This study investigated the mechanism by which Jianpi Bushen Yiqi Decoction promotes acetylcholine receptor(AChR) clustering in myasthenia gravis through the Agrin/low-density lipoprotein receptor-related protein 4(LRP4)/muscle-specific receptor tyrosine kinases(MuSK) signaling pathway. A total of 114 female C57BL/6J mice were divided into the normal group, modeling group, and solvent control group. The normal group and the solvent control group were immunized with phosphate-buffered saline(PBS), while the modeling group was established as an experimental autoimmune myasthenia gravis(EAMG) model using the murine-derived AChR-α subunit R97-116 peptide fragment. After successful modeling, the mice were randomly assigned to the model group, the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups, and the prednisone group. After four weeks of continuous treatment, muscle strength was assessed using Lennon scores and grip strength tests. Immunofluorescence staining was conducted on differentiated C2C12 myotubes incubated with a drug-containing serum to observe the number of AChR clusters. The integrity of AChR on myofilaments in mouse gastrocnemius muscles was further assessed by immunofluorescence staining. Hematoxylin-Eosin(HE)staining was applied to examine pathological changes in the gastrocnemius muscles of EAMG mice treated with Jianpi Bushen Yiqi Decoction. Western blot was utilized to detect the expression of key proteins in the Agrin/LRP4/MuSK signaling pathway in both C2C12 myotubes and mouse gastrocnemius muscles. The results demonstrated that compared to the model group, the prednisone group exhibited a significant decrease in the body weights of mice, whereas no significant differences in the body weights of mice were observed among the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups. All treatment groups showed significantly improved grip strength and Lennon scores. Additionally, the formula promoted AChR clustering on myotubes and enhanced AChR integrity in gastrocnemius myofilaments and reduced inflammatory infiltration between muscle tissue and fibrous hyperplasia. Furthermore, Jianpi Bushen Yiqi Decoction upregulated the protein expression of AChRα1, Agrin, and p-MuSK in C2C12 myotubes and increased the protein expression of AChRα1, Agrin, MuSK, p-MuSK, LRP4, and docking protein 7(Dok-7)in gastrocnemius tissue. In conclusion, Jianpi Bushen Yiqi Decoction may promote AChR clustering by targeting key proteins in the Agrin/LRP4/MuSK signaling pathway, thereby improving neuromuscular junction function and enhancing muscle strength.
Animals ; Agrin/genetics* ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Receptors, Cholinergic/genetics* ; Female ; Mice, Inbred C57BL ; Receptor Protein-Tyrosine Kinases/genetics* ; Neuromuscular Junction/metabolism* ; Myasthenia Gravis, Autoimmune, Experimental/physiopathology* ; Humans ; LDL-Receptor Related Proteins

Objective To investigate the effect of FUNDC1 tyrosine phosphorylation site mutations on mitophagy in H9c2 myocardial cells by constructing tyrosine site mutant plasmids (Y11 and Y18) of the FUN14 domain-containing protein 1 (FUNDC1). Methods The mutant plasmids constructed by whole-gene synthesis were transfected into rat myocardial H9c2 cells and divided into five groups: empty plasmid group, FUNDC1 overexpression group, Y11 mutant group, Y18 mutant group, and Y11 combined with Y18 mutant group. The viability of H9c2 cells was assessed using the CCK-8 assay. Additionally, tetramethylrhodamine ethyl ester (TMRE) staining was utilized to detect mitochondrial membrane potential. The protein expression levels of FUNDC1, translocase of the outer mitochondrial membrane 20 (TOM20), and cytochrome c oxidase IV (COX IV) were detected by Western blot analysis. Confocal microscopy was used to evaluate transfection efficiency as well as the co-localization of mitochondria and lysosomes. Results The FUNDC1 overexpression, Y11, Y18, and Y11 combined with Y18 mutant plasmids were successfully constructed. After plasmid transfection, widespread GFP fluorescence expression was observed under confocal microscopy. Compared with the empty plasmid group, FUNDC1 protein expression levels were significantly increased in the FUNDC1 overexpression group, Y11 mutation group, Y18 mutation group, and Y11 combined with Y18 mutation group, while cell viability and mitochondrial membrane potential showed no significant changes. Compared to the empty plasmid group, cells transfected with Y18 and Y11 combined with Y18 mutant plasmids showed increased TOM20 and COX IV expression levels and decreased mitochondrial-lysosomal co-localization. Conclusion Transfection with FUNDC1 Y18 or Y11 combined with Y18 mutant plasmids inhibited mitophagy in H9c2 myocardial cells.
Animals ; Rats ; Mitophagy/genetics* ; Myocytes, Cardiac/cytology* ; Mitochondrial Proteins/metabolism* ; Mutation ; Phosphorylation ; Tyrosine/genetics* ; Cell Line ; Membrane Proteins/metabolism* ; Membrane Potential, Mitochondrial

OBJECTIVES: To study clinical manifestations and gene mutation features of neonates with centronuclear myopathy. METHODS: A retrospective analysis was conducted on the medical data of 5 neonates with centronuclear myopathy diagnosed in the Neonatal Intensive Care Unit of Children's Hospital, Zhejiang University School of Medicine from January 2020 to August 2024. The data included gender, gestational age, birth weight, Apgar score, clinical manifestations, creatine kinase level, electromyography, genetic testing results and the outcomes of the infants. RESULTS: All 5 male neonates had a history of postpartum asphyxia and resuscitation. They all presented with hypotonia, myasthenia, and respiratory failure; two neonates also had swallowing dysfunction. Of the five neonates, three had normal creatine kinase levels, while two had slightly elevated levels. Electromyography was performed for three neonates, among whom two had myogenic damage. MTM1 gene mutations were identified by genetic testing in all five neonates, including two nonsense mutations and three missense mutations, among which one variant had not been previously reported. Four mutations were inherited from the mother, and the other one was a de novo mutation. The five neonates showed no clinical improvement following treatment, failed weaning from mechanical ventilation, and ultimately died after withdrawal of life-sustaining therapy. CONCLUSIONS Centronuclear myopathy caused by MTM1 gene mutation often has a severe phenotype and a poor prognosis, and it should be considered for neonates with hypotonia and myasthenia after birth. Genetic testing should be performed as soon as possible.
Humans ; Myopathies, Structural, Congenital/genetics* ; Male ; Infant, Newborn ; Retrospective Studies ; Mutation ; Female ; Protein Tyrosine Phosphatases, Non-Receptor/genetics*

OBJECTIVE: To analyze the molecular genetic spectrum of children with acute myeloid leukemia (AML), and explore its correlation with clinical characteristics and prognosis. METHODS: The clinical and molecular genetic data of 116 children with newly diagnosed AML in Wuhan Children's Hospital from September 2015 to August 2022 were retrospectively analyzed. The Fisher's exact test was used to analyze the correlation of gene mutations with clinical features, and Kaplan-Meier curve was used to analyze the influences of gene mutations on the prognosis. RESULTS: NRAS (22%), KRAS (14.9%), and KIT (14.7%) mutations were the most common genetic abnormalities in 116 children with AML. Children with KIT, CEBPA and GATA2 mutations showed a higher median onset-age than those without mutations (all P < 0.05). Children with FLT3-ITD mutation exhibited a higher white blood cell count at initial diagnosis compared to those without mutations (P < 0.05). Children with ASXL2 mutation had lower platelet count and hemoglobin at initial diagnosis than those without mutations (both P < 0.05). KIT mutations were often co-occurred with t(8;21)(q22;q22). There was no significant relationship between gene mutation and minimal residual disease (MRD) remission rate after the first and second induction therapy (P >0.05). KIT and NRAS mutations were not associated with prognosis significantly (P >0.05). The overall survival (OS) rates of children with CEBPA and FLT3-ITD mutations were superior to those without mutations, but the differences were not statistically significant (P >0.05). The 3-year OS rate of 61 children treated by allogeneic hematopoietic stem cell transplantation was 89.8%, which was significantly higher than 55.2% of those only treated by chemotherapy (P < 0.001). CONCLUSIONS Gene mutations are common in children with AML, and next-generation sequencing can significantly improve the detection rate of gene mutations, which can guide the risk stratification therapy. In addition, FLT3-ITD and KIT mutations may no longer be poor prognostic factors.
Humans ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Prognosis ; Retrospective Studies ; fms-Like Tyrosine Kinase 3/genetics* ; Child ; Proto-Oncogene Proteins c-kit/genetics* ; Male ; Female ; CCAAT-Enhancer-Binding Proteins/genetics* ; Membrane Proteins/genetics* ; Child, Preschool ; Adolescent ; GATA2 Transcription Factor/genetics* ; GTP Phosphohydrolases/genetics* ; Proto-Oncogene Proteins p21(ras)/genetics*

Lung cancer represents one of the most prevalent malignant tumors globally, and its treatment has entered the era of targeted therapy. The epidermal growth factor receptor (EGFR) mutation is a common type of genetic mutation in non-small cell lung cancer (NSCLC), while c-ros oncogene 1 receptor tyrosine kinase (ROS-1) fusion mutation is a rare mutation site. Currently, there are few case reports on the coexistence of EGFR and ROS-1 gene mutations. This study reports a case of NSCLC with coexisting EGFR and ROS-1 gene mutations, aiming to provide relevant treatment strategies for clinical practice.
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Humans ; Carcinoma, Non-Small-Cell Lung/enzymology* ; Lung Neoplasms/enzymology* ; ErbB Receptors/genetics* ; Mutation ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins/genetics* ; Male ; Middle Aged ; Female

The cooccurrence of NPM1, FLT3-ITD, and DNMT3A mutations (i.e., triple mutation) is related to dismal prognosis in patients with acute myeloid leukemia (AML) receiving chemotherapy alone. In this multicenter retrospective cohort study, we aimed to identify whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) could overcome the poor prognosis of DNMT3A^mutNPM1^mutFLT3-ITD^mut AML across four transplant centers in China. Fifty-three patients with triple-mutated AML receiving allo-HSCT in complete remission were enrolled. The 1.5-year probabilities of relapse, leukemia-free survival, and overall survival after allo-HSCT were 11.9%, 80.3%, and 81.8%, respectively. Multivariate analysis revealed that more than one course of induction chemotherapy and allo-HSCT beyond CR1 were associated with poor survival. To our knowledge, this work is the largest study to explore the up-to-date undefined role of allo-HSCT in patients with triple-mutated AML. Our real-world data suggest that allo-HSCT could overcome the poor prognosis of DNMT3A^mutNPM1^mutFLT3-ITD^mut in AML.
Humans ; Nucleophosmin ; Leukemia, Myeloid, Acute/mortality* ; Hematopoietic Stem Cell Transplantation/methods* ; Male ; Female ; DNA Methyltransferase 3A ; Adult ; China ; Retrospective Studies ; DNA (Cytosine-5-)-Methyltransferases/genetics* ; Middle Aged ; Prognosis ; fms-Like Tyrosine Kinase 3/genetics* ; Mutation ; Young Adult ; Transplantation, Homologous ; Nuclear Proteins/genetics* ; Adolescent ; Aged

L-tyrosine is one of the 20 amino acids that make up proteins and is an essential amino acid for mammals, often used as a nutritional supplement. The conventional methods for synthesizing L-tyrosine have some problems such as the production of many by-products, high requirements for production conditions, and environmental pollution. In this study, we designed and constructed a multi-enzyme cascade for the synthesis of L-tyrosine with alanine, glutamate, ammonium chloride, and phenol as substrates. Initially, the sources of glutamate oxidase, alanine aminotransferase, and tyrosine phenol lyase were screened and analyzed, which was followed by the identification of the rate-limiting enzyme in the reaction process. A colorimetric screening method was established, and the rate-limiting enzyme DbAlaA was engineered to enhance its activity by 40.0%. Subsequently, the reaction conditions, including temperature, pH, cell concentration, and surfactant and coenzyme dosages, were optimized. After optimization, the yield of L-tyrosine reached 9.93 g/L, with a alanine conversion rate of 54.90%. Finally, a feed-batch fermentation strategy was adopted, and the yield of L-tyrosine reached 56.07 g/L after 24 h, with a alanine conversion rate of 65.22%. This study provides a reference for the whole-cell catalytic synthesis of L-tyrosine and its industrialization.
Tyrosine/biosynthesis* ; Escherichia coli/metabolism* ; Tyrosine Phenol-Lyase/genetics* ; Multienzyme Complexes/metabolism* ; Fermentation

Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Based on the focal adhesion kinase(FAK)/steroid receptor coactivator(Src)/extracellular regulated protein kinase(ERK) pathway, this study explored the effects of Xihuang Pills on angiogenesis, invasion, and metastasis in prostate cancer. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) was used to analyze and identify the active ingredients of Xihuang Pills. Bioinformatics techniques, including R language and Perl programs, were employed to analyze the interactions between prostate cancer-related targets and the potential targets of Xihuang Pills. A subcutaneous transplantation tumor model of prostate cancer was established in nude mice using PC3 cells to verify the efficacy and molecular mechanisms of Xihuang Pills. In vitro cellular experiments, including cell proliferation assays(CCK-8), Transwell assays, scratch assays, real-time quantitative reverse transcription PCR, and Western blot, were used to detect the effects of Xihuang Pills on the proliferation, invasion, and migration of prostate cancer cells, as well as on FAK/Src/ERK pathway-related targets. LC-MS/MS identified 99 active ingredients in Xihuang Pills, including gallic acid, gentisic acid, artemisinin, corilagin, phenylbutazone-glucoside, thujic acid, and arecoic acid B. Network pharmacological analysis of the active ingredients in Xihuang Pills revealed that the FAK/Src/ERK signaling pathway was a key pathway in its anti-prostate cancer effects. In vivo and in vitro experiments confirmed that Xihuang Pills significantly inhibited the proliferation, invasion, and migration of PC3 and LNCaP cells, suppressed the growth of PC3 subcutaneous tumors, and reduced the protein expression levels related to the FAK/Src/ERK signaling pathway. In conclusion, the inhibition of angiogenesis, invasion, and metastasis by regulating the FAK/Src/ERK pathway is one of the mechanisms by which Xihuang Pills exert anti-prostate cancer effects.
Humans ; Male ; Prostatic Neoplasms/enzymology* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Mice ; Cell Proliferation/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; src-Family Kinases/genetics* ; Neovascularization, Pathologic/metabolism* ; Neoplasm Metastasis ; Neoplasm Invasiveness ; Focal Adhesion Kinase 1/genetics* ; Extracellular Signal-Regulated MAP Kinases/genetics* ; MAP Kinase Signaling System/drug effects* ; Focal Adhesion Protein-Tyrosine Kinases/genetics* ; Signal Transduction/drug effects* ; Angiogenesis

BACKGROUND: Thymic carcinomas (TCs) and thymic neuroendocrine neoplasms (TNENs) are two aggressive subtypes of thymic malignancy. Traditional therapy for advanced TCs and TNENs has limited outcome. New genomic profiling of TCs and TNENs might provide insights that contribute to the development of new treatment approaches. METHODS: We used gene panel sequencing technologies to investigate the genetic aberrations of 32 TC patients and 15 TNEN patients who underwent surgery at Shanghai Chest Hospital between 2015 and 2017. Patient samples were sequenced using a 324-gene platform with licensed technologies. In this study, we focused on clinically relevant genomic alterations (CRGAs), which are previously proven to be pathogenic alterations, to identify the pathology-specific mutational patterns, prognostic signatures of TCs and TNENs. RESULTS: The mutational profiles between TCs and TNENs were diverse. The genetic alterations that ranked highest in TCs were in CDKN2A, TP53, ASXL1, CDKN2B, PIK3C2G, PTCH1, and ROS1 , while those in TNENs were in MEN1, MLL2, APC, RB1 , and TSC2 . Prognostic analysis showed that mutations of ROS1, CDKN2A, CDKN2B, BRAF, and BAP1 were significantly associated with worse outcomes in TC patients, and that mutation of ERBB2 indicated shortened disease-free survival (DFS) and overall survival (OS) in TNEN patients. Further investigation found that the prognosis-related genes were focused on signal pathways of cell cycle control, chromatin remodeling/DNA methylation, phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase (MAPK) signaling. CONCLUSION We profiled the mutational features of 47 Chinese patients with thymic malignancy of diverse pathologic phenotypes to uncover the integrated genomic landscape of these rare tumors, and identified the pathology-specific mutational patterns, prognostic signatures, and potential therapeutic targets for TCs and TNENs.
Humans ; Thymoma ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins/genetics* ; China ; Thymus Neoplasms/pathology* ; Prognosis ; Neuroendocrine Tumors/pathology* ; Mutation/genetics*