Integrative single-cell analysis of LUAD: elucidating immune cell dynamics and prognostic modeling based on exhausted CD8+ T cells.

Background: The tumor microenvironment (TME) plays a pivotal role in the progression and metastasis of lung adenocarcinoma (LUAD). However, the detailed characteristics of LUAD and its associated microenvironment are yet to be extensively explored. This study aims to delineate a comprehensive profile of the immune cells within the LUAD microenvironment, including CD8+ T cells, CD4+ T cells, and myeloid cells. Subsequently, based on marker genes of exhausted CD8+ T cells, we aim to establish a prognostic model for LUAD. Method: Utilizing the Seurat and Scanpy packages, we successfully constructed an immune microenvironment atlas for LUAD. The Monocle3 and PAGA algorithms were employed for pseudotime analysis, pySCENIC for transcription factor analysis, and CellChat for analyzing intercellular communication. Following this, a prognostic model for LUAD was developed, based on the marker genes of exhausted CD8+ T cells, enabling effective risk stratification in LUAD patients. Our study included a thorough analysis to identify differences in TME, mutation landscape, and enrichment across varying risk groups. Moreover, by integrating risk scores with clinical features, we developed a new nomogram. The expression of model genes was validated via RT-PCR, and a series of cellular experiments were conducted, elucidating the potential oncogenic mechanisms of GALNT2. Results: Our study developed a single-cell atlas for LUAD from scRNA-seq data of 19 patients, examining crucial immune cells in LUAD's microenvironment. We underscored pDCs' role in antigen processing and established a Cox regression model based on CD8_Tex-LAYN genes for risk assessment. Additionally, we contrasted prognosis and tumor environments across risk groups, constructed a new nomogram integrating clinical features, validated the expression of model genes via RT-PCR, and confirmed GALNT2's function in LUAD through cellular experiments, thereby enhancing our understanding and approach to LUAD treatment. Conclusion: The creation of a LUAD single-cell atlas in our study offered new insights into its tumor microenvironment and immune cell interactions, highlighting the importance of key genes associated with exhausted CD8+ T cells. These discoveries have enabled the development of an effective prognostic model for LUAD and identified GALNT2 as a potential therapeutic target, significantly contributing to the improvement of LUAD diagnosis and treatment strategies.

Prediction of High-Risk Gastrointestinal Stromal Tumor Recurrence Based on Delta-CT Radiomics Modeling: A 3-Year Follow-up Study After Surgery.

Background: Medium- to high-risk classification-gastrointestinal stromal tumors (MH-GIST) have a high recurrence rate and are difficult to treat. This study aims to predict the recurrence of MH-GIST within 3 years after surgery based on clinical data and preoperative Delta-CT Radiomics modeling. Methods: A retrospective analysis was conducted on clinical imaging data of 242 cases confirmed to have MH-GIST after surgery, including 92 cases of recurrence and 150 cases of normal. The training set and test set were established using a 7:3 ratio and time cutoff point. In the training set, multiple prediction models were established based on clinical data of MH-GIST and the changes in radiomics texture of enhanced computed tomography (CT) at different time periods (Delta-CT radiomics). The area under curve (AUC) values of each model were compared using the Delong test, and the clinical net benefit of the model was tested using decision curve analysis (DCA). Then, the model was externally validated in the test set, and a novel nomogram predicting the recurrence of MH-GIST was finally created. Results: Univariate analysis confirmed that tumor volume, tumor location, neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), diabetes, spicy hot pot, CT enhancement mode, and Radscore 1/2 were predictive factors for MH-GIST recurrence (P < .05). The combined model based on these above factors had significantly higher predictive performance (AUC = 0.895, 95% confidence interval [CI] = [0.839-0.937]) than the clinical data model (AUC = 0.735, 95% CI = [0.6 62-0.800]) and radiomics model (AUC = 0.842, 95% CI = [0.779-0.894]). Decision curve analysis also confirmed the higher clinical net benefit of the combined model, and the same results were validated in the test set. The novel nomogram developed based on the combined model helps predict the recurrence of MH-GIST. Conclusions: The nomogram of clinical and Delta-CT radiomics has important clinical value in predicting the recurrence of MH-GIST, providing reliable data reference for its diagnosis, treatment, and clinical decision-making.

Monochasma Savatieri Aqueous Extract inhibits Human Breast Cancer Cell Line Migration and Adhesion Without Generating Toxicity.

BACKGROUND: Monochasma savatieri, is a rare and endangered plant used to treat cancer in Chinese traditional medicine. OBJECTIVE: To evaluate the anti-cancer activity of M. savatieri aqueous extract by determining its cytotoxicity, anti-migratory, and anti-adhesion effects on breast cancer cells. METHODS: Cell viability, migration, adhesion, circularity, and cell cycle were evaluated by crystal violet (CV) staining, wound-healing, and transwell assays and flow cytometry in MCF7 and MDA-MB-231 cells. Caveolin-1, snail, vimentin and activated Erk and Akt expression were determined by western blot in MDA-MB-231 cells. Immunofluorescent assays confirmed caveolin-1 expression in MDA-MB-231 cells. RESULTS: Survival and cell cycle of MCF7 and MDA-MB-231 cells were not modified by doses up to 500 µg/mL of the extract. The extract inhibited cell migration and adhesion of MDA-MB-231 cells. When cells were exposed to the extract, there was a slight decrease in protein expression of factors related to epithelial-to-mesenchymal transition (snail and vimentin) and a strong decrease in the expression of the oncogenic membrane protein caveolin- 1. Furthermore, the levels of phosphorylated Erk and Akt were also decreased. The content of acteoside, a phenylpropanoid glycoside with reported anti-cancer activity present in M. savatieri, was almost 5 times as much as isoacteoside. CONCLUSION: M. savatieri possesses anti-cancer activity without exerting cytotoxicity on breast cancer cells. The extract exhibited anti-migratory and anti-adhesion effects on breast cancer cells by regulating Erk and Akt signaling pathways and the expression of caveolin-1. In addition, acteoside present in M. savatieri could be responsible for the observed effects.

Engineering Phage Nanocarriers Integrated with Bio-Intelligent Plasmids for Personalized and Tunable Enzyme Delivery to Enhance Chemodynamic Therapy.

Customizable and number-tunable enzyme delivery nanocarriers will be useful in tumor therapy. Herein, a phage vehicle, T4-Lox-DNA-Fe (TLDF), which adeptly modulates enzyme numbers using phage display technology to remodel the tumor microenvironment (TME) is presented. Regarding the demand for lactic acid in tumors, each phage is engineered to display 720 lactate oxidase (Lox), contributing to the depletion of lactic acid to restructure the tumor's energy metabolism. The phage vehicle incorporated dextran iron (Fe) with Fenton reaction capabilities. H2O2 is generated through the Lox catalytic reaction, amplifying the H2O2 supply for dextran iron-based chemodynamic therapy (CDT). Drawing inspiration from the erythropoietin (EPO) biosynthetic process, an EPO enhancer is constructed to impart the EPO-Keap1 plasmid (DNA) with tumor hypoxia-activated functionality, disrupting the redox homeostasis of the TME. Lox consumes local oxygen, and positive feedback between the Lox and the plasmid promotes the expression of kelch ECH Associated Protein 1 (Keap1). Consequently, the downregulation of the antioxidant transcription factor Nrf2, in synergy with CDT, amplifies the oxidative killing effect, leading to tumor suppression of up to 78%. This study seamlessly integrates adaptable T4 phage vehicles with bio-intelligent plasmids, presenting a promising approach for tumor therapy.

Palladium-Catalyzed Directed Carbon-Carbon Bond Activation of Aryl Nitriles for Cyano Transfer.

Herein, we report the C-H cyanation of indoles via a palladium-catalyzed directed C-CN activation reaction using aryl nitrile as a cyano source. The employment of the phenoxy-oriented group is the key to the cleavage of the C-CN bond. This protocol features a broad substrate scope, good efficiency, and high regioselectivity. Furthermore, the practical application of this protocol was showcased in the late-stage functionalization and synthesis of indole derivatives, which were derived from drugs and natural products, through the process of cyanation.

miR-26b-5p Affects the Progression of Acute Myeloid Leukemia by Regulating the USP48-Mediated Wnt/ß-Catenin Pathway.

Acute myeloid leukemia (AML) is a highly heterogeneous disease. Exploring the pathogenesis of AML is still an important topic in the treatment of AML. The expression levels of miR-26b-5p and USP48 were measured by qRT-PCR. The expression levels of related proteins were detected by Western blot. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Coimmunoprecipitation was used to examine the interaction between USP48 and Wnt5a. Bioinformatics analysis showed that high levels of miR-26b-5p and low levels of USP48 were associated with poor prognosis in AML. miR-26b-5p can negatively regulate the expression of USP48. Downregulation of miR-26b-5p inhibited EMT, cell viability and proliferation of AML cells and accelerated apoptosis. Furthermore, the influence of miR-26b-5p inhibition and USP48 knockdown on AML progression could be reversed by a Wnt/ß-catenin signaling pathway inhibitor. This study revealed that miR-26b-5p regulates AML progression, possibly by targeting the USP48-mediated Wnt/ß-catenin molecular axis to affect AML cell biological behavior.

Photopolymerizable and Antibacterial Hydrogels Loaded with Metabolites from Lacticaseibacillus rhamnosus GG for Infected Wound Healing.

In response to increasing antibiotic resistance and the pressing demand for safer infected wound care, probiotics have emerged as promising bioactive agents. To address the challenges associated with the safe and efficient application of probiotics, this study successfully loaded metabolites from Lacticaseibacillus rhamnosus GG (LGG) into a gelatin cross-linked macromolecular network by an in situ blending and photopolymerization method. The obtained LM-GelMA possesses injectability and autonomous healing capabilities. Importantly, the incorporation of LGG metabolites endows LM-GelMA with excellent antibacterial properties against Staphylococcus aureus and Escherichia coli, while maintaining good biocompatibility. In vivo assessments revealed that LM-GelMA can accelerate wound healing by mitigating infections induced by pathogenic bacteria. This is accompanied by a reduction in the expression of key proinflammatory cytokines such as TNF-α, IL-6, VEGFR2, and TGF-ß, leading to increased re-epithelialization and collagen formation. Moreover, microbiological analysis confirmed that LM-GelMA can modulate the abundance of beneficial wound microbiota at family and genus levels. This study provides a facile strategy and insights into the functional design of hydrogels from the perspective of wound microenvironment regulation.

Targeting DNA2 overcomes metabolic reprogramming in multiple myeloma.

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover mechanisms through which MM cells overcome DNA damage, we investigate how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting Interleukin enhancer binding factor 2 (ILF2), a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identify the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage. Our study reveals a mechanism of vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells.

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ T cells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ T cells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Severe pediatric COVID-19: a review from the clinical and immunopathophysiological perspectives.

BACKGROUND: Coronavirus disease 2019 (COVID-19) tends to have mild presentations in children. However, severe and critical cases do arise in the pediatric population with debilitating systemic impacts and can be fatal at times, meriting further attention from clinicians. Meanwhile, the intricate interactions between the pathogen virulence factors and host defense mechanisms are believed to play indispensable roles in severe COVID-19 pathophysiology but remain incompletely understood. DATA SOURCES: A comprehensive literature review was conducted for pertinent publications by reviewers independently using the PubMed, Embase, and Wanfang databases. Searched keywords included "COVID-19 in children", "severe pediatric COVID-19", and "critical illness in children with COVID-19". RESULTS: Risks of developing severe COVID-19 in children escalate with increasing numbers of co-morbidities and an unvaccinated status. Acute respiratory distress stress and necrotizing pneumonia are prominent pulmonary manifestations, while various forms of cardiovascular and neurological involvement may also be seen. Multiple immunological processes are implicated in the host response to COVID-19 including the type I interferon and inflammasome pathways, whose dysregulation in severe and critical diseases translates into adverse clinical manifestations. Multisystem inflammatory syndrome in children (MIS-C), a potentially life-threatening immune-mediated condition chronologically associated with COVID-19 exposure, denotes another scientific and clinical conundrum that exemplifies the complexity of pediatric immunity. Despite the considerable dissimilarities between the pediatric and adult immune systems, clinical trials dedicated to children are lacking and current management recommendations are largely adapted from adult guidelines. CONCLUSIONS: Severe pediatric COVID-19 can affect multiple organ systems. The dysregulated immune pathways in severe COVID-19 shape the disease course, epitomize the vast functional diversity of the pediatric immune system and highlight the immunophenotypical differences between children and adults. Consequently, further research may be warranted to adequately address them in pediatric-specific clinical practice guidelines.

Identification and Validation of Lipid Metabolism Gene FASN-Associated miRNA in Wilms Tumor.

miRNA has been a research hotspot in recent years and its scope of action is very wide, involving the regulation of cell proliferation, differentiation, apoptosis, and other biological behaviors. This study intends to explore the role of miRNA in the lipid metabolism and development of Wilms tumor (WT) by detecting and analyzing the differences in the expression profiles of miRNAs between the tumor and adjacent normal tissue. Gene detection was performed in tumor tissues and adjacent normal tissues of three cases of WT to screen differentially expressed miRNAs (DEMs). According to our previous research, FASN, which participates in the lipid metabolism pathway, may be a target of WT. The starBase database was used to predict FASN-targeted miRNAs. The above two groups of miRNAs were intersected to obtain FASN-targeted DEMs and then GO Ontology (GO) functional enrichment analysis of FASN-targeted DEMs was performed. Finally, the FASN-targeted DEMs were compared and further verified by qRT‒PCR. Through gene sequencing and differential analysis, 287 DEMs were obtained, including 132 upregulated and 155 downregulated miRNAs. The top ten DEMs were all downregulated. Fourteen miRNAs targeted by the lipid metabolism-related gene FASN were predicted by starBase. After intersection with the DEMs, three miRNAs were finally obtained, namely, miR-107, miR-27a-3p, and miR-335-5p. GO enrichment analysis was mainly concentrated in the Parkin-FBXW7-Cul1 ubiquitin ligase complex and response to prostaglandin E. Further experimental verification showed that miR-27a-3p was significantly correlated with WT (P = 0.0018). Imbalanced expression of miRNAs may be involved in the occurrence and development of WT through lipid metabolism. The expression of miR-27a-3p is related to the malignant degree of WT, and it may become the target of diagnosis, prognosis, and treatment of WT in the later stage.

An IROA Workflow for correction and normalization of ion suppression in mass spectrometry-based metabolomic profiling data.

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and signal-to-noise sensitivity. Here we report a new method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) plus novel companion algorithms to 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We have evaluated the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reverse phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibited ion suppression ranging from 1% to 90+% and coefficient of variations ranging from 1% to 20%, but the Workflow and companion algorithms were highly effective at nulling out that suppression and error. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

Tryptophan Metabolites And Their Predicted Microbial Sources In Fecal Samples From Healthy Individuals.

Gut microbiota produce tryptophan metabolites (TMs) important to homeostasis. However, measuring TM levels in stool and determining their microbial sources can be difficult. Here, we measured TMs from the indole pathway in fecal samples from 21 healthy adults with the goal to: 1) determine fecal TM concentrations in healthy individuals; 2) link TM levels to bacterial abundance using 16S and whole genome shotgun (WGS) sequencing data; and 3) predict likely bacterial sources of TM production. Within our samples, we identified 151 genera (16S) and 592 bacterial species (WGS). Eight TMs were found in ≥17 fecal samples, including four in all persons. To our knowledge, we are the first to report fecal levels for indole-3-lactate, indole-3-propionate, and 3-indoleacrylate levels in healthy persons. Overall, indole, indole-3-acetate (IAA), and skatole accounted for 86% of the eight TMs measured. Significant correlations were found between seven TMs and 29 bacterial species.  Predicted multiple TM sources support the notion of a complex network of TM production and regulation. Further, the data suggest key roles for Collinsella aerofaciens and IAA, a metabolite reported to maintain intestinal homeostasis through enhanced barrier integrity and anti-inflammatory/antioxidant activities. These findings extend our understanding of TMs and their relationship to the microbial species that act as effectors and/or regulators in the healthy intestine and may lead to novel strategies designed to manipulate tryptophan metabolism to prevent disease and/or restore health to the dysbiotic gut.

N-acetylaspartate from fat cells regulates postprandial body temperature.

N-acetylaspartate (NAA), the brain's second most abundant metabolite, provides essential substrates for myelination through its hydrolysis. However, activities and physiological roles of NAA in other tissues remain unknown. Here, we show aspartoacylase (ASPA) expression in white adipose tissue (WAT) governs systemic NAA levels for postprandial body temperature regulation. Proteomics and mass spectrometry revealed NAA accumulation in WAT of Aspa knockout mice stimulated the pentose phosphate pathway and pyrimidine production. Stable isotope tracing confirmed higher incorporation of glucose-derived carbon into pyrimidine metabolites in Aspa knockout cells. Additionally, serum NAA positively correlates with the pyrimidine intermediate orotidine and this relationship predicted lower body mass index in humans. Using whole-body and tissue-specific knockout mouse models, we demonstrate that fat cells provided plasma NAA and suppressed postprandial body temperature elevation. Furthermore, exogenous NAA supplementation reduced body temperature. Our study unveils WAT-derived NAA as an endocrine regulator of postprandial body temperature and physiological homeostasis.

Palladium-Catalyzed Deuteration of Arylketone Oxime Ethers.

We report herein the development of palladium-catalyzed deacylative deuteration of arylketone oxime ethers. This protocol features excellent functional group tolerance, heterocyclic compatibility, and high deuterium incorporation levels. Regioselective deuteration of some biologically important drugs and natural products are showcased via Friedel-Crafts acylation and subsequent deacylative deuteration. Vicinal meta-C-H bond functionalization (including fluorination, arylation, and alkylation) and para-C-H bond deuteration of electro-rich arenes are realized by using the ketone as both directing group and leaving group, which is distinct from aryl halide in conventional dehalogenative deuteration.

TiO2 nanorod arrays@PDA/Ag with biomimetic polydopamine as binary mediators for duplex SERS detection of illegal food dyes.

Based on TiO2 nanorod arrays@PDA/Ag (TNRs@PDA/Ag), a better surface-enhanced Raman scattering (SERS) sensor with effective enrichment and enhancement was investigated for duplex SERS detection of illicit food dyes. Biomimetic PDA functions as binary mediators by utilizing the structural characteristics of polydopamine (PDA), which include the conjugated structure and abundant hydrophilic groups. One PDA functioned as an electron transfer mediator to enhance the efficiency of electron transfer, and the other as an enrichment mediator to effectively enrich rhodamine B (RhB) and crystal violet (CV) through hydrogen bonding, π-π stacking, and electrostatic interactions. Individual and duplex detection of illicit food dyes (RhB and CV) was performed using TNRs@PDA/Ag to estimate SERS applications. Their linear equations and limits of detection of 1 nM for RhB and 5 nM for CV were derived. Individual and duplex food colour detection was successfully accomplished even in genuine chili meal with good results. The bifunctional TNRs@PDA/Ag-based highly sensitive and duplex SERS dye detection will have enormous potential for food safety monitoring.

The Auricular Injection Technique: A New Treatment for Lying Ear.

SUMMARY: Lying-ear deformity refers to an auricle that bends backward excessively, is excessively folded against the head, and has a very prominent antihelix. It usually requires experienced surgeons to perform surgical treatment and a prolonged postoperative recovery process. This article proposes a simple and effective hyaluronic acid injection technique that significantly improves the shape of the outer ear and enhances perceived facial aesthetics. Twenty patients underwent treatment with multiple injections. Measure-related parameters were used to evaluate the postoperative effect, and the results were graded using a visual analog scale. Interrater reliability among graders was evaluated using intraclass correlation coefficients. After treatment, no serious complications, such as infection or embolism, occurred. Six months after the procedure, the average auriculocephalic angle increased from 25.11 ± 9.46 to 32.72 ± 8.29 degrees, the average conchoscaphal angle increased from 87.69 ± 9.06 to 95.94 ± 7.11 degrees, and patients' average visual analog scale score increased from 4.40 ± 1.14 to 8.57 ± 0.68. Interrater reliability was fair to good for visual analog scale before injection and 6 months after injection (intraclass correlation coefficients, 0.49 and 0.45, respectively; both P < 0.001). The patients were satisfied with the injection process and results. This injection protocol improved the shape of the outer ear, resulting in excellent postoperative outcomes.