictive power. The risk score showed a universal negative correlation with immunotherapy checkpoint genes and the cytotoxic T cells infiltration. The mutation rates of EGFR in low-risk group was significantly higher than that of high-risk group. In conclusion, we developed a risk score based on key genes associated with platinum-based therapy and polyamine metabolism, which provide a new perspective for prognosis prediction of LUAD.

miR-185-5p May Modulate the Chemosensitivity of LUSC to Cisplatin via Targeting PCDHA11: Multi-omics Analysis and Experimental Validation.

Drug resistance is the major difficulty in treatment of lung squamous cell carcinoma (LUSC). This study aims to explore drug response-related miRNAs (DRmiRNAs) based on multi-omics research. We identified DRmiRNAs of LUSC with a multi-omics integrated system that combines expression data of microRNA, lncRNA, mRNA, methylation levels, somatic mutations. After identifying DRmiRNAs, we screened and validated of the target mRNAs of DRmiRNAs through Targetscan and the miRDB database. Then, Real-time PCR and Western blot assays were used to estimate the expression of DRmiRNAs and target protein, and the dual-luciferase assays were used to confirm the interaction of DRmiRNAs and target mRNA. Furthermore, CCK-8 (Cell Counting Kit-8) assays were used to evaluate cell proliferation and drug sensitivity. After integrated analysis, hsa-miR-185-5p was identified as DRmiRNA based on multi-omics data. Through Targetscan and miRDB database, the possible target mRNAs were obtained and PCDHA11 was validated as a target mRNA of miR-185-5p by real-time PCR, Western blot assays and dual-luciferase assays. CCK-8 assays and clone formation assays showed that the proliferation of miR-185-5p mimics was significantly slower than that of miR-185-5p inhibitors, which means overexpression of miR-185-5p enhanced the anticancer effects of cisplatin, whereas the downregulation of miR-185-5p reduced the effects. Furthermore, the proliferation of silencing PCDHA11 was significantly slower than that of overexpression of PCDHA11, which means PCDHA11 overexpression weakened the anticancer effects of cisplatin, and silencing PCDHA11 enhanced the effects. This study demonstrated that miR-185-5p was involved in chemoresistance of LUSC cells to cisplatin partly via down-regulating PCDHA11, which may promote understanding the underlying molecular mechanisms of drug response.

The cognitive-linguistic profiles and academic performances of Chinese children with dyslexia across cultures: Beijing, Hong Kong, and Taipei.

This study examined the cognitive-linguistic and literacy-related correlates of dyslexia in three Chinese cities and the English word reading and mathematics performances of Chinese children with dyslexia. Chinese children with/without dyslexia were measured with an equivalent test battery of literacy and mathematics in Beijing, Hong Kong, and Taipei. Univariate analysis results suggested that phonological sensitivity distinguished those with and without dyslexia across all three cities in group comparisons. In Taipei and Hong Kong, morphological awareness, delayed copying, and spelling also distinguished the groups. Logistic regression analyses demonstrated that Chinese character reading, as directly compared to Chinese word reading, also distinguished the groups particularly well. In addition, in Beijing and Hong Kong, children with dyslexia performed significantly less well in English word reading than those without dyslexia. In Hong Kong and Taipei, children with dyslexia also had difficulties in mathematics performance. Findings highlight the fundamental importance of some cognitive-linguistic skills for explaining Chinese dyslexia across cultures, the utility of recognizing the individual Chinese character as a foundational unit of analysis in Chinese across cultures, and the generalizability of the comorbidity of both English as a second language (L2) and mathematics with dyslexia in Chinese children in both Beijing and Hong Kong.

FSTL3 is associated with prognosis and immune cell infiltration in lung adenocarcinoma.

PURPOSE: FSTL3 expression is altered in various types of cancer. However, the role and mechanism of action of FSTL3 in lung adenocarcinoma development and tumor immunity are unknown. We investigated the association between FSTL3 expression and clinical characteristics and immune cell infiltration in lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA) and a separate validation set from our hospital. METHODS: Data on immune system infiltration, gene expression, and relevant clinical information were obtained by analyzing lung adenocarcinoma sample data from TCGA database. Using online tools like GEPIA, the correlations between FSTL3 expression and prognosis, clinical stage, survival status, and tumor-infiltrating immune cells were examined. In a validation dataset, immunohistochemistry was performed to analyze FSTL3 expression and its related clinical characteristics. RESULTS: FSTL3 expression was markedly reduced in patients with lung adenocarcinoma. N stage, pathological stage, and overall survival were significantly correlated with FSTL3 expression. According to GSEA, FSTL3 is strongly linked to signaling pathways such as DNA replication and those involved in cell cycle regulation. Examination of TCGA database and TIMER online revealed a correlation between FSTL3 and B cell, T cell, NK cell, and neutrophil levels. The prognosis of patients with lung adenocarcinoma was significantly affected by six genes (KRT6A, VEGFC, KRT14, KRT17, SNORA12, and KRT81) related to FSTL3. CONCLUSION: FSTL3 is significantly associated with the prognosis and progression of lung adenocarcinoma and the infiltration of immune cells. Thus, targeting FSTL3 and its associated genes in immunotherapy could be potentially beneficial for the treatment of lung adenocarcinoma.

Kondo Effect of Co-Porphyrin: Remarkable Sensitivity to Adsorption Sites and Orientations.

We investigated the Kondo effect of cobalt(II)-5-15-bis(4'-bromophenyl)-10,20-bis(4'-iodophenyl)porphyrin (CoTPPBr2I2) molecules on Au(111) with low-temperature scanning tunneling microscopy under ultrahigh vacuum conditions. The molecules exhibit four adsorption configurations at the top and bridge sites of the surface with different molecular orientations. The Kondo resonance shows extraordinary sensitivity to the adsorption configuration. By switching the molecule between different configurations, the Kondo temperature is varied over a wide range from ≈8 up to ≈250 K. Density functional theory calculations reveal that changes of the adsorption configuration lead to distinct variations of the hybridization between the molecule and the surface. Furthermore, we show that surface reconstruction plays a significant role for the molecular Kondo effect.

A risk score model based on lipid metabolism-related genes could predict response to immunotherapy and prognosis of lung adenocarcinoma: a multi-dataset study and cytological validation.

BACKGROUND: Lipid metabolism is a key factor in tumorigenesis and drug resistance, and models related to lipid metabolism have shown potential to predict survival and curative effects of adjuvant therapy in various cancers. However, the relationship between lipid metabolism and prognosis and treatment response of lung adenocarcinoma (LUAD) are still unclear. METHODS: We enrolled seven bulk RNA-sequence datasets (GSE37745, GSE19188, GSE30219, GSE31547, GSE41271, GSE42127, and GSE72094) from the GEO database and one single-cell RNA-sequencing dataset (GSE117570) from the TISCH2 database. Non-negative matrix factorization (NMF) was utilized to construct the risk score model based on lipid score calculated by GSVA algorithm. Phs000452.v3, PMID: 26359337, PMID: 32472114, PRJEB23709 datasets were used to test the response to immunotherapy. Drug sensitivity analysis was assessed according to the GDSC database, and immunotherapy response was evaluated using the Wilcoxon test. Cellular function assays including clone formation, EDU assays and flow cytometry were implemented to explore the phenotype alteration caused by the knockdown of PTDSS1, which is one of key gene in risk score model. RESULTS: We analyzed both bulk and single-cell RNA sequencing data to establish and validate a risk score model based on 18 lipid metabolism-related genes with significant impact on prognosis. After divided the patients into two groups according to risk score, we identified differences in lipid-related metabolic processes and a detailed portrait of the immune landscapes of high- and low-risk groups. Moreover, we investigated the potentials of our risk score in predicting response to immunotherapy and drug sensitivity. In addition, we silenced PTDSS1 in LUAD cell lines, and found that the proliferation of the cells was weakened, and the apoptosis of the cells was increased. CONCLUSION: Our study highlights the crucial roles of lipid metabolism in LUAD and provides a reliable risk score model, which can aid in predicting prognosis and response to immunotherapy. Furthermore, we investigated the roles of PTDSS1 in LUAD carcinogenesis, which showed that PTDSS1 regulated proliferation and apoptosis of LUAD cells.

Prognostic and immune correlation analysis of mitochondrial autophagy and aging-related genes in lung adenocarcinoma.

PURPOSE: Mitophagy and aging (MiAg) are very important pathophysiological mechanisms contributing to tumorigenesis. MiAg-related genes have prognostic value in lung adenocarcinoma (LUAD). However, prognostic, and immune correlation studies of MiAg-related genes in LUAD are lacking. METHODS: MiAg differentially expressed genes (DEGs) in LUAD were obtained from public sequencing datasets. A prognostic model including MiAg DEGs was constructed according to patients divided into low- and high-risk groups. Gene Ontology, gene set enrichment analysis, gene set variation analysis, CIBERSORT immune infiltration analysis, and clinical characteristic correlation analyses were performed for functional annotation and correlation of MiAgs with prognosis in patients with LUAD. RESULTS: Seven MiAg DEGs of LUAD were identified: CAV1, DSG2, DSP, MYH11, NME1, PAICS, PLOD2, and the expression levels of these genes were significantly correlated (P < 0.05). The RiskScore of the MiAg DEG prognostic model demonstrated high predictive ability of overall survival of patients diagnosed with LUAD. Patients with high and low MiAg phenotypic scores exhibited significant differences in the infiltration levels of eight types of immune cells (P < 0.05). The multi-factor DEG regression model showed higher efficacy in predicting 5-year survival than 3- and 1-year survival of patients with LUAD. CONCLUSIONS: Seven MiAg-related genes were identified to be significantly associated with the prognosis of patients diagnosed with LUAD. Moreover, the identified MiAg DEGs might affect the immunotherapy strategy of patients with LUAD.

Global, regional, and national time trends in cancer mortality attributable to high fasting plasma glucose: an age-period cohort analysis.

BACKGROUND: High fasting plasma glucose (HFPG) is the fastest-growing risk factor for cancer deaths worldwide. We reported the cancer mortality attributable to HFPG at global, regional, and national levels over the past three decades and associations with age, period, and birth cohort. METHODS: Data for this study were retrieved from the Global Burden of Disease Study 2019, and we used age-period-cohort modelling to estimate age, cohort and period effects, as well as net drift (overall annual percentage change) and local drift (annual percentage change in each age group). RESULTS: Over the past 30 years, the global age-standardized mortality rate (ASMR) attributable to HFPG has increased by 27.8%. The ASMR in 2019 was highest in the male population in high sociodemographic index (SDI) areas (8.70; 95% CI, 2.23-18.04). The net drift for mortality was highest in the female population in low SDI areas (2.33; 95% CI, 2.12-2.55). Unfavourable period and cohort effects were found across all SDI quintiles. Cancer subtypes such as "trachea, bronchus, and lung cancers", "colon and rectal cancers", "breast cancer" and "pancreatic cancer" exhibited similar trends. CONCLUSIONS: The cancer mortality attributable to HFPG has surged during the past three decades. Unfavourable age-period-cohort effects on mortality were observed across all SDI quintiles, and the cancer mortality attributable to HFPG is expected to continue to increase rapidly in the future, particularly in lower SDI locations. This is a grim global public health issue that requires immediate attention.

Evaluation of Male Breast Cancer and the Application of Sentinel Lymph Node Biopsy: A Multicenter Retrospective Study.

Sentinel lymph node biopsy (SLNB) is currently used as a routine treatment for patients with breast cancer. However, it may not be applicable for patients with male breast cancer (MBC), because they have notably different clinicopathological features from those occurring in females. There is a lack of evidence of SLNB application and safe exemption from axillary lymph node dissection (ALND) in patients with MBC. This study aimed to evaluate the application of SLNB to provide information for the standardized treatment of patients with MBC. The MBC patient records from 4 institutions ranging from January 2001 to November 2020 were retrospectively reviewed. There were 220 patients with MBC with a median age of 60 (range 24-88) years and an average tumor size of 2.3 cm (range 0.5 cm-6.5 cm). Sixty-six percent of patients underwent SLNB, and 39% of them showed positive results. A total of 157 patients underwent ALND, while only half of them had positive nodes, causing unnecessary complications. For patients in the clinical early stage, we found that the SLNB showed a noninferiority to the ALND treatment in DFS (P = .18) and OS (P = .055). In conclusion, there are certain obstacles to the broad application of SLNB due to the lower proportion of patients with clinically negative lymph nodes. However, it is undeniable that SLNB can safely and effectively exempt patients with MBC at early stage with clinically negative nodes from ALND to reduce subsequent complications. It is still an ideal criterion for the axillary staging of patients with MBC.

Martynoside rescues 5-fluorouracil-impaired ribosome biogenesis by stabilizing RPL27A.

Martynoside (MAR), a bioactive component in several well-known tonic traditional Chinese herbs, exhibits pro-hematopoietic activity during 5-fluorouracil (5-FU) treatment. However, the molecular target and the mechanism of MAR are poorly understood. Here, by adopting the mRNA display with a library of even-distribution (md-LED) method, we systematically examined MAR-protein interactions in vitro and identified the ribosomal protein L27a (RPL27A) as a key cellular target of MAR. Structural and mutational analysis confirmed the specific interaction between MAR and the exon 4,5-encoded region of RPL27A. MAR attenuated 5-FU-induced cytotoxicity in bone marrow nucleated cells, increased RPL27A protein stability, and reduced the ubiquitination of RPL27A at lys92 (K92) and lys94 (K94). Disruption of MAR binding at key residues of RPL27A completely abolished the MAR-induced stabilization. Furthermore, by integrating label-free quantitative ubiquitination proteomics, transcriptomics, and ribosome function assays, we revealed that MAR restored RPL27A protein levels and thus rescued ribosome biogenesis impaired by 5-FU. Specifically, MAR increased mature ribosomal RNA (rRNA) abundance, prevented ribosomal protein degradation, facilitated ribosome assembly, and maintained nucleolar integrity. Collectively, our findings characterize the target of a component of Chinese medicine, reveal the importance of ribosome biogenesis in hematopoiesis, and open up a new direction for improving hematopoiesis by targeting RPL27A.

Human SAMD9 is a poxvirus-activatable anticodon nuclease inhibiting codon-specific protein synthesis.

As a defense strategy against viruses or competitors, some microbes use anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAPhe), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAPhe depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAPhe-specific 2'-O-methylation at the wobble position, making virtually all eukaryotic tRNAPhe susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.

Outcomes and prognosis of non-small cell lung cancer patients who underwent curable surgery: a protocol for a real-world, retrospective, population-based and nationwide Chinese National Lung Cancer Cohort (CNLCC) study.

INTRODUCTION: Surgery is one of the main approaches for the comprehensive treatment of early and locally advanced non-small cell lung cancer (NSCLC). This study conducts a nationwide multicentre study to explore factors that could influence the outcomes of patients with I-IIIA NSCLC who underwent curable surgery in real-world scenarios. METHODS AND ANALYSIS: All patients diagnosed with NSCLC between January 2013 and December 2020 will be identified from 30 large public medical services centres in mainland China. The algorithm of natural language processing and artificial intelligence techniques were used to extract data from electronic health records of enrolled patients who fulfil the inclusion criteria. Six categories of parameters are collected and stored from the electronic records, then the parameters will be structured as a high-quality structured case report form. The code book will be compiled and each parameter will be classified and designated a code. In addition, the study retrieves the survival status and causes of death of patients from the Chinese Centre for Disease Control and Prevention. The primary endpoints are overall survival and the secondary endpoint is disease-free survival. Finally, an online platform is formed for data queries and the original records will be stored as secure electronic documents. ETHICS AND DISSEMINATION: The study has been approved by the Ethical Committee of the Chinese Academy of Medical Sciences. Study findings will be disseminated via presentations at conferences and publications in open-access journals. This study has been registered in the Chinese Trial Register (ChiCTR2100052773) on 11 May 2021, http://www.chictr.org.cn/showproj.aspx?proj=136659. TRIAL REGISTRATION NUMBER: ChiCTR2100052773.

Establishment and validation of a multigene model to predict the risk of relapse in hormone receptor-positive early-stage Chinese breast cancer patients.

BACKGROUND: Breast cancer patients who are positive for hormone receptor typically exhibit a favorable prognosis. It is controversial whether chemotherapy is necessary for them after surgery. Our study aimed to establish a multigene model to predict the relapse of hormone receptor-positive early-stage Chinese breast cancer after surgery and direct individualized application of chemotherapy in breast cancer patients after surgery. METHODS: In this study, differentially expressed genes (DEGs) were identified between relapse and nonrelapse breast cancer groups based on RNA sequencing. Gene set enrichment analysis (GSEA) was performed to identify potential relapse-relevant pathways. CIBERSORT and Microenvironment Cell Populations-counter algorithms were used to analyze immune infiltration. The least absolute shrinkage and selection operator (LASSO) regression, log-rank tests, and multiple Cox regression were performed to identify prognostic signatures. A predictive model was developed and validated based on Kaplan-Meier analysis, receiver operating characteristic curve (ROC). RESULTS: A total of 234 out of 487 patients were enrolled in this study, and 1588 DEGs were identified between the relapse and nonrelapse groups. GSEA results showed that immune-related pathways were enriched in the nonrelapse group, whereas cell cycle- and metabolism-relevant pathways were enriched in the relapse group. A predictive model was developed using three genes ( CKMT1B , SMR3B , and OR11M1P ) generated from the LASSO regression. The model stratified breast cancer patients into high- and low-risk subgroups with significantly different prognostic statuses, and our model was independent of other clinical factors. Time-dependent ROC showed high predictive performance of the model. CONCLUSIONS: A multigene model was established from RNA-sequencing data to direct risk classification and predict relapse of hormone receptor-positive breast cancer in Chinese patients. Utilization of the model could provide individualized evaluation of chemotherapy after surgery for breast cancer patients.

Functional preference of the left inferior parietal lobule to second language reading.

Additional neural substance for reading in a second language has been reported by prior studies. However, to date, there has been little investigation into whether and how the brain's adaptation to a second language is induced by specific linguistic tasks or is a general effect during reading in a new language. To address this issue, our study investigated Chinese children learning English as a second language by combining cross-sectional and longitudinal Functional Magnetic Resonance Imaging (fMRI) studies. We compared brain activation across four reading tasks, orthographic tasks and phonological tasks in Chinese (the first language, L1) and English (the second language, L2). By comparing the activation pattern across languages, we observed greater activation in the left inferior parietal lobule (LIPL) in English compared to Chinese, suggesting a functional preference of the LIPL to L2. In addition, greater correlation between LIPL-related FC and L2 was mainly observed in the phonological task, indicating that LIPL could be associated with phonological processing. Moreover, a proportion of the children were enrolled in an 8-week phonological-based reading-training program. We observed significant functional plasticity of the LIPL elicited by this training program only in the English phonological task and not in the orthographic task, further substantiating that the additional requirements of the LIPL in L2 are mainly associated with phonological processing. The findings provide new insights into understanding the functional contribution of the LIPL to reading in a second language.

Controlling the Spin States of FeTBrPP on Au(111).

Spin-flip excitations of iron porphyrin molecules on Au(111) are investigated with a low-temperature scanning tunneling microscope. The molecules adopt two distinct adsorption configurations on the surface that exhibit different magnetic anisotropy energies. Density functional theory calculations show that the different structures and excitation energies reflect unlike occupations of the Fe 3d levels. We demonstrate that the magnetic anisotropy energy can be controlled by changing the adsorption site, the orientation, or the tip-molecule distance.

Rational Protein Design Yields a CD20 CAR with Superior Antitumor Efficacy Compared with CD19 CAR.

Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.