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🏛️ Key Research Academy41 Indexed Works

Shanghai Jiao Tong University

Verified scientific contributions, CAS laboratory outputs, clinical trial papers, and engineering breakthroughs produced by researchers and faculty affiliated with Shanghai Jiao Tong University.

Stem Cell Research & Therapy•2026

Editorial Expression of Concern: Co-encapsulation of HNF4α overexpressing UMSCs and human primary hepatocytes ameliorates mouse acute liver failure

Authors: Defu Kong, Huiming Xu, Mo Chen, Yeping Yu, Yongbing Qian, Tian Qin, Ying Tong, Qiang Xia, Hualian Hang

The Editor-in-Chief is issuing an Editorial Expression of Concern to alert readers about concerns regarding the reporting of animal ethics approval in this article. The article cites approval number SYXK 2008 0050, which was noted to appear in multiple publications describing different experiments. The authors have explained that this number refers to an Experimental Animal Use License for the animal facility rather than a study specific ethics approval and have provided documentation indicating that separate ethical approval was obtained for this study. Despite this, the reporting of animal use approval in the article and the use of a general approval instead of a specific one is inadequate. Readers are therefore advised to interpret the information regarding animal ethics approval with caution.

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Stem Cell Research & Therapy•2026

A novel OTUD5 variant disrupts neural progenitor cell homeostasis: mechanistic insights from HEK293T cell-based analyses

Authors: Na Xu, Shihao Wang, Tingting Yang, Meiping Yu, Yu Sun, Yongkun Zhan, Yongguo Yu

Background: Variants in OTUD5 are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel OTUD5 variant (c.697G>A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of OTUD5 in neurodevelopment. Methods: The OTUD5 variant was identified in two NDD patients via exome sequencing. Patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9-corrected isogenic controls were generated. NPC proliferative activity was assessed by Ki67 immunofluorescence staining, cell-cycle distribution was analyzed by flow cytometry, and neuronal differentiation was evaluated by Tuj1/MAP2 immunofluorescence staining. Substrate screening was conducted in HEK293T cells using co-immunoprecipitation (Co-IP) and mass spectrometry. Deubiquitination capacity and protein stability were validated through ubiquitination assays and cycloheximide (CHX) chase experiments. Results: The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the OTUD5 protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67+ cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj+ cells). Mechanistically, wild-type OTUD5 stabilized GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%. Conclusion: This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in OTUD5-related NDDs.

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Acta Biochimica et Biophysica Sinica•2026

Super-resolution imaging reveals higher-order structures within common fragile sites in human mitotic chromosomes

Authors: Jiabin Wang, Daniel M. Czajkowsky, Zhifeng Shao

Common fragile sites (CFSs) are large genomic loci that are frequently deleted under replication stress and are thought to play a role in carcinogenesis as well as developmental disorders during early embryogenesis. They often appear as gaps or breaks in mitotic chromosomes by conventional optical microscopy and are also loci that are replicated in mitosis in a process called mitotic DNA synthesis (MiDAS). However, we still have a very poor understanding of the potential mechanisms underlying their genomic instability. We recently found that in normal mitotic chromosomes, there is a two-level hierarchy in the higher-order chromatin structure, with mechanically resistant ~90 nm Mitotic NanoDomains (MNDs) assembling into ~125 nm compact granules (CGs). In this work, we combine multiple super-resolution imaging techniques, including binding-activated localization microscopy (BALM), atomic force microscopy (AFM), and stochastic optical reconstruction microscopy (STORM), to characterize CFSs. Our super-resolution data indicate that sites that appear as gaps by conventional microscopy can be filled with chromatin of the size and shape of MNDs but not CGs. Moreover, we find that MiDAS loci only form an array of MNDs with no CGs. Taken together, our work suggests that under replication stress, CFS loci can not only be replicated but also assembled into higher-order chromatin. However, this organization is limited to the level of MNDs, which suggests that a failure to progress beyond MNDs to form CGs constitutes a key structural deficiency at these loci.

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Chinese Journal of Tissue Engineering Research•2026

Oxidative stress and osteoporosis: a bibliometric analysis of literature from SCI core database

Authors: GUO Jun, LU Zheng, YU Jinling, HAO Yuanyuan, LIU Kaishun, LIU Xuexia, HUANG Yourong

BACKGROUND: Oxidative stress, representing an imbalance between oxidative and antioxidant systems in the body, plays a crucial role in the pathogenesis of osteoporosis. However, a systematic analysis of the current research status and trends in the field of oxidative stress and osteoporosis is lacking. OBJECTIVE: To analyze the current research status, hot topics, and trends in the field of oxidative stress and osteoporosis using bibliometric methods. METHODS: The Web of Science core database was searched using “oxidative stress” and “osteoporosis” as search terms, with the language limited to “English” and the document type limited to “article” and “review article.” The search period was from January 1, 1999 to December 31, 2024. After screening the literature according to the inclusion and exclusion criteria, CiteSpace (6.3.R1) and VOSviewer (1.6.20) software were used for data analysis and visualization of publication volume, country, institution, author, journal, and keywords. RESULTS AND CONCLUSION: (1) Publication volume analysis: A total of 2 558 articles were retrieved, with 2 416 articles included. From 1999 to 2024, the number of publications in the field of oxidative stress and osteoporosis showed a significant increasing trend, especially after 2011, reflecting the gradual deepening and rising popularity of research in this field. (2) Country analysis: China ranked first with 1 088 publications, but the average citations per article were relatively low. The United States ranked second with 353 publications, but the average citations per article were as high as 74.62, demonstrating international influence in research quality. (3) Institution analysis: Chinese institutions dominated in publication volume, but the level of international cooperation needs improvement. Among them, Shanghai Jiao Tong University and Soochow University had extensive cooperation and high citation counts. (4) Author and co-cited author analysis: A few core authors such as Almeida and Manolagas had significant influence in this field, with extremely high citation counts and diverse collaboration models, dominated by international authors. (5) Journal analysis: American journals such as the Journal of Bone and Mineral Research occupied a core position in the field of osteoporosis and oxidative stress, with significant academic authority and influence. (6) Keyword analysis: The research core focused on the interaction mechanism between “osteoporosis” and “oxidative stress.” High-frequency keywords included “reactive oxygen species,” “inflammation,” “osteoblasts,” and “osteoclasts.” Keyword clustering analysis showed that research hotspots concentrated on inflammation, oxidative stress and bone metabolic imbalance, population and clinical studies, and the development and application of antioxidant therapy. (7) The field of oxidative stress and osteoporosis is growing rapidly. Although China occupies a dominant position, its international influence needs improvement. Future research should deeply analyze the oxidative stress signaling network, explore cross-disease interactions, develop novel antioxidants and therapeutic methods, promote precision medicine and multi-omics technology applications, and strengthen international cooperation and exchange, in order to provide more scientific and effective solutions for the prevention and treatment of osteoporosis.

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Chinese Journal of Tissue Engineering Research•2026

Exosomes and neuropathic pain: visualization analysis on literature

Authors: MA Jing, HAN Jing, SHI Linyu, WU Yuwei, YUAN Haiguang, LI Yongfeng

BACKGROUND: Neuropathic pain has a complex pathogenesis and limited clinical intervention outcomes. In recent years, exosomes have gradually emerged as a focal point in the study of neuropathic pain due to their unique intercellular communication functions and molecular delivery capabilities. OBJECTIVE: To systematically review research progress on exosomes in the field of neuropathic pain using bibliometric methods, summarize the knowledge framework and research hotspots, and provide theoretical foundations and translational strategies for advancing this field. METHODS: Based on the Web of Science Core Collection (WOSCC) database, literature on exosomes and neuropathic pain was retrieved from 2012-01-01 to 2025-03-31. VOSviewer and CiteSpace software were used for keyword co-occurrence, cluster analysis, burst detection, and collaboration network visualization. RESULTS AND CONCLUSION: A total of 313 articles were included. Among 42 countries or regions, the United States and China made significant contributions. Shanghai Jiao Tong University and Nantong University were the most prolific institutions. The most productive and co-cited journals were Neural Regeneration Research and International Journal of Molecular Sciences, respectively. A total of 382 authors were identified; Zhang Zhenggang had the most articles, and Zhang Yi had the most citations. Key high-frequency keywords included 'nerve regeneration', 'neuroinflammation', 'schwann cell', 'regenerative medicine', and 'spinal cord injury', which are key research areas for future development. Through bibliometric analysis, this study mapped the research trends of exosomes in neuropathic pain. Exosomes hold broad prospects in elucidating basic mechanisms and developing novel precision therapies for neuropathic pain. Future efforts should integrate multi-omics and engineered exosome platforms to advance this field.

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Chinese Journal of Tissue Engineering Research•2026

Function and molecular mechanism of physcion in regulating bone homeostasis

Authors: Qi Yuxin, Dang Yifan, Dai Liming, Zhang Xiaoling

BACKGROUND: Although physcion has been shown to have protective effects against osteoporosis, the exact mechanism is not fully understood. OBJECTIVE: Through multidimensional analysis of the regulatory effect of physcion on the AKT signaling pathway, the molecular mechanism of its regulation on osteoclast induced differentiation and osteogenic function induced differentiation is revealed. METHODS: (1) RAW264.7 cells and C3H10T1/2 cells were cultured in vitro and subsequently exposed to 0, 10, 20, 30, 40, 50, and 60 µmol/L physcion, respectively. The cytotoxicity of physcion was detected by cell counting kit-8 assay. (2) RAW264.7 cells and C3H10T1/2 cells were treated with different concentrations (0, 20, 40 µmol/L) of physcion during osteoclast and osteoblast differentiation, respectively. Differentiation ability was assessed by qPCR, Western Blot, and alkaline phosphatase staining. (3) Network pharmacology was used to analyze the regulation of physcion on osteoclast differentiation and related signaling pathways, and molecular docking was performed for target proteins. (4) Western Blot was used to verify the phosphorylation level of AKT in the downstream target signaling pathway AKT axis regulated by physcion. RESULTS AND CONCLUSION: (1) At concentrations of 0-60 µmol/L, cell viability in all groups was greater than 90%, indicating no significant cytotoxicity. (2) Physcion significantly inhibited the expression of osteoclast differentiation-related genes, with Acp5, CTSK, DC-STAMP, and Nfatc1 showing downregulation, but had no significant effect on osteoblast differentiation-related genes COL1A1, Runx2, OSX expression or alkaline phosphatase staining intensity. (3) Network pharmacology and molecular docking suggested that physcion affects osteoclast differentiation and regulates the PI3K-AKT pathway, with a binding energy of -10.72 kJ/mol to AKT1, indicating strong binding activity. (4) During osteoclast differentiation, the p-AKT/AKT ratio in RAW264.7 cells increased (n=3, P=0.0063), while physcion decreased this ratio. These findings indicate that physcion inhibits osteoclast differentiation by regulating the AKT signaling pathway, thereby modulating bone homeostasis.

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Chinese Journal of Tissue Engineering Research•2026

Neutrophils and the repair of hard-to-heal wounds

Authors: LIU Yufei, CHANG Jinxia, MI Baolai, YUAN Liang, GU Hancheng, CAO Jianchun

BACKGROUND: Dysfunction of neutrophils in the microenvironment of refractory wounds and their abnormal interactions with other immune cells and repair cells have become a central research focus for understanding the pathophysiology of chronic wounds and developing novel intervention strategies. OBJECTIVE: To investigate the current research status, hotspots, frontiers, and development trends regarding neutrophils in the field of hard-to-heal wound repair. METHODS: Relevant literature regarding neutrophils in the repair of hard-to-heal wounds published between 2004 and 2024 was retrieved from the Web of Science Core Collection database. Bibliometric visualization analysis methods were employed for analysis and visualization, revealing the research landscape, major hotspots, and frontier trends from dimensions including publication volume, countries/regions, institutions, authors, journals, references, and keywords. RESULTS AND CONCLUSION: A total of 1 062 relevant articles concerning neutrophils in hard-to-heal wound repair were included. The annual publication output in this field showed a significant growth trend, especially after 2014, with the number of publications exceeding 100 in 2023, indicating increasing academic attention. The United States led globally with 378 publications and extensive international collaboration networks; China ranked second with 189 publications, showing strong research vitality, though international collaboration and citations per paper still have room for improvement. Shanghai Jiao Tong University in China was the institution with the most publications, while the University of Illinois and Harvard University in the United States were centers of citation frequency. Journal analysis showed that Wound Repair and Regeneration was the core journal with the highest publication and citation counts. Research hotspots focused on inflammation, angiogenesis, diabetic foot ulcers, etc. Keyword clustering timeline and burst analysis revealed that neutrophil extracellular trap formation and neutrophil-macrophage interactions are frontier research trends. The results indicate that the field has formed a clear development path from clinical problems to molecular mechanisms and then to immune intervention strategies, and future precise regulation of neutrophil function is expected to open new avenues for the treatment of hard-to-heal wounds.

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Chinese Journal of Tissue Engineering Research•2026

Regulatory effects of optimized extraction processes for chlorella-derived peptides on key pathological links in rheumatoid arthritis

Authors: ZHANG Yiwei, FANG Ya, SUN Xin, YANG Han, LIN Haiyang, CHEN Zhouhao, ZHENG Yue, FU Jingke, WANG Jinwu

BACKGROUND: Recent studies have shown that Chlorella possesses potential value in treating rheumatoid arthritis. The pathological progression of rheumatoid arthritis is closely associated with an imbalance in oxidative stress, abnormal macrophage polarization, aggressive activation of fibroblast-like synoviocytes, and disturbances in the vascular endothelial system. However, the optimization of extraction processes for peptides derived from Chlorella and their regulatory effects on key pathological links of rheumatoid arthritis remain to be systematically validated. OBJECTIVE: To optimize the extraction process of antioxidant peptides from Chlorella, clarify their antioxidant activity and biosafety, and explore their regulatory effects on pathological phenotypes of rheumatoid arthritis-related cells (RAW 264.7 mouse monocyte macrophage leukemia cells, fibroblast-like synoviocytes, and human umbilical vein endothelial cells), providing experimental evidence for the treatment of rheumatoid arthritis with Chlorella peptides. METHODS: (1) Chlorella peptide extract was prepared by bromelain enzymatic hydrolysis combined with phosphomolybdic acid precipitation. Using peptide yield as the evaluation index, the extraction process parameters were optimized by single-factor experiments, including solid-liquid ratio, enzymatic hydrolysis time, and reaction system pH. (2) The peptide content was determined by BCA method, antioxidant capacity was detected by ABTS method, and biosafety of peptides on RAW 264.7 cells, fibroblast-like synoviocytes, and human umbilical vein endothelial cells was evaluated by CCK-8 method. (3) An inflammatory model of RAW 264.7 cells induced by lipopolysaccharide was established. The effects of peptides on intracellular reactive oxygen species levels and M1/M2 polarization phenotypes were detected by DCFH-DA staining, flow cytometry, and real-time fluorescence quantitative reverse transcription polymerase chain reaction. (4) An activation model of fibroblast-like synoviocytes induced by tumor necrosis factor-alpha was established. The effects of peptides on migration, proliferation, invasion, and related gene expression of fibroblast-like synoviocytes were detected by wound healing assay, EdU proliferation assay, Transwell invasion assay, and real-time fluorescence quantitative reverse transcription polymerase chain reaction. (5) An abnormal activation model of human umbilical vein endothelial cells induced by vascular endothelial growth factor A was established. The effects of peptides on migration, tube formation, and expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor A genes were detected by wound healing assay, Transwell assay, tube formation assay, and real-time fluorescence quantitative reverse transcription polymerase chain reaction. RESULTS AND CONCLUSION: (1) The optimal extraction process for Chlorella peptides was solid-liquid ratio of 2:1 (g:100 mL), enzymatic hydrolysis time of 60 minutes, and reaction system pH of 6.5, yielding the highest peptide yield. (2) Chlorella peptides exhibited concentration-dependent antioxidant activity and showed no obvious toxicity to RAW 264.7 cells, fibroblast-like synoviocytes, and human umbilical vein endothelial cells in the concentration range of 1-10 μg/mL, indicating good biocompatibility. (3) Chlorella peptides dose-dependently inhibited lipopolysaccharide-induced reactive oxygen species generation in RAW 264.7 cells, downregulated M1 pro-inflammatory genes such as interleukin-1 beta and tumor necrosis factor-alpha, upregulated M2 anti-inflammatory genes such as interleukin-10 and arginase 1, and promoted macrophage polarization from M1 to M2 phenotype. (4) Chlorella peptides significantly inhibited tumor necrosis factor-alpha-induced migration, proliferation, and invasion of fibroblast-like synoviocytes, and downregulated the expression of interleukin-6, matrix metalloproteinase 13, tumor necrosis factor receptor superfamily member 11A, and C-X-C motif chemokine ligand 12. (5) Chlorella peptides effectively inhibited vascular endothelial growth factor A-induced migration and tube formation of human umbilical vein endothelial cells, and reduced the expression of hypoxia-inducible factor 1 alpha and vascular endothelial growth factor A genes. These results indicate that Chlorella peptides regulate multiple pathological links of rheumatoid arthritis through anti-oxidative stress, regulation of macrophage polarization, inhibition of aggressive phenotype of fibroblast-like synoviocytes, and improvement of vascular endothelial disorders, suggesting potential therapeutic value for rheumatoid arthritis.

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Stem Cell Research & Therapy•2026

Editorial Expression of Concern: Co-encapsulation of HNF4α overexpressing UMSCs and human primary hepatocytes ameliorates mouse acute liver failure

Authors: Defu Kong, Huiming Xu, Mo Chen, Yeping Yu, Yongbing Qian, Tian Qin, Ying Tong, Qiang Xia, Hualian Hang

The Editor-in-Chief is issuing an Editorial Expression of Concern to alert readers about concerns regarding the reporting of animal ethics approval in this article [1]. The article cites approval number SYXK 2008 0050, which was noted to appear in multiple publications describing different experiments. The authors have explained that this number refers to an Experimental Animal Use License for the animal facility rather than a study specific ethics approval and have provided documentation indicating that separate ethical approval was obtained for this study. Despite this, the reporting of animal use approval in the article and the use of a general approval instead of a specific one is inadequate. Readers are therefore advised to interpret the information regarding animal ethics approval with caution.

Peer ReviewedView Paper→

Stem Cell Research & Therapy•2026

A novel OTUD5 variant disrupts neural progenitor cell homeostasis: mechanistic insights from HEK293T cell-based analyses

Authors: Na Xu, Shihao Wang, Tingting Yang, Meiping Yu, Yu Sun, Yongkun Zhan, Yongguo Yu

Background Variants in OTUD5 are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel OTUD5 variant (c.697G > A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of OTUD5 in neurodevelopment. Methods The OTUD5 variant was identified in two NDD patients via exome sequencing. Patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9-corrected isogenic controls were generated. NPC proliferative activity was assessed by Ki67 immunofluorescence staining, cell-cycle distribution was analyzed by flow cytometry, and neuronal differentiation was evaluated by Tuj1/MAP2 immunofluorescence staining. Substrate screening was conducted in HEK293T cells using co-immunoprecipitation (Co-IP) and mass spectrometry. Deubiquitination capacity and protein stability were validated through ubiquitination assays and cycloheximide (CHX) chase experiments. Results The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the OTUD5 protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67+ cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj+ cells). Mechanistically, wild-type OTUD5 stabilized GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%. Conclusion This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in OTUD5-related NDDs.

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Acta Biochimica et Biophysica Sinica•2026

Development of an in vitro TurboID labeling assay for the detection of protoRAG-interacting proteins in the amphioxus

Authors: Qingyi Lu, Jie Xu, Junye Hong, Enfan Xiao, Qiuzhu Wei, Yuhe Sun, Zihan Zhao, Yuhang Zhang, Guangrui Huang

The recombination-activating gene (RAG)-mediated V(D)J rearrangement mechanism is essential for generating the diversity of the adaptive immune system in jawed vertebrates. The RAG1 and RAG2 proteins form a tetrameric complex that recognizes and cleaves pairs of recombination signal sequences (RSSs). In jawed vertebrates, the N-terminal region of RAG1 and the C-terminal region of RAG2 are critical for the regulation of physiological function and enhancement of recombination accuracy. Moreover, the intracellular activity of RAG1/2 is rigorously regulated and is thought to represent the aspect shaped by host coevolution. However, these regulatory mechanisms remain incompletely understood, impeding our deeper understanding of the physiological functions of RAG. Previous studies have shown that jawed vertebrate RAG evolved from invertebrate RAG-like (RAGL) transposases and underwent key structural changes during evolution. RAGL genes have been identified in multiple invertebrate species, but from Branchiostoma belcheri (Bb), the RAG-like protein in Bb amphioxus (BbRAG1L) and the RAG2-like protein in Bb amphioxus (BbRAG2L) are among the most extensively characterized to date. Lancelets (amphioxus) represent the most basal extant chordates (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates). Consequently, amphioxus has been considered a “living fossil” for studying the origins and evolution of adaptive immunity. Homologs of the core structural domain of RAG1 were discovered in the amphioxus genome in 2005, and the homologs were subsequently shown to exhibit recombinase activity in cooperation with murine-derived RAG2. A large number of transposon genes were then identified in the amphioxus genome, followed by the identification of target site duplication (TSD) and terminal inverted repeat (TIR) sequences in the amphioxus genome. Notably, BbRAG2L lacks the C-terminal region present in murine RAG2, including the acidic hinge and PHD domain. BbRAG1L coupled with BbRAG2L displays significant transposition activity in vitro and in human cell lines. In contrast, vertebrate RAG exhibits limited transposon activity in vitro and extremely low transposon activity in vivo. This leads to the question of how RAG lost its transposase activity during evolution. Structural comparison and functional experiments of BbRAGL (protoRAG) and vertebrate RAG have revealed that vertebrates undergo essential adaptive changes to eliminate the primitive transposase activity of protoRAG. Research on BbRAGL has therefore provided strong support for the hypothesis that RAG originated from a domesticated transposon. Given the similarity in both structure and function between BbRAG1L and vertebrate RAG and the fact that BbRAGL proteins are the only RAGL proteins that can currently be efficiently expressed in vertebrate cells, characterizing the binding partners of BbRAGL involved in amphioxus is of great significance for understanding the regulation of BbRAGL and its adaptation and evolution. Parallel comparison of BbRAGL and RAG interaction networks may further clarify how BbRAGL transitioned from a transposase to a recombinase during host coevolution and reveal general principles of vertebrate intracellular RAG regulation. In recent years, proximity labeling techniques have greatly facilitated approaches to identifying RAG cofactors. For example, RAG1 fused to biotin ligase RAG1 fused to a biotin ligase found in Escherichia coli (BirM) has been used to define dynamic interaction networks of RAG-associated proteins. Inspired by these advances, we sought to apply a similar approach to identify the BbRAGL cofactors in amphioxus and build a regulatory network of BbRAGL that could shed light on its relationship with host evolution. However, endogenous BbRAGL expression is undetectable in the adult amphioxus (data not shown), and the generation of transgenic amphioxus lines expressing proximity-based labeling proteins remains technically challenging. These constraints make it difficult to analyze native cofactors of endogenous BbRAGL in vivo. Because BbRAGL can be expressed in vertebrate cell lines, we instead developed an in vitro proximity-labeling strategy. We fused TurboID (TbID), an engineered biotin ligase, to the N-terminus of BbRAG1L or BbRAG2L (Figure 1A), purified the fusion proteins, and incubated them with protein lysates prepared from the hepatic cecum and colons of adult B. belcheri amphioxus in vitro to label the interacting proteins. The proteins were finally analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 1B). A pTT5 vector was used to express plasmids encoding maltose-binding protein (MBP) and TurboID with or without full-length BbRAG1L or BbRAG2L, yielding pTT5-MBP-TurboID, pTT5-MBP-TurboID-BbRAG1L, and pTT5-MBP-TurboID-BbRAG2L. Plasmids expressing MBP-BbRAG1L and MBP-BbRAG2L were also generated.

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Acta Biochimica et Biophysica Sinica•2026

Super-resolution imaging reveals higher-order structures within common fragile sites in human mitotic chromosomes

Authors: Jiabin Wang, Daniel M. Czajkowsky, Zhifeng Shao

Common fragile sites (CFSs) are large genomic loci that are frequently deleted under replication stress and are thought to play a role in carcinogenesis as well as developmental disorders during early embryogenesis. They often appear as gaps or breaks in mitotic chromosomes by conventional optical microscopy and are also loci that are replicated in mitosis in a process called mitotic DNA synthesis (MiDAS). However, we still have a very poor understanding of the potential mechanisms underlying their genomic instability. We recently found that in normal mitotic chromosomes, there is a two-level hierarchy in the higher-order chromatin structure, with mechanically resistant ~90 nm Mitotic NanoDomains (MNDs) assembling into ~125 nm compact granules (CGs). In this work, we combine multiple super-resolution imaging techniques, including binding-activated localization microscopy (BALM), atomic force microscopy (AFM), and stochastic optical reconstruction microscopy (STORM), to characterize CFSs. Our super-resolution data indicate that sites that appear as gaps by conventional microscopy can be filled with chromatin of the size and shape of MNDs but not CGs. Moreover, we find that MiDAS loci only form an array of MNDs with no CGs. Taken together, our work suggests that under replication stress, CFS loci can not only be replicated but also assembled into higher-order chromatin. However, this organization is limited to the level of MNDs, which suggests that a failure to progress beyond MNDs to form CGs constitutes a key structural deficiency at these loci.

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Acta Biochimica et Biophysica Sinica•2026

Targeting USP2 induces degradation of PML-RARα with or without drug-resistant mutations in acute promyelocytic leukemia

Authors: Jie Zhang, Wenxuan Wu, Yun Wang, Youping Zhang, Yingying Wang, Wenhui Bai, Zhenge Zhang, Chujiao Zhu, Yunzhao Wu, Ziwei Zhang, Li Yang, Hu Lei, Hanzhang Xu, Li Zhou, Yingli Wu

Despite the high efficacy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL), approximately 10%–20% of patients develop drug resistance due to mutations in PML-RARα and other factors. Here, we find that inhibition of USP2 with ML364 or USP2 silencing reduces PML-RARα protein levels in both ATRA-sensitive and ATRA-resistant APL cells, and this effect is reversed by proteasome inhibition. Conversely, USP2 overexpression enhances PML-RARα stability. Mechanistically, USP2 interacts with and deubiquitinates PML-RARα, including its drug-resistant mutants. Consistent with PML-RARα degradation, ML364 treatment significantly induces apoptosis in APL cell lines and primary leukemia cells. In conclusion, this study identifies USP2 as a novel deubiquitinating enzyme for PML-RARα and highlights USP2 inhibition as a potential therapeutic strategy for APL with PML-RARα mutations.

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Acta Biochimica et Biophysica Sinica•2026

ADD domain added new binding partners for the nuclear hub protein ATRX

Authors: Yan Chen, Yang Luo, Jielin Sun, Shouhua Wang, Bingbing Wan

ATRX is a large, multi-domain nuclear protein that functions as a crucial ATP-dependent chromatin remodeler, transcriptional regulator, and guardian of telomeric and genomic integrity. As a member of the SWI/SNF family of chromatin remodeling proteins, a primary and well-defined function of ATRX is to facilitate the replication-independent deposition of the histone variant H3.3 at specific genomic loci, predominantly repetitive sequences such as telomeres, pericentromeric heterochromatin, and ribosomal DNA (rDNA). Mutations in the ATRX gene are associated with a severe X-linked neurodevelopmental disorder and alpha-thalassemia. Moreover, ATRX dysfunction can lead to genomic instability, contributing to the development and progression of various cancers, including gliomas and pancreatic neuroendocrine tumors (PanNETs). ATRX orchestrates chromatin dynamics through its modular domains. Its N-terminal ADD domain and a PxVxL-like motif recognize histone H3K9me3 and interact with the heterochromatin-binding protein HP1α, respectively. Collaborating with the histone chaperone DAXX, ATRX then utilizes its C-terminal ATPase/Helicase domain to provide the energy needed to remodel chromatin and deposit the histone variant H3.3 into repetitive DNA regions. The Chen lab and other two groups previously elucidated the minimal elements for DAXX interaction, demonstrating that a short 1260‒1289 residues motif (DAXX-binding motif, DBM) of ATRX is solely responsible for ATRX-DAXX heterodimer formation. Beyond its role in chromatin remodeling, ATRX employs its RBR (RNA-binding region) to engage the telomeric long non-coding RNA (lncRNA) TERRA, thereby regulating TERRA-mediated R-loops and telomeric G-quadruplex (G4) structures. Interestingly, this same RBR also binds the muscle-specific lncRNA ChRO1 to coordinate constitutive heterochromatin reorganization and regulate cell differentiation. Notably, nearly half of the disease-causing mutations in ATRX, leading to a severe neurodevelopmental disorder, are clustered within the ADD domain, highlighting this relatively small domain’s critical functional importance and warranting more intense investigation. This Research Highlight discusses recent findings by Yan et al. that the histone variant macroH2A binds the ATRX ADD domain, expanding the known binding partners of this domain and providing structural insights into the interaction.

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Acta Biochimica et Biophysica Sinica•2026

Proximity-based proteomic profiling uncovers distinct interactome of human RAG1 and RAG2

Authors: HONG Junye, ZHENG Xueming, WEI Kunyu, LU Qingyi, HUANG Guangrui, ZHANG Yuhang

The recombination-activating gene (RAG) complex initiates adaptive immunity by catalyzing V(D)J recombination to generate diverse antigen receptors. While the catalytic function of the RAG core is well defined, its regulatory interactions and physiological roles remain poorly understood due to limited knowledge of RAG-associated proteins. The RAG complex forms a heterotetramer of two RAG1 and RAG2 subunits, yet the individual contributions of each subunit remain unclear. Here, we use TurboID-mediated proximity labelling to map the human RAG interactome. By fusing TurboID to RAG1 or RAG2, we identify 88 RAG1- and 146 RAG2-associated proteins, with only 23 shared proteins, indicating distinct sets of proximal proteins. Although RAG1 and RAG2 are thought to exert their physiological functions by forming a complex, they display distinct potential interaction networks, suggesting subunit-specific functions and revealing their spatial proximity to each subunit. These findings uncover distinct RAG1 and RAG2 interaction landscapes and establish a framework for exploring broader RAG functions in immunity.

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Acta Biochimica et Biophysica Sinica•2026

1α,25(OH)2D3 prevents CD19 CAR-T cell exhaustion and differentiation via VDR-dependent transcriptional reprogramming

Authors: Mengke He, Ningzhe Li, Yiwen Cao, Jie Jiang, Fan Zhang, Zeyi Li, Jie Shen, Dehao Zhu, Xiaxin Liu, Qiang Wang, Chenjing Ye, Junmin Li, Zhen Jin, Rufang Xiang

CD19-directed chimeric antigen receptor T (CAR-T) cell therapy is promising for treating relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), yet its long-term efficacy remains limited by CAR-T-cell exhaustion. Given the broad immunomodulatory activities of vitamin D, we investigate whether its active form, 1α,25(OH)2D3, enhances CAR-T-cell functionality and improves therapeutic outcomes. We demonstrate that 1α,25(OH)2D3 treatment significantly mitigates exhaustion and enhances the antitumor activity of CD19 CAR-T cells derived from both healthy donors and DLBCL patients, which is further validated in xenograft mouse models. Mechanistically, we show that 1α,25(OH)2D3 upregulates the expression of the vitamin D receptor (VDR), promoting transcriptional reprogramming associated with memory-like differentiation and downregulation of exhaustion-related genes, thereby reshaping the functional heterogeneity of CAR-T cells under tumor stimulation. Our study highlights 1α,25(OH)2D3 supplementation as a safe and accessible approach to mitigate terminal differentiation and exhaustion of CAR-T cells, offering a promising strategy to enhance the clinical efficacy of CAR-T therapy in patients with R/R DLBCL.

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Acta Biochimica et Biophysica Sinica•2026

DDX11 interacts with PARP1 to facilitate PARylation, thereby promoting gallbladder cancer progression and conferring gemcitabine resistance

Authors: Yuan Gao, Junchi Liu, Xiang Yao, Letian Gong, Shubin Luo, Chaoxian Zhao, Shaofeng Pu, Ganglong Gao

Gemcitabine resistance poses a significant challenge in gallbladder cancer (GBC) treatment, necessitating exploration of its molecular mechanisms. This study focuses on DDX11, which is highly expressed in gemcitabine-resistant GBC cells, suggesting a potential role in DNA damage repair. We establish gemcitabine-resistant GBC cell lines and observe significantly higher DDX11 expression in these cells than in parental cells. Clinical tissue analysis through qRT-PCR, western blot analysis, and immunohistochemistry confirms elevated DDX11 levels in tumors compared with adjacent normal tissues. Functional assays demonstrate that DDX11 knockdown inhibits cell proliferation, colony formation, and tumor growth, while restoring gemcitabine sensitivity. Mechanistically, proteomic analysis and co-immunoprecipitation reveal that the interaction of DDX11 with PARP1 leads to increased poly(ADP-ribosyl)ation (PARylation), which promotes DNA repair and drug resistance. Notably, combining gemcitabine with the PARP inhibitor olaparib has synergistic anti-tumor effects on resistant cells. These findings indicate that DDX11 contributes to GBC progression and chemoresistance by regulating PARP1-mediated PARylation and that targeting this pathway with PARP inhibitors may overcome gemcitabine resistance. This study provides new insights into GBC drug resistance mechanisms and suggests that combining conventional chemotherapy with PARP inhibition is a potential therapeutic strategy for resistant patients. The DDX11-PARP1-PARylation axis represents a promising target for improving GBC treatment outcomes, particularly in gemcitabine-resistant patients.

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Chinese Journal of New Drugs•2025

Development of a Renal Function Assessment and Monitoring System Using Machine Learning and Cloud Computing

Authors: Y. Zhang, L. Wang, H. Li, J. Chen

Chronic kidney disease (CKD) is a global health burden, and early detection is crucial for effective management. This study presents a novel renal function assessment and monitoring system that integrates machine learning algorithms with cloud computing to enable real-time, non-invasive monitoring of renal function. The system utilizes a multi-modal approach, combining clinical biomarkers, patient demographics, and continuous physiological data from wearable sensors. A gradient boosting machine (GBM) model was trained on a large retrospective cohort (n=12,000) and validated on a prospective cohort (n=1,500), achieving an AUC of 0.94 for detecting early-stage CKD. The system also incorporates a cloud-based dashboard for remote monitoring and alerts, facilitating timely interventions. Key innovations include the use of explainable AI (XAI) to provide interpretable predictions, and a federated learning framework to ensure data privacy. The system demonstrated high accuracy, scalability, and usability in clinical settings, suggesting its potential to transform CKD management by enabling proactive, personalized care.

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Chinese Journal of New Drugs•2025

Advanced cholangiocarcinoma therapy: enhanced drug delivery via targeted nanocarriers

Authors: Y. Zhang, L. Wang, H. Li, J. Chen

Cholangiocarcinoma (CCA) is a highly aggressive malignancy with poor prognosis, and conventional chemotherapy is limited by systemic toxicity and drug resistance. This study presents a novel targeted nanocarrier system for the delivery of therapeutic agents to CCA cells, utilizing a specific ligand that binds to overexpressed receptors on tumor cells. The nanocarriers were characterized for size, zeta potential, drug loading, and release profiles. In vitro studies demonstrated enhanced cellular uptake and cytotoxicity against CCA cell lines, while in vivo studies in a mouse model showed significant tumor growth inhibition and reduced systemic toxicity compared to free drug. The findings suggest that this targeted nanocarrier system holds promise for improving the therapeutic efficacy and safety of CCA treatment.

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Chinese Journal of New Drugs•2025

A Study on the Application of Fuzzy Logic in the Optimization of Machining Parameters for Enhanced Surface Quality in CNC Milling

Authors: Y. Zhang, L. Wang, H. Li, X. Chen

This paper presents a comprehensive study on the application of fuzzy logic for optimizing machining parameters in CNC milling processes to enhance surface quality. The proposed fuzzy logic model integrates multiple input parameters such as spindle speed, feed rate, and depth of cut to predict and optimize surface roughness. Experimental validation was conducted on aluminum alloy 6061, demonstrating significant improvements in surface finish compared to conventional methods. The results indicate that the fuzzy logic approach effectively handles the non-linear relationships between machining parameters and surface quality, providing a robust framework for process optimization. The study also discusses the potential of integrating fuzzy logic with other intelligent techniques for real-time adaptive control in smart manufacturing environments.

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Chinese Journal of New Drugs•2025

Enhancing the Mechanical Properties of 3D-Printed Continuous Carbon Fiber Reinforced Polymer Composites via Process Parameter Optimization

Authors: J. Zhang, L. Wang, M. Chen, R. Liu

This study investigates the influence of key process parameters on the mechanical properties of continuous carbon fiber reinforced polymer (CFRP) composites fabricated via fused filament fabrication (FFF). A systematic experimental design was employed to evaluate the effects of layer height, extrusion temperature, and printing speed on tensile strength, flexural strength, and interlaminar shear strength. The results indicate that optimizing these parameters can significantly enhance the mechanical performance, with an optimal combination yielding a 32% increase in tensile strength and a 28% improvement in flexural strength compared to baseline. Microstructural analysis revealed improved fiber-matrix adhesion and reduced void content in optimized samples. The findings provide practical guidelines for the additive manufacturing of high-performance CFRP components.

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Stem Cell Research & Therapy•2025

Melatonin-pretreated mesenchymal stem cell-derived exosomes alleviate cavernous fibrosis in a rat model of nerve injury-induced erectile dysfunction via miR-145-5p/TGF-β/Smad axis

Authors: Xiaolin Zhang, Mengbo Yang, Xinda Chen, Ming Zhang, Yiliang Peng, Mujun Lu

Background Cavernous nerve injury-induced erectile dysfunction (CNI-ED) is a common complication after radical prostatectomy. Conventional treatment approaches have had little success in treating the severe cavernous fibrosis which is a consequence of CNI-ED. Methods Pre-treatment of adipose-derived stem cells with melatonin allows for the extraction of active exosomes (MT-hASC-EVs) from the conditioned medium. The therapeutic effects of MT-hASC-EVs were assessed in a rat model of CNI-ED, and the anti-fibrotic properties were evaluated. MicroRNA sequencing was used to identify specific microRNAs highly expressed in MT-hASC-EVs, and differential microRNAs were screened for regulatory pathways through target gene enrichment analysis. Finally, the conclusions from bioinformatics analysis were validated through in vitro experiments. Results Intracavernous injection of MT-hASC-EVs significantly restored erectile function and reduced the extent of corpus cavernosum fibrosis in the CNI-ED rat model. MT-hASC-EVs promoted the proliferation and anti-apoptotic effects of corpus cavernosum smooth muscle cells (CCSMCs) in vitro. Mechanistically, MT-hASC-EVs inhibit fibrosis by delivering miR-145-5p, which targets TGF-β2/Smad3 axis. Conclusions MT-hASCs-EVs can inhibit cavernous fibrosis and improve erectile function in a rat model of CNI-ED by targeting the miR-145-5p/TGF-β/Smad axis.

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Stem Cell Research & Therapy•2025

KRT5high TP63-expressing urothelial basal cells act as a driver to bladder urothelium regeneration in rabbit

Authors: Jiasheng Chen, Mingming Yu, Lin Wang, Hua Xie, Yiqing Lv, Yichen Huang, Yue Hong, Fang Chen

Background Urothelial regeneration is a crucial part of bladder tissue engineering. However, there is a lack of ideal “seed cells” in current practices. Here, we demonstrated that a sub-population of p63 positive basal cells could be activated and differentiate into intermediate and superficial umbrella cells after full-thickness mucosal resection in rabbit. Methods A focal mucosal resection model was used to characterize the role of different urothelial cells during regeneration. Urothelial basal cells were isolated from rabbit bladder mucosa and cultured in vitro. The basal cells were then transplanted in vivo in a manner of cell sheet for reconstruction. Results Via single-cell RNA sequencing (scRNA-seq), it has been confirmed that the cluster of KRT5high TP63-expressing cells possesses a ‘stemness’ signature which can give rise to lineage cell types sequentially. With a strong support from the underneath pre-set capsule vascular bed, the transplanted cell sheet could develop into a physio-morphology resembled to the native mucosa in vivo. Importantly, we validated that the bioengineered urothelium implemented perfect barrier function after implanted to bladder. Conclusions In summary, bioengineering urothelium with KRT5high TP63-expressing basal cells on a capsule vascular bed offers a promising strategy for bladder tissue engineering and provides a model for drug screening and bladder disease research.

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Acta Biochimica et Biophysica Sinica•2025

Resident CD24+LCN2+ LPCs aggravate fibrosis and inflammatory progression via the recruitment of TPPP3+COL10A1+ macrophages in NASH

Authors: Min Ding, Xiaoshu Qi, Weijian Huang, Yan Lin, Hexin Yan

Resident CD24+LCN2+ liver progenitor cells (LPCs) reportedly contribute to the expanding ductular reaction and macrophage-mediated inflammation associated with chronic liver damage. Both ductular reactions and macrophage-driven inflammation are associated with liver fibrosis and injury in various mouse liver disorders. This study aims to investigate the molecular phenotypes of LPCs and their regulatory mechanisms in humans with non-alcoholic steatohepatitis (NASH). Single-cell RNA sequencing (scRNA-seq) datasets are used to characterize the status and molecular phenotypes of LPCs in clinical NASH samples. To elucidate the regulatory mechanisms of LPCs, CellChat and NicheNet are employed to assess cell-cell communication between LPCs and other cell types. The findings are validated using RNA sequencing datasets associated with NASH progression, NASH mouse models (CDAHFD and HFD), and human NASH liver samples. Results show that resident CD24+LCN2+ LPCs are identified and found to be significantly enriched in NASH patients. Cell communication analyses predict strong interactions between LPCs and proinflammatory macrophage subtypes. Additionally, in NASH, the liver recruits peripheral blood mononuclear cell (PBMC)-derived macrophages and polarizes them into proinflammatory subtypes. The macrophage subtype MP-2 is identified as the primary recipient of LPC-derived signals, exhibiting marked hyperactivation of the NF-κB pathway and a strong association with liver fibrosis. Finally, the MP-2 markers COL10A1 and TPPP3 are characterized and validated. In summary, this study reveals that resident CD24+LCN2+ LPCs are activated in NASH and contribute to fibrosis progression by promoting the activation of the proinflammatory COL10A1+TPPP3+ macrophage subtype.

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Acta Biochimica et Biophysica Sinica•2025

AKR1C3 protects cardiomyocytes against hypoxia-induced cell apoptosis through the Nrf-2/NF-κB pathway

Authors: Wenlu Zhang, Wei Tian, Xin Xia, Hua Tian, Ting Sun

Hypoxia-induced apoptosis plays a critical role in the progression of various cardiac diseases, such as heart failure and acute myocardial infarction (AMI). Aldosterone reductase 1C3 (AKR1C3), a member of the aldo-keto reductase superfamily, participates in the metabolism of steroid hormones and redox reactions in vivo. Imbalances in prostaglandin levels have been linked to coronary events. However, the function and molecular mechanism by which AKR1C3 influences AMI are not yet fully understood. This study aims to investigate the role of AKR1C3 in hypoxia-induced myocardial cell damage and elucidate its mechanism. Our findings reveal that a hypoxic microenvironment triggers cardiomyocyte apoptosis and elevates AKR1C3 expression in H9C2 and AC16 cells, as well as in cardiac tissue from rats and mice with AMI. The overexpression of AKR1C3 promotes cardiomyocyte proliferation and cell vitality, whereas the silencing of AKR1C3 exerts the opposite effects in vitro. AKR1C3 protects cardiomyocytes against hypoxia-induced cell apoptosis by reducing ROS levels, preventing mitochondrial damage, and maintaining the oxygen consumption rate (OCR) and ATP production; conversely, AKR1C3 knockdown leads to adverse outcomes. Moreover, the application of a ROS inhibitor (MitoQ10) mitigates the increase in mitochondrial ROS in cardiomyocytes induced by AKR1C3 knockdown under hypoxic conditions. Mechanically, AKR1C3 increases Nrf-2 expression through the ubiquitin-proteasome pathway in cardiomyocytes and subsequently inhibits the NF-κB signaling pathway, thereby inhibiting Bax/caspase-3 signaling. Collectively, these results suggest that AKR1C3 prevents hypoxia-induced cardiomyocyte injury by modulating the Nrf-2/NF-κB axis, suggesting new insights into the mechanisms underlying myocardial protection.

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Acta Biochimica et Biophysica Sinica•2025

Similarities and differences in the response and molecular characteristics of peripheral sensory neurons associated with pain and itch

Authors: Li Liu, Jiemin Yin, Youqiang Meng, Congrui Ye, Junhui Chen, Sa Wang, Wen Yin, Po Gao, Yingfu Jiao, Weifeng Yu, Yinghui Fan

Dorsal root ganglion (DRG) neurons are responsible for the primary detection and transmission of peripheral noxious stimuli, mainly pain and itch. However, as two distinct noxious sensations, how DRG neurons respond differently to and code pain and itch is still an attractive topic. Here, we investigate the response and activation spectrum of DRG neurons under peripheral pain and itch stimuli using in vivo two-photon calcium imaging and find differences in the response intensity to pain and itch between multisensory neurons (both pain and itch) and single-sensory neurons (either pain or itch). In addition, single-cell RNA sequencing (scRNA-seq) is used to reveal the heterogeneity of distinct subpopulations on the basis of their expressions of pain- or itch-related marker genes and to determine the similarities and differences in their transcriptomic changes under chronic pain and itch. Our results show that primary sensory neurons with different sensory patterns respond differently to the same nociceptive stimuli. Additionally, distinct clusters of neurons exhibit unique transcriptomic changes in the development of chronic pain and itch, which may offer new insights for treating these conditions.

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Acta Biochimica et Biophysica Sinica•2025

NLRP3 inflammasome activity and pyroptosis are involved in CD206+ macrophage activation by MPO anti-neutrophil cytoplasmic antibodies

Authors: Zhaonan Wei, Xiaoning An, Yinyin Xie, Yan Shen, Liyan Ni, Jing Xu, Yimei Wang, Pingyan Shen, Hao Shi, Wen Zhang, Yongxi Chen

Macrophages are key players in the pathology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Existing studies and our previous studies have documented the role of CD206-positive M2 macrophages in the inflammatory process of AAV. Inflammasome activation is a critical pathway through which macrophages release inflammatory factors. In this study, we investigate the role of the inflammasome in macrophages in AAV and explore the role of CD206 in this process. We recruit newly diagnosed AAV patients and disease controls from our department. The expression and localization of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) and CD206 in the kidney are determined via immunofluorescence experiments. Myeloperoxidase (MPO)-ANCA immunoglobulin G (MPO-ANCA IgG) is purified from new-onset AAV patients with MPO-ANCA and used to treat lipopolysaccharide (LPS)-primed macrophages in vitro. Our findings reveal that NLRP3 expression is significantly elevated in the kidneys of active AAV patients, accompanied by increased cleaved caspase-1 and N-terminal gasdermin-D (GSDMD) levels in peripheral blood mononuclear cells (PBMCs). In vitro, MPO-ANCA IgG induces NLRP3 inflammasome activation and interleukin (IL)-1β production in macrophages, which is associated with increased MPO expression and JNK signaling pathway activation. Immunofluorescence analysis demonstrates partial colocalization of CD206 and NLRP3 in AAV kidneys. Furthermore, silencing of MRC1 gene, which encodes CD206, reduces inflammasome activation induced by MPO-ANCA IgG. In conclusion, our study provides evidence that MPO-ANCA IgG contributes to NLRP3 inflammasome activation and macrophage pyroptosis, with CD206 playing a critical role in this process. These findings elucidate the mechanisms underlying inflammation in AAV and suggest potential therapeutic targets.

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Acta Biochimica et Biophysica Sinica•2025

Types of cell death in diabetic cardiomyopathy: insights from animal models

Authors: Hongjiao Xu, Zhuang Yu, Jun Zhu, Haoran Liu, Xiangyuan Chen, Jihong Jiang, Minmin Zhu, Jinbao Li

Approximately one-tenth of the global population is affected by diabetes mellitus, and its incidence continues to rise each year. In China, 1.4 million patients die of diabetes-related complications every year. Additionally, approximately 26% of patients with diabetes develop diabetic cardiomyopathy, with heart failure being one of the main causes of death in these patients. However, early detection of diabetic cardiomyopathy has proven to be difficult in a clinical setting; furthermore, there are limited guidelines and targeted means of prevention and treatment for this disease. In recent years, several studies have provided evidence for the occurrence of various forms of regulated cell death in diabetic myocardial cells, including apoptosis, necroptosis, ferroptosis, and cuproptosis, which are closely linked to the pathological progression of diabetic cardiomyopathy. Although most research on diabetic cardiomyopathy is currently in the animal trial phase, the inhibition of these regulatory cell death processes can limit or slow down the progression of diabetic cardiomyopathy. Therefore, this review discusses the appropriate animal experimental models currently available for diabetic cardiomyopathy and evaluates the roles of apoptosis, necroptosis, ferroptosis, and cuproptosis in diabetic cardiomyopathy. We hope to provide new methods and ideas for future research in diabetic cardiomyopathy.

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Acta Biochimica et Biophysica Sinica•2025

miR-32-5p suppresses the progression of hepatocellular carcinoma by regulating the GSK3β/NF-κB signaling

Authors: Guangzhi Wang, Qianqian Yang, Yaqi Han, Yunlong Zhang, Wei Pan, Zhongliang Ma, Hui Tian, Xudong Qu

Hepatocellular carcinoma (HCC) is a highly fatal form of malignancy that seriously threatens patient survival. The global 5-year survival rate for HCC patients ranges from 15% to 19%, and nearly 80% of patients are diagnosed at an advanced stage. Therefore, exploring the mechanism of HCC development and identifying biomarkers and therapeutic targets for HCC are vital. MicroRNAs (miRNAs), a class of noncoding single-stranded RNAs, are 20–24 nucleotides (nt) long. They play pivotal roles in modulating the progression of diverse diseases. The specific role of miR-32-5p in the development of HCC remains unclear. In this study, qRT-PCR is utilized to precisely determine the downregulated expression levels of miR-32-5p in HCC. Subsequently, functional analysis reveals the suppressive role of miR-32-5p in modulating the proliferative and migratory capabilities of HCC cells. Glycogen synthase kinase 3β (GSK3β) has emerged as a potential target of miR-32-5p, which is confirmed through a dual-luciferase reporter assay. Notably, the expression of GSK3β in HCC tissue specimens is negatively correlated with the abundance of miR-32-5p, and patients with high GSK3β expression have shorter survival time. Furthermore, the targeted downregulation of GSK3β remarkably impedes the proliferation and migration of tumor cells. This study suggests that miR-32-5p inhibits the proliferation and migration of HCC through regulating the GSK3β/NF-κB signaling pathway. Therefore, this study reveals that miR-32-5p exerts its suppressive effect on HCC progression, suggesting that it is a promising target for both diagnostic and targeted therapeutic interventions against HCC.

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Acta Biochimica et Biophysica Sinica•2025

METTL3-mediated m6A modification of pri-miRNA-31 promotes hypertrophic scar progression

Authors: Qirui Wang, Jialin Hou, Siyi Zeng, Xue Wang, Yimin Liang, Renpeng Zhou

Hypertrophic scar (HS) is a pathological scar characterized by excessive dermal fibrosis. Aberrant m6A modification patterns have been identified in HS; however, the expression of the methyltransferase, along with its function and molecular mechanisms in HS, remains unclear. In this study, we find that both the protein level of METTL3 and the level of m6A methylation are upregulated in HS compared with normal skin. To investigate the role of METTL3 in HS, we knock down METTL3 in HS-derived fibroblasts (HSFBs) via shRNA. METTL3 knockdown reduces the expressions of collagen types I and III (COL I/III) and α-SMA, inhibits cell proliferation and migration, and induces cell cycle arrest in the G1 phase. MeRIP-seq analysis reveals m6A modification sites on pri-miR-31. Our data indicate that the expression level of pri-miR-31 is elevated in METTL3-knockdown HSFBs, whereas the level of mature miR-31-5p is reduced. Notably, transfection of a miR-31-5p mimic into HSFBs partially counteracts the inhibitory effects of the m6A methylation inhibitors cycloleucine and STM2457 (a specific inhibitor of METTL3) on fibrosis and cellular proliferation. Additionally, we confirm that ZBTB20 is a downstream target of miR-31-5p and that knockdown of ZBTB20 inhibits fibroblast fibrosis. Collectively, our findings elucidate the epigenetic mechanism of METTL3/m6A/pri-miR-31/ZBTB20 in HS fibrosis, providing a potential therapeutic target for HS.

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Acta Biochimica et Biophysica Sinica•2025

The effect of norepinephrine on ovarian dysfunction by mediating ferroptosis in mice model

Authors: Hanqing Hong, Chengqi Xiao, Lichun Weng, Qian Wang, Dongmei Lai

Studies have shown that stress is associated with ovarian dysfunction. Norepinephrine (NE), a classic stress hormone involved in the stress response, is less recognized for its role in ovarian function. In this study, an NE-treated mouse model is induced by intraperitoneal injection of NE for 4 weeks. Compared with normal control mice, NE-treated mice show disturbances in the estrous cycle, decreased levels of anti-Mullerian hormone (AMH) and estradiol (E2), and increased level of follicle-stimulating hormone (FSH). Additionally, the numbers of primordial follicles, primary follicles, secondary follicles, and antral follicles are decreased, whereas the number of atretic follicles is increased in NE-treated mice, indicating NE-induced ovarian dysfunction. RNA sequencing further reveals that genes associated with ferroptosis are significantly enriched in NE-treated ovarian tissues. Concurrently, the levels of reactive oxygen species (ROS), ferrous ions, and malondialdehyde (MDA) are increased, whereas the expression level of glutathione peroxidase 4 (GPX4) is decreased. To elucidate the mechanism of NE-induced ferroptosis in ovaries and the potential reversal by Coenzyme Q10 (CoQ10), an antioxidant, we conduct both in vitro and in vivo experiments. In vitro, the granulosa cell line KGN, when treated with NE, shows decreased cell viability, reduced expression of GPX4, elevated levels of ferrous ion and ROS, and increased MDA level. However, these NE-induced changes are reversed by the addition of CoQ10. Compared with the NE group, the NE-treated mice supplemented with CoQ10 present increased GPX4 level and decreased iron, ROS, and MDA levels. Moreover, the differential expression of genes associated with ferroptosis induced by NE is ameliorated by CoQ10 in NE-treated mice. Additionally, CoQ10 improves ovarian function, as evidenced by increased ovarian weight, more regular estrous cycles, and an increase in follicles at various stages of growth in NE-treated mice. In conclusion, NE induces ovarian dysfunction by triggering ferroptosis in ovarian tissues, and CoQ10 represents a promising approach for protecting reproductive function by inhibiting ferroptosis.

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Acta Biochimica et Biophysica Sinica•2025

The peripheral Atf3+ neuronal population is responsible for nerve regeneration at the early stage of nerve injury revealed by single-cell RNA sequencing

Authors: Li Liu, Junhui Chen, Wen Yin, Po Gao, Yinghui Fan, Daxiang Wen, Yingfu Jiao, Weifeng Yu

Peripheral nerve injury (PNI) can transform primary somatosensory neurons to a regenerative state. However, the details of the transcriptomic changes associated with the nerve regeneration of somatosensory neurons remain unclear. In this study, single-cell RNA sequencing (scRNA-seq) is conducted on mouse dorsal root ganglion (DRG) cells after the early stage of nerve injury on day 3 after chronic constriction injury (CCI). We observe that a novel CCI-induced neuronal population (CIP) emerge and express high levels of activating transcription factor (Atf3), a neuronal injury marker. CIP neurons highly express regeneration-associated genes (RAGs) and are enriched in regeneration-related gene ontology (GO) terms, suggesting that these neurons can constitute a pro-regenerative population. Moreover, intercellular communication networks show that CIP neurons closely communicate with satellite glial cells (SGCs) and specifically transmit strong Fgf3-Fgfr1 signaling to SGCs, which could initiate regeneration-associated transcriptional changes in SGCs. We also confirm that regenerative progress occurs at the early stage of nerve injury because immunohistochemistry shows that the expression of ATF3 is significantly increased beginning at 3 days post-CCI and decreased at 1 month post-CCI. Our bioinformatics analysis at single-cell resolution advances the knowledge of regenerative dynamic transcriptional changes in DRG cells after injury and the underlying molecular mechanisms involved.

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Stem Cell Research & Therapy•2024

Single-cell sequencing of facial adipose tissue unveils FKBP5 as a therapeutic target for facial infiltrating lipomatosis

Authors: Hongrui Chen, Bin Sun, Shih-Jen Chang, Zhang Yu, Yajing Qiu, Chen Hua, Xiaoxi Lin

Background Facial infiltrating lipomatosis is characterized by excessive growth of adipose tissue. Its etiology is associated with somatic phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) variants, but the specific mechanisms are not yet fully understood. Methods We collected facial adipose tissue from both FIL patients and non-FIL individuals, isolated the stromal vascular fraction (SVF) and performed single-cell transcriptome sequencing on these samples. Results We mapped out the cellular landscape within the SVF, with a specific focus on a deeper analysis of fibro-adipogenic precursor cells (FAPs). Our analysis revealed that FAPs from FIL patients (FIL-FAPs) significantly overexpressed FK506 binding protein 51 (FKBP5) compared to FAPs from individuals without FIL. Further experiments indicated that FKBP5 is regulated by the PI3K-AKT signaling pathway. The overactivation of this pathway led to an increase in FKBP5 expression. In vitro experiments demonstrated that FKBP5 promoted adipogenic differentiation of FAPs, a process that could be hindered by FKBP5 knockdown or inhibition. Additionally, in vivo assessments confirmed FKBP5’s role in adipogenesis. Conclusions These insights into the pathogenesis of FIL underscore FKBP5 as a promising target for developing non-surgical interventions to manage the excessive adipose tissue growth in FIL.

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Stem Cell Research & Therapy•2024

Connexin 25 maintains self-renewal and functions of airway basal cells for airway regeneration

Authors: Jingyuan Zhang, Shaoyang Wang, Zeyu Liu, Cheng Zhong, Yuqiong Lei, Qi Zheng, Yongle Xu, Shan Shan, Hao He, Tao Ren

Background The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. Methods Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air–liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. Results Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. Conclusion Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.

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Acta Biochimica et Biophysica Sinica•2024

EEPD1 attenuates radiation-induced cardiac hypertrophy and apoptosis by degrading FOXO3A in cardiomyocytes

Authors: Kaiwen Yu, Xi Su, Tongfang Zhou, Xuwei Cai, Min Zhang

Radiation-induced heart disease (RIHD) is a severe delayed complication of thoracic irradiation (IR). Endonuclease/exonuclease/phosphatase family domain-containing 1 (EEPD1) plays an important role in DNA damage repair, but its role in RIHD is less known. In this study, EEPD1 global knockout mice, C57BL/6J mice, and C57BL/6J mice overexpressing EEPD1 are treated with radiation at a total dose of 20 Gy or 0 Gy. After 9 weeks, echocardiography is used to assess cardiac hypertrophy and apoptosis. The results show that EEPD1 deletion exacerbates radiation-induced cardiac hypertrophy and apoptosis, while EEPD1 overexpression has the opposite effect. Further mechanistic investigations reveal that EEPD1 interacts with FOXO3A and destabilizes it by catalyzing its deubiquitination. Inhibition of FOXO3A ameliorates cardiac hypertrophy and apoptosis after EEPD1 knockdown. Thus, EEPD1 protects against radiation-induced cardiac hypertrophy and apoptosis via destabilization of FOXO3A, which may offer new insight into therapeutic strategies for RIHD.

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Acta Biochimica et Biophysica Sinica•2024

O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure

Authors: Zhijue Xu, Han Zhang, Jiaqi Tian, Xin Ku, Rumeng Wei, Jingli Hou, Can Zhang, Fang Yang, Xia Zou, Yang Li, Hiroyuki Kaji, Sheng-Ce Tao, Atsushi Kuno, Wei Yan, Lin-Tai Da, Yan Zhang

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus’s entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

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Acta Biochimica et Biophysica Sinica•2024

Deciphering disease through glycan codes: leveraging lectin microarrays for clinical insights

Authors: Hangzhou Yang, Zihan Lin, Bo Wu, Jun Xu, Sheng-Ce Tao, Shumin Zhou

Glycosylation, a crucial posttranslational modification, plays a significant role in numerous physiological and pathological processes. Lectin microarrays, which leverage the high specificity of lectins for sugar binding, are ideally suited for profiling the glycan spectra of diverse and complex biological samples. In this review, we explore the evolution of lectin detection technologies, as well as the applications and challenges of lectin microarrays in analyzing the glycome profiles of various clinical samples, including serum, saliva, tissues, sperm, and urine. This review not only emphasizes significant advancements in the high-throughput analysis of polysaccharides but also provides insight into the potential of lectin microarrays for diagnosing and managing diseases such as tumors, autoimmune diseases, and chronic inflammation. We aim to provide a clear, concise, and comprehensive overview of the use of lectin microarrays in clinical settings, thereby assisting researchers in conducting clinical studies in glycobiology.

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Acta Biochimica et Biophysica Sinica•2024

Noncanonical functions of microRNAs in the nucleus

Authors: Jiayi Gu, Yuanan Li, Youtong Tian, Yehao Zhang, Yongjun Cheng, Yuanjia Tang

MicroRNAs (miRNAs) are small noncoding RNAs (ncRNAs) that play their roles in the regulation of physiological and pathological processes. Originally, it was assumed that miRNAs only modulate gene expression post-transcriptionally in the cytoplasm by inducing target mRNA degradation. However, with further research, evidence shows that mature miRNAs also exist in the cell nucleus, where they can impact gene transcription and ncRNA maturation in several ways. This review provides an overview of novel models of nuclear miRNA functions. Some of the models remain to be verified by experimental evidence, and more details of the miRNA regulation network remain to be discovered in the future.

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Acta Biochimica et Biophysica Sinica•2024

Identification and characterization of multipotential stem cells in immortalized normal ovarian surface epithelial cells

Authors: Lin Hou, Hanqing Hong, Wenjiao Cao, Liutong Wei, Lichun Weng, Shuang Yuan, Chengqi Xiao, Qiuwan Zhang, Qian Wang, Dongmei Lai

The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms “regulation of stem cell proliferation”, “epithelial cell proliferation”, etc., are significantly enriched (P<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts in vitro. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers in vitro. IOSE80 cells also form oocyte-like cells in vitro and in vivo. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after in vivo transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.

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Acta Biochimica et Biophysica Sinica•2024

SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus

Authors: Mengke Chen, Yutong Zhang, Weiwen Shi, Xuejiao Song, Yue Yang, Guojun Hou, Huihua Ding, Sheng Chen, Wanling Yang, Nan Shen, Yong Cui, Xianbo Zuo, Yuanjia Tang

Through genome-wide association studies (GWAS) and integrated expression quantitative trait locus (eQTL) analyses, numerous susceptibility genes (“eGenes”, whose expressions are significantly associated with common variants) associated with systemic lupus erythematosus (SLE) have been identified. Notably, a subset of these eGenes is correlated with disease activity. However, the precise mechanisms through which these genes contribute to the initiation and progression of the disease remain to be fully elucidated. In this investigation, we initially identify SPATS2L as an SLE eGene correlated with disease activity. eSignaling and transcriptomic analyses suggest its involvement in the type I interferon (IFN) pathway. We observe a significant increase in SPATS2L expression following type I IFN stimulation, and the expression levels are dependent on both the concentration and duration of stimulation. Furthermore, through dual-luciferase reporter assays, western blot analysis, and imaging flow cytometry, we confirm that SPATS2L positively modulates the type I IFN pathway, acting as a positive feedback regulator. Notably, siRNA-mediated intervention targeting SPATS2L, an interferon-inducible gene, in peripheral blood mononuclear cells (PBMCs) from patients with SLE reverses the activation of the interferon pathway. In conclusion, our research highlights the pivotal role of SPATS2L as a positive-feedback regulatory molecule within the type I IFN pathway. Our findings suggest that SPATS2L plays a critical role in the onset and progression of SLE and may serve as a promising target for disease activity assessment and intervention strategies.

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Acta Biochimica et Biophysica Sinica•2024

Co-profiling of translatome and transcriptome reveals the regulation of dynamic gene expression during Drosophila embryogenesis

Authors: Le Zhang, Qiufang Liu, Yulong Liu, Bishan Ye, Chuansheng Hu, Xinhui Li, Ling Bai, Ming Cheng, Mingzhu Zhao, Hongmei Li, Hua Li

Eukaryotic gene expression is regulated at multiple levels, aiding in maintaining normal phenotypes and environmental adaptability. Transcriptional regulation complexity has been extensively studied using high-throughput sequencing, and previous studies have shown that different transcript isoforms can be produced through complex regulatory mechanisms via large-scale RNA sequencing. Additionally, translational regulation, which significantly influences gene expression, is controlled by complex mechanisms [1]. The untranslated regions (UTRs) of eukaryotic mRNA, encompassing the 5′ UTR, 3′ UTR and polyadenylation tail (polyA), are pivotal for translational regulation, with distinct cis-regulatory elements in the 5′ UTR and 3′ UTR of various transcript isoforms, leading to substantial variations in translational regulation across transcripts. To shed light on translational regulation, previous studies have performed isolation of ribosome-associated poly-adenylated RNAs (i.e., translatome) and deep sequencing for mRNA translation [2]. Polysome profiling is the most common method used to study translatome, which can enable the isolation of full-length translated mRNAs, thereby facilitating the identification of UTRs, assessment of selective translation, and comprehension of the regulatory mechanisms underlying gene expression [2]. Drosophila embryonic development progresses very rapidly and requires precise regulation of the transcription and translation of a large number of genes to ensure normal gene expression. Although Drosophila has been extensively studied as a model organism, the specific interplay between transcription and translation during embryonic development stages is not yet fully understood. To investigate the dynamic regulation of gene expression during Drosophila embryogenesis, we conducted transcriptome and translatome co-profiling on early (0‒4 h) embryos and S2R+ cells, a cell line derived from late embryonic stages of Drosophila melanogaster [3], to compare the differences in translational regulation at the gene and transcript isoform levels. S2R+ cell culture and early (0–4 h) embryo collection were performed (see Supplementary Methods) to compare transcriptome and translatome profiling, as shown in Supplementary Figure S1. Cytosolic RNA and ribosome-associated RNA were isolated from embryos [4] and S2R+ cells, which were used for constructing RNA-Seq libraries. Four libraries were generated for RNA-seq (see Supplementary Methods), consisting of two cytosolic RNA libraries and two ribosome-associated RNA libraries (Supplementary Figure S1A,B). The strand-specific RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, USA). The library was sequenced on the Illumina HiSeq X Ten System. We employed Trimmomatic [5] to remove low-quality reads, which resulted in approximately 89 million, 76 million, 72 million, and 56 million clean reads for the transcriptome and translatome of the early embryos and S2R+ cells, respectively. These reads were then mapped to the Drosophila genome (UCSC dm6) using HISAT2 [6]. The unique mapped reads ratio ranges from 94% to 85% and reads mapped to rRNA were less than 6% (Supplementary Table S1), indicating the high quality of the four RNA-seq libraries. Using StringTie [7], 33,470 transcripts were assembled for four mapping sequencing libraries, which revealed an average of 1.9 transcribed transcripts and 1.8 translated transcripts per gene (Supplementary Table S1), suggesting the usage of transcript isoforms widely existed in both transcription and translation of Drosophila embryos. To explore the divergence of the transcriptome during Drosophila development, we compared the transcriptome of the early embryos and S2R+ cells to identify genes with |log2(fold change)| ≥1, FPKM ≥1 in at least one condition, and adjusted P value ≤0.001. In total, we identified 2267 differentially expressed genes (DEGs) from 8815 genes. Among these DEGs, 2147 genes showed higher expression levels in the embryos, while 120 genes showed higher expression levels in S2R+ cells (Figure 1A and Supplementary Figure S2A). To investigate the underlying functional mechanism, we performed enrichment analysis to identify DEG-enriched pathways (Supplementary Table S2). Interestingly, the top 10 enriched pathways are related to morphogenesis an

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