Detection and Quantification of 5moU RNA Modification from Direct RNA Sequencing Data.

Background: Chemically modified therapeutic mRNAs have gained momentum recently. In addition to commonly used modifications (e.g., pseudouridine), 5moU is considered a promising substitution for uridine in therapeutic mRNAs. Accurate identification of 5-methoxyuridine (5moU) would be crucial for the study and quality control of relevant in vitro-transcribed (IVT) mRNAs. However, current methods exhibit deficiencies in providing quantitative methodologies for detecting such modification. Utilizing the capabilities of Oxford nanopore direct RNA sequencing, in this study, we present NanoML-5moU, a machine-learning framework designed specifically for the read-level detection and quantification of 5moU modification for IVT data. Materials and Methods: Nanopore direct RNA sequencing data from both 5moU-modified and unmodified control samples were collected. Subsequently, a comprehensive analysis and modeling of signal event characteristics (mean, median current intensities, standard deviations, and dwell times) were performed. Furthermore, classical machine learning algorithms, notably the Support Vector Machine (SVM), Random Forest (RF), and XGBoost were employed to discern 5moU modifications within NNUNN (where N represents A, C, U, or G) 5-mers. Results: Notably, the signal event attributes pertaining to each constituent base of the NNUNN 5-mers, in conjunction with the utilization of the XGBoost algorithm, exhibited remarkable performance levels (with a maximum AUROC of 0.9567 in the "AGTTC" reference 5-mer dataset and a minimum AUROC of 0.8113 in the "TGTGC" reference 5-mer dataset). This accomplishment markedly exceeded the efficacy of the prevailing background error comparison model (ELIGOs AUC 0.751 for site-level prediction). The model's performance was further validated through a series of curated datasets, which featured customized modification ratios designed to emulate broader data patterns, demonstrating its general applicability in quality control of IVT mRNA vaccines. The NanoML-5moU framework is publicly available on GitHub (https://github.com/JiayiLi21/NanoML-5moU). Conclusion: NanoML-5moU enables accurate read-level profiling of 5moU modification with nanopore direct RNA-sequencing, which is a powerful tool specialized in unveiling signal patterns in in vitro-transcribed (IVT) mRNAs.

The chemically stable analogue of resolvin D1 ameliorates experimental autoimmune encephalomyelitis by mediating the resolution of inflammation.

While Resolvin D1 (RvD1) shows promise in resolving inflammation in experimental autoimmune encephalomyelitis (EAE), its pro-resolving roles on dendritic cells (DCs) remain unknown, and the chemical instability of RvD1 poses significant challenges to its drug development. This study aims to investigate whether 4-(2'-methoxyphenyl)-1-[2'-[N-(2″-pyridinyl)-p-fluorobenzamido]ethyl]piperazine (p-MPPF), a novel chemically stable analogue of RvD1, can play a pro-resolving role in EAE, particularly on DCs, and if p-MPPF could serve as a potential substitute for RvD1. We showed that both RvD1 and p-MPPF mediated the resolution of inflammation in EAE, as evidenced by ameliorated EAE progression, attenuated pathological changes in the spinal cord, altered cytokine expression profile in serum, and reduced proportion of pro-inflammatory immune cells in the spleen. Utilizing DCs derived from both the spleen and bone marrow of EAE, our investigation showed that RvD1 and p-MPPF prevented DC maturation, decreased pro-inflammatory cytokine secretion, shifted DCs away from a pro-inflammatory phenotype, increased the phagocytosis capacity of DCs, and suppressed their ability to induce differentiation of CD4+ T cells into Th1 and Th17 subsets. For underlying intracellular mechanisms, we found that RvD1 and p-MPPF down-regulated the lactate dehydrogenase A signaling pathways. Comparisons between RvD1 and p-MPPF showed that they exerted overlapped pro-resolving effects to a large extent. This study demonstrates that both RvD1 and p-MPPF exert therapeutic effects on EAE by mediating inflammation resolution, which is closely associated with modulating DC immune function towards a tolerogenic phenotype. SPM mimetics may serve as a more promising therapeutic drug.

Targeting mitochondrial damage: shining a new light on immunotherapy.

Mitochondrial damage has a particular impact on the immune system and tumor microenvironment, which can trigger cell stress, an inflammatory response, and disrupt immune cell function, thus all of which can accelerate the progression of the tumor. Therefore, it is of essence to comprehend how the immune system function and the tumor microenvironment interact with mitochondrial dysfunction for cancer treatment. Preserving the integrity of mitochondria or regulating the function of immune cells, such as macrophages, may enhance the efficacy of cancer therapy. Future research should concentrate on the interactions among mitochondria, the immune system, and the tumor microenvironment to identify new therapeutic strategies.

A branched polymer-based agent for efficient and precise targeting of fibrosis diseases by magnetic resonance imaging.

Herein, we synthesized and characterized gadolinium-based hyperbranched polymers, POADGd and PODGd, through RAFT polymerization as magnetic resonance imaging (MRI) contrast agents for detecting fibrosis. POADGd and PODGd contain biocompatible short-chain OEGMA to prolong blood circulation, and they can be decomposed in response to ROS after MRI examination to prevent potential accumulation. The relaxivities of POADGd and PODGd are 9.81 mM-1 s-1 and 9.58 mM-1 s-1 respectively, which are significantly higher than that of DTPA-Gd, a clinically used agent (3.74 mM-1 s-1). In comparison with PODGd, POADGd can specifically target allysine in fibrosis tissues through its oxyamine groups. Therefore, it displays a sharp spatial resolution and a high signal-to-noise ratio in the liver and lung fibrosis tissue at a field strength of 3.0 T or 7.0 T, and the morphology of these fibrosis tissues is accurately delineated. Our MRI diagnosis results based on POADGd are highly aligned with those from pathological examinations, while MRI diagnosis could avoid invasive biopsy. In addition, POADGd shows excellent biosafety and low toxicity. Therefore, POADGd could be applied to non-invasively and accurately diagnose liver and lung fibrosis diseases.

Enhancement of sludge dewaterability using combined technology of bioleaching and Fenton: Microscopic structure and hydrophilic/hydrophobic properties of sludge particles.

Bioleaching and Fenton technology are commonly used preconditioning techniques for sludge dewatering. This study compared the dewatering mechanisms of different conditioning technologies. The results showed that bound water, specific resistance to filtration (SRF), and capillary suction time decreased from 3.95 g/g, 6.16 × 1012 m/kg, and 130.6 s to 3.15 g/g, 2.81 × 1011 m/kg, and 33 s, respectively, under combined treatment condition. Moreover, the free radicals, including ·OH, O2-·and Fe (Ⅳ), further damaged the cell structure, thus increasing the concentration of DNA in the S-EPS layer. This intense degradation sludge particle size decreased by 15.6% and significantly increased zeta potential. Under the combined technology, the α-helix and ß-sheet decreased by 42.2% and 56.5%, respectively, destabilizing the spatial structure of proteins and promoting the release of bound water. In addition, the combined technology decreased (Ala/Lys) ratio in the TB-EPS layer by 67.6%, indicating the weakening of protein water-holding capacity. Moreover, the conversion of oxygen-containing compounds to nonpolar hydrocarbons increased the hydrophobicity of the sludge under a combined treatment, thus enhancing dewatering performance.

Hinokiflavone exerts dual regulation on apoptosis and pyroptosis via the SIX4/Stat3/Akt pathway to alleviate APAP-induced liver injury.

Hinokiflavone (HF), classified as a flavonoid, is a main bioactive compound in Platycladus orientalis and Selaginella. HF exhibits activities including anti-HIV, anti-inflammatory, antiviral, antioxidant and anti-tumor effects. The study aimed to explore the function and the mechanisms of HF on acetaminophen (APAP)-induced acute liver injury. Results indicated that HF treatment mitigated the impact of APAP on viability and restored levels of MDA, GSH and SOD on HepG2 cells. The accumulation of reactive oxygen species (ROS) mitochondrial membrane potential (MMP) in HepG2 cells stimulated by APAP were also blocked by HF. HF reduced the levels of pro-apoptotic and pro-pyroptotic proteins. Flow cytometry analysis and fluorescence staining results were consistent with western blot analysis. Following HF treatment in the APAP-induced cell model, there was observed an augmentation in the phosphorylation of Stat3 and an increase in the expression of SIX4. However, not only silenced the SIX4 protein in HepG2 cells by siRNA, but also adding the Stat3 inhibitor (Stattic), attenuated the anti-apoptotic and anti-pyroptotic effects of HF significantly. Furthermore, HF alleviated liver damage in C57BL/6 mice model. Overall, our study demonstrated that HF mitigates apoptosis and pyroptosis induced by APAP in drug-induced liver injury (DILI) through the SIX4/Akt/Stat3 pathway in vivo and in vitro. HF may have promising potential for for the treatment of DILI.

Association between obesity phenotypes and dietary patterns: A two-step cluster analysis based on the China multi-ethnic cohort study.

OBJECTIVE: This study aimed to explore obesity phenotypes and investigate their association with dietary patterns. METHODS: Data were obtained from the baseline survey conducted in the China Multi-Ethnic Cohort Study from July 2018 to August 2019. All participants with a body mass index of at least 24 kg/m2 were enrolled and underwent a questionnaire survey, physical examination, and clinical laboratory tests. A two-step cluster analysis was employed to classify the participants into phenotypes. Dietary information was collected using the food frequency questionnaire, and principal component analysis was conducted to identify distinct dietary patterns. RESULTS: We analyzed the data of 8757 participants. They were categorized based on demographic characteristics, biochemical indicators, and anthropometric measurements into two distinct clusters identified as metabolically healthy obesity and metabolically unhealthy obesity (MUO). Key predictors included serum uric acid, sex, and diastolic blood pressure. Subgroup analysis by sex identified three distinct clusters within both male and female participants. The MUO group had the highest prevalence of a range of chronic noncommunicable diseases. The analysis uncovered three unique dietary patterns among participants classified as the premium protein, rice-oil-red meat, and oil-salt patterns. Notably, the MUO subgroup demonstrated significantly higher factor scores for both the rice-oil-red meat and oil-salt patterns. CONCLUSIONS: Obesity phenotypes are closely related to metabolic and demographic characteristics, with serum uric acid being a significant factor in categorizing the metabolic states of obesity. The rice-oil-red meat and oil-salt patterns may be related to the metabolic status of individuals with obesity.

Biomedical Utility of Non-Enzymatic DNA Amplification Reaction: From Material Design to Diagnosis and Treatment.

Nucleic acid nanotechnology has become a promising strategy for disease diagnosis and treatment, owing to remarkable programmability, precision, and biocompatibility. However, current biosensing and biotherapy approaches by nucleic acids exhibit limitations in sensitivity, specificity, versatility, and real-time monitoring. DNA amplification reactions present an advantageous strategy to enhance the performance of biosensing and biotherapy platforms. Non-enzymatic DNA amplification reaction (NEDAR), such as hybridization chain reaction and catalytic hairpin assembly, operate via strand displacement. NEDAR presents distinct advantages over traditional enzymatic DNA amplification reactions, including simplified procedures, milder reaction conditions, higher specificity, enhanced controllability, and excellent versatility. Consequently, research focusing on NEDAR-based biosensing and biotherapy has garnered significant attention. NEDAR demonstrates high efficacy in detecting multiple types of biomarkers, including nucleic acids, small molecules, and proteins, with high sensitivity and specificity, enabling the parallel detection of multiple targets. Besides, NEDAR can strengthen drug therapy, cellular behavior control, and cell encapsulation. Moreover, NEDAR holds promise for constructing assembled diagnosis-treatment nanoplatforms in the forms of pure DNA nanostructures and hybrid nanomaterials, which offer utility in disease monitoring and precise treatment. Thus, this paper aims to comprehensively elucidate the reaction mechanism of NEDAR and review the substantial advancements in NEDAR-based diagnosis and treatment over the past five years, encompassing NEDAR-based design strategies, applications, and prospects.

MiR-494-3p regulates skin fibroblast activities by mediating fibromodulin production.

Skin wound healing is a well-coordinated process in which various cells and factors participate, during which fibroblast exhibits a critical role by exerting its multiple activities, including proliferation, migration, invasion, and differentiation. Previous studies have identified that fibromodulin (FMOD) could enhance dermal wound healing by promoting skin fibroblast activities, but little is known about its upstream regulator. We occasionally found that FMOD expression was downregulated in skin fibroblast by transforming growth factor-ß1 treatment. It was hypothesized that microRNAs (miRNA) in skin fibroblast could downregulate FMOD production and blocking them would increase FMOD expression, as well as promote skin wound healing. Here, by utilizing combined analysis of miRNA microarray from the Gene Expression Omnibus database and miRNA targets prediction, we successfully identified a miRNA, termed miR-494-3p, could regulate FMOD production in human skin fibroblast (BJ fibroblast). The functional analysis revealed that miR-494-3p mimics could inhibit BJ fibroblast migration and invasion but not proliferation and differentiation, while miR-494-3p inhibition markedly promotes migration, invasion, and differentiation of BJ fibroblast. Moreover, we established FMOD overexpression (OE) and knockout BJ fibroblast. We found that FMOD OE could rescue the inhibitory effects of miR-494-3p mimics on the migration and invasion of BJ fibroblast. In contrast, the miR-494-3p inhibitor transfection could not enhance migration, invasion, and differentiation of FMOD KO BJ fibroblast. Together, our results suggest that miR-494-3p may be a potential target for skin wound management via regulating FMOD production.

[Clinical features and genetic analysis of a case with CHARGE syndrome due to variant of CHD7 gene].

OBJECTIVE: To explore the genetic basis for child with CHARGE syndrome. METHODS: A child who was diagnosed at Ningbo Women and Children's Hospital on September 29, 2022 was selected as the study subject. Relevant clinical data were collected. The child and her parents were subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor a de novo c.2972T>C (p.L991S) missense variant of the CHD7 gene, which was detected in neither of her parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted to be likely pathogenic (PM6+PM2_Supporting+PP2+PP3+PP4). Bioinformatic analysis predicted that amino acid 991 is highly conserved among various species, and a hydrogen bond has formed between Asp993 and the mutant Ser991. CONCLUSION: The heterozygous c.2972T>C (p.L991S) missense variant of the CHD7 gene probably underlay the pathogenesis of CHARGE syndrome in this child. Above finding has also enriched the mutational spectrum for CHARGE syndrome.

Insights into ACO genes across Rosaceae: evolution, expression, and regulatory networks in fruit development.

BACKGROUND: ACO (1-aminocyclopropane-1-carboxylic acid) serves as a pivotal enzyme within the plant ethylene synthesis pathway, exerting influence over critical facets of plant biology such as flowering, fruit ripening, and seed development. OBJECTIVE: This study aims to identify ACO genes from representative Rosaceae genomes, reconstruct their phylogenetic relationships by integrating synteny information, and investigate their expression patterns and networks during fruit development. METHODS: we utilize a specialized Hidden Markov Model (HMM), crafted on the sequence attributes of ACO gene-encoded proteins, to systematically identify and analyze ACO gene family members across 12 representative species within the Rosaceae botanical family. Through transcriptome analysis, we delineate the expression patterns of ACO genes in six distinct Rosaceae fruits. RESULTS: Our investigation reveals the presence of 62 ACO genes distributed among the surveyed Rosaceae species, characterized by hydrophilic proteins predominantly expressed within the cytoplasm. Phylogenetic analysis categorizes these ACO genes into three discernible classes, namely Class I, Class II, and Class III. Further scrutiny via collinearity assessment indicates a lack of collinearity relationships among these classes, highlighting variations in conserved motifs and promoter types within each class. Transcriptome analysis unveils significant disparities in both expression levels and trends of ACO genes in fruits exhibiting respiratory bursts compared to those that do not. Employing Weighted Gene Co-Expression Network Analysis (WGCNA), we discern that the co-expression correlation of ACO genes within loquat fruit notably differs from that observed in apples. Our findings, derived from Gene Ontology (GO) enrichment results, signify the involvement of ACO genes and their co-expressed counterparts in biological processes linked to terpenoid metabolism and carbohydrate synthesis in loquat. Moreover, our exploration of gene regulatory networks (GRN) highlights the potential pivotal role of the GNAT transcription factor (Ejapchr1G00010380) in governing the overexpression of the ACO gene (Ejapchr10G00001110) within loquat fruits. CONCLUSION: The constructed HMM of ACO proteins offers a precise and systematic method for identifying plant ACO proteins, facilitating phylogenetic reconstruction. ACO genes from representative Rosaceae fruits exhibit diverse expression and regulative patterns, warranting further function characterizations.

Association Between the Copper-to-Zinc Ratio and Cardiovascular Disease Among Chinese Adults: A China Multi-ethnic Cohort (CMEC) Study.

The impact of metal exposure on cardiovascular diseases has become an increasingly concerning topic. To date, few studies have investigated the relationship between the copper-to-zinc ratio and CVD (Cardiovascular disease). This China multi-ethnic cohort study explored the association between the copper-to-zinc ratio and CVD in Chinese adults. The study included a sample size of 9878 people. Logistic regression analysis was used to examine the correlation between urinary copper, urinary zinc, and the copper-to-zinc ratio and CVD prevalence. Restricted cubic spline (RCS) analysis was used to investigate the potential dose-response relationships among copper-to-zinc ratio, urinary copper, urinary zinc, and CVD prevalence. In addition, the least absolute shrinkage and selection operator (LASSO) regression method was used to identify significant risk factors associated with CVD, leading to the development of a nomogram. The predictive performance of the nomogram model for CVD was assessed using the receiver operating characteristic (ROC) curve and the area under the curve (AUC). Compared with the copper-to-zinc ratio in Q1, the copper-to-zinc ratio in Q4 was associated with CVD after adjusting for all potential confounders (Model 3) (Q4, odds ratio [OR] 0.608, 95% confidence interval [CI] 0.416-0.889, P = 0.010). After adjusting for all potential confounders (Model 3), urinary copper levels in Q4 were associated with CVD (Q4, odds ratio [OR] 0.627, 95% confidence interval [CI] 0.436-0.902, P = 0.012). No significant difference was found between urinary zinc levels and CVD. The RCS showed a linear dose-response relationship between the copper-to-zinc ratio and CVD (P for overall = 0.01). The nomogram based on the influencing factors examined with LASSO showed good predictive power, and the AUC was 76.3% (95% CI 73.7-78.9%). Our results suggest that there is a significant linear negative correlation between the copper-to-zinc ratio and CVD in Chinese adults and that it has good predictive value for CVD.

Achieving dendrite-free growth of Zn anode by electrodepositing on zincophilic gold-furnished mesh for aqueous zinc-ion batteries.

Here, we report a gold-furnished mesh as the current collector for Zn electrodeposition, which is used as the anode in aqueous zinc-ion batteries. The anode exhibits excellent cycling performance without obvious dendrite growth, and the full cell shows an outstanding specific capacity and long-term durability, surpassing those of bare Zn.

Enantioselective Synthesis of Spiro[cyclopentane-1,3'-oxindole] Scaffolds with Five Consecutive Stereocenters.

A highly diastereo- and enantioselective cascade annulation reaction of Morita-Baylis-Hillman (MBH) maleimides of isatins with ortho-hydroxychalcones was achieved by a chiral N,N'-dioxide/Mg(II) complex Lewis acid catalyst. This strategy provides a concise and efficient route to densely functionalized spiro[cyclopentane-1,3'-oxindole] compounds with five consecutive stereocenters. The reaction itself features mild conditions, good functional group compatibility, and broad substrate scope (62 examples, up to 99% yield, up to >20:1 dr, 97% ee). In addition, an obvious ligand acceleration effect and chiral amplification effect were observed. DFT calculations were performed to elucidate the stereoselectivity observed. The gram-scale synthesis and the inhibitory effect of two products on the viability of A549 cells demonstrate the potential utility of the current method.

Unveiling the Younger Face of Gastric Cancer: A Comprehensive Review of Epidemiology, Risk Factors, and Prevention Strategies.

Gastric cancer poses a significant global health challenge, with high incidence and mortality rates each year. Despite advancements in screening and treatment, late detection remains a critical issue. Efforts to address this include raising public awareness and implementing targeted screening programs for high-risk populations. The increasing incidence of gastric cancer among younger individuals underscores the need for lifestyle adjustments and targeted interventions to mitigate risks and improve outcomes. Understanding the various factors contributing to gastric cancer risk is essential for effective prevention strategies, including Helicobacter pylori eradication, lifestyle modifications, and regular screening for high-risk groups. A comprehensive approach addressing both individual behaviors and broader societal factors is crucial in the fight against gastric cancer. This review provides an in-depth examination of gastric cancer epidemiology, risk factors, preventive measures, and screening initiatives, with a particular focus on the rising incidence among younger demographics. Emphasizing the importance of early detection and intervention, the review highlights the need for proactive screening to improve patient outcomes and reduce mortality rates. By addressing these aspects comprehensively, this paper aims to enhance the understanding of gastric cancer dynamics, particularly its incidence among younger individuals, and to inform future strategies for prevention and control.

Thymol improves ischemic brain injury by inhibiting microglia-mediated neuroinflammation.

BACKGROUND: Microglia-mediated inflammation is a critical factor in the progression of ischemic stroke. Consequently, mitigating excessive microglial activation represents a potential therapeutic strategy for ischemic injury. Thymol, a monophenol derived from plant essential oils, exhibits diverse beneficial biological activities, including anti-inflammatory and antioxidant properties, with demonstrated protective effects in various disease models. However, its specific effects on ischemic stroke and microglial inflammation remain unexplored. METHODS: Rodent transient middle cerebral artery occlusion (tMCAO) model was established to simulate ischemic stroke. TTC staining, modified neurological function score (mNSS), and behavioral tests were used to assess the severity of neurological damage. Then immunofluorescence staining and cytoskeleton analysis were used to determine activation of microglia. Lipopolysaccharide (LPS) was utilized to induce the inflammatory response of primary microglia in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to exam the expression of inflammatory cytokines. And western blot was used to investigate the mechanism of the anti-inflammatory effect of thymol. RESULTS: In this study, we found that thymol treatment could ameliorate post-stroke neurological impairment and reduce infarct volume by mitigating microglial activation and pro-inflammatory response (IL-1ß, IL-6, and TNF-α). Mechanically, thymol could inhibit the phosphorylation of phosphatidylinositol-3-kinase (PI3K), sink serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR), thereby suppressing the activation of nuclear factor-κB (NF-κB). CONCLUSIONS: Our study demonstrated that thymol could reduce the microglial inflammation by targeting PI3K/Akt/mTOR/NF-κB signaling pathway, ultimately alleviating ischemic brain injury. These findings suggest that thymol is a promising candidate as a neuroprotective agent against ischemic stroke.

Photophysical Properties of Submicrometer Ultrathin Perovskite Single-Crystal Films.

Organic-inorganic hybrid perovskite (OIHP) has attracted a great deal of interest with respect to diverse optoelectronic devices. However, the photophysical properties of the OIHP require further understanding because most of the investigations have been conducted with polycrystalline perovskites, which contain high-density structural defects. Here, diverse photophysical properties, including structural characterization, spectroscopic features, and photoexcited products, are studied in submicrometer CH3NH3PbBr3 ultrathin single-crystal (UTSC) films. Unlike polycrystalline films and large single crystals, the UTSC film provides a unique platform for studying spectroscopic characteristics of single-crystal perovskites. Compared with the polycrystalline film, the UTSC film presents an atomically flat morphology and near-perfect lattice with a lower defect density, leading to an isotropic system that can be applied in the construction of high-performance optoelectronic devices. Furthermore, a long lifetime emissive channel assigned to the trion is indicated, which is scarcely found in perovskite polycrystalline films. Our results profoundly improve our understanding of their photophysical properties and expand the horizons for perovskite materials in photonic applications.

Safety assessment for autonomous vehicles: A reference driver model for highway merging scenarios.

Driver models are crucial for the safety assessment of autonomous vehicles (AVs) because of their role as reference models. Specifically, an AV is expected to achieve at least the same level of safety performance as a careful and competent driver model. To make this comparison possible, quantitative modeling of careful and competent driver models is essential. Thus, the UNECE Regulation No. 157 proposes two driver models as benchmarks for AVs, enabling safety assessment of AV longitudinal behaviors. However, these two driver models are unable to be applied in non-car-following scenarios, limiting their applications in scenarios such as highway merging. To this end, we propose a careful and competent driver model for highway merging (CCDM2) scenarios using interpretable reinforcement learning-based decision-making and safety constraint control. We compare our model's safe driving capabilities with human drivers in challenging merging scenarios and demonstrate the "careful" and "competent" characteristics of our model while ensuring its interpretability. The results indicate the model's capability to handle merging scenarios with even better safety performance than human drivers. This model is of great value for AV safety assessment in merging scenarios and contributes to future reference driver models to be included in AV safety regulations.

Analysis of the resistance profile of real-world alectinib first-line therapy in patients with ALK rearrangement-positive advanced non-small cell lung cancer using organoid technology in one case of lung cancer.

Background: Alectinib has achieved excellent therapeutic efficacy in anaplastic lymphoma kinase (ALK) fusion gene-positive non-small cell lung cancer (NSCLC) patients, however, patients eventually develop resistance to it. Exploring the gene variant mapping after alectinib resistance provides a basis for the whole management of ALK-positive advanced NSCLC. This study aimed to characterize the mutation profiles of real-world ALK rearrangement-positive advanced NSCLC patients after first-line alectinib treatment resistance. The research also investigated the treatment options and coping strategies after resistance. Methods: Clinical data of patients with advanced NSCLC who received first-line alectinib treatment in the First Affiliated Hospital of Guangzhou Medical University between November 2018 and April 2022 were collected. Moreover, next-generation sequencing (NGS) data of the patient's baseline and post-resistance tissues were gathered. One patient underwent lung cancer organoid culture and drug sensitivity testing. Results: Out of 35 first-line alectinib-treated patients with advanced NSCLC, 31 are presently in progression-free survival (PFS; 4.3-35.0 months). Four patients experienced progressive disease, and all of them were sequentially treated with ceritinib. Tissue NGS results before sequential treatment in three patients indicated an echinoderm microtubule-associated protein-like 4-ALK fusion that remained at the original baseline, and the PFS for ceritinib treatment was 0.5-1.3 months. One patient developed acquired resistance mutations in the structural domain of ALK protein kinase (V1180L and E1161D), and the PFS for ceritinib treatment was 6.7 months. For one patient who maintained original baseline ALK rearrangement positive without acquired mutation after progression of ceritinib resistance, lung cancer-like organ culture with sequential brigatinib and lorlatinib led to a PFS of 3.2 and 1.9 months, respectively, which aligned with the corresponding drug susceptibility testing results for this patient. Conclusions: For ALK rearrangement-positive patients, blind sequencing of other second-generation tyrosine kinase inhibitors (TKIs) or third-generation lorlatinib may not guarantee satisfactory tumor suppression following first-line second-generation ALK-TKI alectinib administration for treatment progression. NGS testing of patients' blood or tissue samples after disease progression may provide insight into the etiology of alectinib resistance. Patient-sourced drug sensitivity testing of lung cancer-like organs selects drug-sensitive medications based on NGS results and provides a reference for subsequent drug therapy for patients after drug resistance, particularly those who remain ALK rearrangement-positive at baseline.