JOURNAL OF CONTROLLED RELEASE
·
2025
·
IF 11.5
Enze Li,Liewei Wen,Caiyun Yin,Nan Wang,Suleixin Yang,Wenzheng Feng,Meiwan Chen
Cuproptosis as a copper-dependent cell death modality driven by pathological aggregation of lipoylated proteins and proteotoxic stress resulting from destabilization of iron-sulfur (Fe S) cluster proteins, represents a promising therapeutic strategy for cancer. However, the therapeutic efficacy of cuproptosis can be compromised by the intrinsic compensatory mechanisms within cancers, particularly low intracellular copper ion concentration and protective autophagy, which facilitates cellular adaptation and survival under stress. To overcome this limitation, a self-amplifying cuproptosis nanoregulator (SHK-Cu/ TK PF/pATG5@HA, abbreviated as SC/TpA@HA) for CD44-targeted delivery is developed that integrate shikonin‑copper (SHK-Cu) coordination complexes with CRISPR/Cas9 plasmids targeting ATG5, condensed by fluorinated polyethyleneimine ( TK PF)-condensed to enhance cancer therapy. Briefly, Cu + is released from the dissociated SHK-Cu complex upon intracellular GSH activation induces dihydrolipoamide S -acetyltransferase (DLAT) oligomerization and reduces Fe S cluster proteins, triggering tricarboxylic acid (TCA) cycle collapse and irreversible mitochondrial damage. Concurrently, CRISPR/Cas9-mediated ATG5 knockout prevents autophagosome formation, creating an autophagic flux trap that accumulates copper-damaged mitochondria. Notably, such mitochondrial dysfunction as induced by copper overload combined with impaired cellular damage clearance from autophagy blockade elevates cuproptosis. In addition, the immunogenic cell death through cuproptosis in cancer cells, as validated by the exposure of calreticulin and the extracellular release of HMGB1, triggers a potent anti-tumor immune response. This response is enhanced through autophagy inhibition, as ATG5 deletion blocks the downstream signaling of copper-activated Unc-51-like autophagy activating kinase 1/2 (ULK1/2), ultimately amplifying cytotoxic T lymphocyte infiltration. Therefore, this dual intervention through copper overload and autophagy blockade potentiates both cuproptosis and anti-tumor immune effect, representing an innovative strategy of cuproptosis treatment.
研究领域:
肿瘤学
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分子生物学
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纳米医学
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免疫学
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细胞死亡机制
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基因编辑
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代谢调控
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转化医学
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DOI
PubMed
高影响因子
PLoS Genetics
·
2022
·Vol. 18
·期 3
·e1009841
·
IF 6.02
Fei Liu,Yayun Qin,Yuwen Huang,Pan Gao,Jingzhen Li,Shanshan Yu,Danna Jia,Xiang Chen,Yuexia Lv,Jiayi Tu,Kui Sun,Yunqiao Han,James Reilly,Xinhua Shu,Qunwei Lu,Zhaohui Tang,Chengqi Xu,Daji Luo,Mugen Liu
Neural retina leucine zipper ( NRL ) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish ( Danio rerio ), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl -dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl . Besides, we identified the mafba gene as a novel regulator of the nrl -independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl / mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.
Vision is mediated by two types of light-sensing cells named rod and cone photoreceptors in animal eyes. Abnormal generation, dysfunction or death of photoreceptor cells all cause irreversible vision problems. NRL is an essential gene for the generation and function of rod cells in mice and humans. Surprisingly, we found that in the zebrafish, a popular animal model for human diseases and therapeutic testing, there are two types of rod cells, and eliminating the function of nrl gene affects the rod cell formation at the embryonic stage but not at the juvenile and adult stages. The rod cell formation at the post-embryonic is driven by the mafba gene, which has not been reported to play a role in rod cells. In addition to the reduced number of rod cells, deletion of nrl also results in the emergence of rod/green-cone hybrid cells and an increased number of green cones. The ensuing cellular and molecular alterations collectively lead to retinal degeneration. These findings expand our understanding of photoreceptor development and maintenance and highlight the underlying conserved and species-specific regulatory mechanisms.
研究领域:
发育生物学
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神经科学
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遗传学
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分子生物学
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DOI
PubMed
高影响因子
Neuroscience Bulletin
·
2021
·Vol. 37
·期 9
·1271-1288
·
IF 5.203
Li Hao,Wu Shihao,Ma Xia,Li Xiao,Cheng Tianlin,Chen Zhifang,Wu Jing,Lv Longbao,Li Ling,Xu Liqi,Wang Wenchao,Hu Yingzhou,Jiang Haisong,Yin Yong,Qiu Zilong,Hu Xintian
Whether direct manipulation of Parkinson’s disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6–10 months), and thus provides a practical transgenic monkey model for future PD studies.
研究领域:
神经科学
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分子生物学
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遗传学
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衰老研究
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疾病模型
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DOI
PubMed
高影响因子
Journal of Biological Engineering
·
2026
·Vol. 20
·期 1
·29
·
IF 6.5
Ye Changchuan,Zhang Yan,Zhang Jie,Shi Menglei,Nie Feixue,Liu Qinghua
Background Vitamin K 2 (VK 2 ), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK 2 and the reduction of its production cost have become the focus of attention. Results In this work, an E. coli strain with high VK 2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli strain BW-T7/MU to produce menaquinol-8 (MKH 2 -8, a reduced form of VK 2 ) by overexpressing menA and ubiE genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24 h and 48 h of fermentation, this strain BW-T7/MU reach a titer of 303 mg/L and 232 mg/L. Secondly, we overexpressed different related genes wrbA (oxidative stress mitigation), qorB (reduction of quinones) and menF (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing menA , ubiE and wrbA genes) reached the highest titer of VK 2 after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK 2 . Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK 2 . The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK 2 titer of 724 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859 mg/L in a shake flask and 1360 mg/L in a 5 L fermenter after 48 h cultivation. Conclusions The stepwise engineering strategy raised the VK 2 titer from the initial 303 mg/L to 859 mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK 2 titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.
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高影响因子
TOXICOLOGY
·
2025
·
IF 4.6
Zhisen Zhuang,Lijuan You,Jianbo Kong,Yaru Tian,Yongning Li,Yuan Zhi,Bo Zhang,Yi Wan,Xudong Jia,Hui Yang
Phthalate esters (PAEs) and per- and polyfluoroalkyl substances (PFAS) are ubiquitous pollutants tied to metabolic and immune disorders. The peroxisome proliferator-activated receptor (PPAR) pathway has been indicated to mediate their toxic effects, but the specific functions of PPAR subtypes and their mediating roles remain unclear. In this study, we generated PPARα-, δ-, and γ-specific knockout THP-1 cell lines by using CRISPR/Cas9 system and then differentiated them into interleukin-4 (IL-4) and interleukin-13 (IL-13)-polarized macrophages (alternative activation). During the induction process, the cells were exposed to 0, 6.25, 12.5, 25, 50, 100 μM of five PAE metabolites: Mono-(2-ethylhexyl) phthalate (MEHP), Monocyclohexyl phthalate (MCHP), Monoisonyl phthalate (MINP), Monoisobutyl phthalate (MIBP), and Monobenzyl phthalate (MBzP); and five PFAS: Perfluoroundecanoic acid (PFUnDA), Perfluorodecanoic acid (PFDA), Perfluorooctanoic acid (PFOA), Potassium perfluorooctanesulfonate (PFOS-K), and Potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F53B) for 48 h. The results showed that PPARδ deletion abolished CD209 expression, confirming its essential role, whereas PPARα deletion reduced and PPARγ deletion enhanced CD209, indicating PPARα promotes and PPARγ restrains alternative activation. Compounds displayed subtype-selective actions: MEHP activated PPARα/γ; MBzP/MCHP inhibited PPARδ yet activated PPARγ; MINP activated PPARγ only. Among PFAS, PFOS-K activated PPARδ/γ; F53B inhibited PPARα; PFOA activated PPARγ; PFDA inhibited PPARα/δ. Transcriptomics revealed compound-specific enrichments—cholesterol (MEHP), fatty-acid (MCHP), glycolysis (PFOS-K), TCA cycle (PFOA)—despite common PPAR pathway engagement. In conclusion, PAEs and PFAS disrupt macrophage plasticity through distinct PPAR-subtype signatures, providing molecular landmarks for future hazard assessment of environmental pollutants.
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PubMed
高影响因子
Nano Today
·
2025
·
IF 10.9
Suleixin Yang,Ruie Chen,Peng Hua,Yi Wu,Meiwan Chen
Photothermal therapy (PTT) demands efficient cancer ablation at relative low temperatures and minimal thermal damage to normal tissues, but suffers from both the protective autophagy-related thermal resistance in cancer cell and reactive oxygen species (ROS)-induced damage to normal cells. Here, we screened out curcumin-Fe ultrasmall nanoparticles (Cur-Fe) that manifested efficient photothermal conversion efficiency (η = 43.38 %) and ROS scavenging ability. Additionally, CRISPR/Cas9 plasmids (pCas-ATG5/ATG7) were also constructed to safely and precisely knockdown the protective autophagy for thermal resistance alleviation. The core Cur-Fe/ATG@ TK PF (CFA@T), which was composed of anionic C ur- F e and pCas- A TGs, was encapsulated by cationic thioketal-crosslinked and fluorinated polyethyleneimine ( T K PF) via electrostatic interaction. Further, CFA@TC was formed by CFA@T coated with an acidic pH-responsive shell OH C -PEG-CHO via Schiff base. Attributed to its dual responsiveness to pH and ROS, CFA@TC exhibited efficient tumor targeting and uptake following intravenous injection. Upon irradiation with a 652 nm laser, CFA@TC demonstrated enhanced efficacy in eradicating cancer cells by inhibiting autophagy, while concurrently mitigating inflammatory responses through intracellular ROS scavenging in vivo and in vitro . Taken together, our study provides a proof-of-concept that CRISPR can be effective for autophagy inhibition, and its integration with ROS-induced inflammatory responses relief can further improve PTT.
研究领域:
癌症治疗
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纳米技术
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分子生物学
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生物医学工程
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产品关联
DOI
高影响因子
FUNCTIONAL & INTEGRATIVE GENOMICS
·
2021
·Vol. 21
·期 5
·557-570
·
IF 3.41
Sun Shouxiang,Cao Xiaojuan,Castro L. Filipe C.,Monroig Óscar,Gao Jian
Obesity is a rapidly growing health pandemic, underlying a wide variety of disease conditions leading to increases in global mortality. It is known that the phosphorylation of various proteins regulates sterol regulatory element-binding transcription factors 1 ( srebf1 ), a key lipogenic transcription factor, to cause the development of obesity. To detect the key protein kinases for regulating srebf1 in lipid deposition, we established the srebf1 knockout model in zebrafish (KO, srebf1 −/− ) by CRISPR/Cas9. The KO zebrafish exhibited a significant reduction of total free fatty acid content (fell 60.5%) and lipid deposition decrease compared with wild-type (WT) zebrafish. Meanwhile, srebf1 deletion in zebrafish eliminated lipid deposition induced by high-fat diet feeding. Compared with WT zebrafish, a total of 697 differentially expressed proteins and 316 differentially expressed phosphoproteins with 439 sites were identified in KO by differential proteomic and phosphoproteomic analyses. A significant number of proteins identified were involved in lipid and glucose metabolism. Moreover, some protein kinases critical for regulating srebf1 in lipid deposition, including Cdk2, Pkc, Prkceb, mTORC1, Mapk12, and Wnk1, were determined by network analyses. An in vitro study was performed to verify the network analysis results. Our findings provide potential targets (kinases) for human obesity treatments.
研究领域:
分子生物学
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遗传学
·
蛋白质组学
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代谢
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营养学
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肥胖研究
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产品关联
DOI
PubMed
高影响因子
Frontiers in Cell and Developmental Biology
·
2022
·
IF 6.081
Deng Changping,Hu Fabiao,Zhao Zhangting,Zhou Yiwen,Liu Yuping,Zhang Tong,Li Shihui,Zheng Wenyun,Zhang Wenliang,Wang Tianwen,Ma Xingyuan
Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.
研究领域:
癌症生物学
·
分子生物学
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基因工程
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药理学
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产品关联
DOI
PubMed
高影响因子
Cell Death Discovery
·
2021
·Vol. 7
·期 1
·1-10
·
IF 5.241
Bian Wan-Ping,Pu Shi-Ya,Xie Shao-Lin,Wang Chao,Deng Shun,Strauss Phyllis R.,Pei De-Sheng
MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17 −/− zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17 −/− larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17 −/− zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17 −/− zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17 −/− zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17 −/− zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.
研究领域:
发育生物学
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分子遗传学
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细胞生物学
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DOI
PubMed
高影响因子
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
·
2023
·Vol. 120
·期 37
·e2308685120
·
IF 11.1
Yu Shi,Qingqing Liu,Wenjie Lu,Jing Yuan,Yihua Yang,John Oakeshott,Yidong Wu
Here, we provide mechanistic support for the involvement of the CYP9A subfamily of cytochrome P450 monooxygenases in the detoxification of host plant defense compounds and chemical insecticides in Spodoptera exigua and Spodoptera frugiperda. Our comparative genomics shows that a large cluster of CYP9A genes occurs in the two species but with significant differences in its contents, including several species-specific duplicates and substantial sequence divergence, both between orthologs and between duplicates. Bioassays of CRISPR-Cas9 knockouts of the clusters show that, collectively, the CYP9As can detoxify two furanocoumarin plant defense compounds (imperatorin and xanthotoxin) and insecticides representing three different chemotypes (pyrethroids, avermectins, and oxadiazines). However, in vitro metabolic assays of heterologously expressed products of individual genes show several differences between the species in the particular CYP9As with activities against these compounds. We also find that the clusters show tight genetic linkage with high levels of pyrethroid resistance in field strains of the two species. We propose that their divergent amplifications of the CYP9A subfamily have not only contributed to the development of the broad host ranges of these species over long evolutionary timeframes but also supplied them with diverse genetic options for evolving resistance to chemical insecticides in the very recent past.
研究领域:
昆虫学
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分子生物学
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基因组学
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毒理学
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DOI
PubMed
高影响因子
Journal of Orthopaedic Surgery and Research
·
2024
·Vol. 19
·期 1
·1-15
·
IF 2.8
Xiao Gongyi,Li Xiangwei,Yang Huiping,Zhang Ruobin,Huang Junlan,Tian Yu,Nie Mao,Sun Xianding
Background The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR , the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. Materials and results This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout ( mTOR -KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca 2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. Conclusion In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.
研究领域:
发育生物学
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分子生物学
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再生医学
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DOI
PubMed
高影响因子
Biomaterials Science
·
2022
·Vol. 10
·期 21
·6230-6243
·
IF 7.59
Yuanji Feng,Zhaopei Guo,Jie Chen,Sijia Zhang,Jiayan Wu,Huayu Tian,Xuesi Chen
To improve the efficiency of nucleic acid and protein delivery by cationic polymers, there is a trade-off between increasing the positive charge density of cationic polymers and decreasing cytotoxicity. In this work, a strategy to introduce multiple interactions between the cell membrane and a delivery system based on cationic polymers was proposed. A novel delivery system consisting of PEI1.8k and an enhancer (LA-RT) was fabricated. The introduction of LA-RT contributed to multiple interactions between the delivery system and the cell membrane including electrostatic interactions, hydrogen bonding, hydrophobic interaction, and dynamic sulfur exchange reactions, which enabled efficient intracellular delivery of nucleic acids and proteins. For nucleic acid delivery, plasmid DNA and mRNA were loaded to realize CRISPR/Cas 9 gene editing in vivo and protein expression in vivo, respectively. For protein delivery, the delivery system carrying OVA protein and CpG formed a nano-vaccine, which induced enhanced humoral and cellular immunity in vivo. In addition, the delivery system based on PEI1.8k revealed negligible cytotoxicity. This work provided a novel strategy to prepare efficient delivery systems based on cationic polymers via the introduction of a multifunctional enhancer.
研究领域:
药学
·
分子生物学
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生物技术
·
免疫学
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DOI
PubMed
高影响因子
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