Frontiers in Bioengineering and Biotechnology
·
2022
·
IF 6.064
Yang Xiangke,Zhao Lili,Chen Qiling,Wang Nan,Shi Kan,Liu Shuwen
Organic acid metabolism by lactic acid bacteria plays a significant role in improving wine quality. During this process, the uptake of extracellular organic acids by the transporters is the first rate-limiting step. However, up to now, there is very little published research on the functional verification of organic acid transporter genes in wine lactic acid bacteria. In this study, a predicted citrate transporter gene JKL54_04345 (citP) by protein homology analysis was knocked out using a CRISPR/Cas9-based gene-editing system, and then complemented using the modified pMG36e vectors in a major wine lactic acid bacterium, Lactiplantibacillus plantarum XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-ΔcitP lost the ability to utilize citric acid, while the gene complement mutant XJ25-ΔcitP-pMG36ek11-citP fully recovered the ability of citric acid utilization. Meanwhile, citP knockout and complement barely affected the utilization of l-malic acid. These indicated that citP in L. plantarum functioned as a citrate transporter and was the only gene responsible for citrate transporter. In addition, two modified plasmid vectors used for gene supplement in L. plantarum showed distinct transcription efficiency. The transcription efficiency of citP in XJ25-ΔcitP-pMG36ek11-citP mutant was 4.01 times higher than that in XJ25-ΔcitP-pMG36ek-citP mutant, and the utilization rate of citric acid in the former was 3.95 times higher than that in the latter, indicating that pMG36ek11 can be used as a high-level expression vector in lactic acid bacteria.
研究领域:
微生物学
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遗传学
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食品科学
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分子生物学
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PubMed
高影响因子
Nature Communications
·
2018
·Vol. 9
·期 1
·1-8
·
IF 12.353
Wang Huidong,Shi Yu,Wang Lu,Liu Shuai,Wu Shuwen,Yang Yihua,Feyereisen René,Wu Yidong
The cotton bollworm Helicoverpa armigera , is one of the world’s major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.
研究领域:
昆虫学
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遗传学
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分子生物学
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农业科学
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DOI
PubMed
高影响因子
Journal of Biological Engineering
·
2026
·Vol. 20
·期 1
·29
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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
Neuroscience Bulletin
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2021
·Vol. 37
·期 9
·1271-1288
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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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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
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IF 8
Jialin Zhang,Ming Zhou,Biyao Mao,Bin Huang,Herui Wen,Jiali Ren
Rapid, sensitive, and low-cost foodborne pathogens detection is crucial for dealing with foodborne diseases caused by bacteria. In this work, we designed a new CRISPR/Cas9-mediated electrochemical biosensor for ultrasensitive detection of Escherichia coli ( E. coli ) based on multifunctional covalent organic framework-based nanocomposite (GOx-AuNPs-COF-H2) and hyperbranched rolling circle amplification (HB-RCA) reaction. When specific DNA targets were present in the system, the targets could be recognized and cleaved by CRISPR-Cas9 system, which triggered strand displacement reaction (SDR) and HB-RCA reaction. By introducing GOx-AuNPs-COF-H2 nanocomposite, H2 probe could hybridize with RCA products to bind to the electrode surface. After added glucose solution, GOx could catalyze it in the detection system and thus offered significant current signals for bacteria detection. This approach showed high analytical performance for assay of E. coli with the detection limit (LOD) of 10 CFU/mL. Furthermore, real samples were detected by this approach, demonstrating its promising potential for foodborne pathogens detection in food monitoring and clinical diagnosis.
研究领域:
生物传感器
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分子生物学
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分析化学
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食品安全
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DOI
高影响因子
Pharmaceutics
·
2024
·Vol. 16
·期 2
·213
·
IF 4.9
Jiahui Ding,Handan Zhang,Tianli Dai,Xueqin Gao,Zhongyuan Yin,Qiong Wang,Mengqi Long,Songwei Tan
Poly (β-amino ester) (PBAE) is an exceptional non-viral vector that is widely used in gene delivery, owing to its exceptional biocompatibility, easy synthesis, and cost-effectiveness. However, it carries a high surface positive charge that may cause cytotoxicity. Therefore, hydrophilic d-α-tocopherol polyethylene glycol succinate (TPGS) was copolymerised with PBAE to increase the biocompatibility and to decrease the potential cytotoxicity of the cationic polymer-DNA plasmid polyplex nanoparticles (NPs) formed through electrostatic forces between the polymer and DNA. TPGS-b-PBAE (TBP) copolymers with varying feeding molar ratios were synthesised to obtain products of different molecular weights. Their gene transfection efficiency was subsequently evaluated in HEK 293T cells using green fluorescent protein plasmid (GFP) as the model because free GFP is unable to easily pass through the cell membrane and then express as a protein. The particle size, ζ-potential, and morphology of the TBP2-GFP polyplex NPs were characterised, and plasmid incorporation was confirmed through gel retardation assays. The TBP2-GFP polyplex NPs effectively transfected multiple cells with low cytotoxicity, including HEK 293T, HeLa, Me180, SiHa, SCC-7 and C666-1 cells. We constructed aMUC2(Mucin2)-targeting CRISPR/cas9 gene editing system in HEK 293T cells, with gene disruption supported by oligodeoxynucleotide (ODN) insertion in vitro. Additionally, we developed anLMP1(latent membrane protein 1)-targeting CRISPR/cas9 gene editing system in LMP1-overexpressing SCC7 cells, which was designed to cleave fragments expressing the LMP1 protein (related to Epstein–Barr virus infection) and thus to inhibit the growth of the cells in vivo. As evidenced by in vitro and in vivo experiments, this system has great potential for gene therapy applications.Keywords:poly (β-amino ester);gene transfection;D-α-tocopherol polyethylene glycol succinate;copolymer;CRISPR/cas9 gene editing;gene therapy
研究领域:
生物技术
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基因治疗
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药学
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分子生物学
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DOI
PubMed
高影响因子
ACS Nano
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2024
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IF 15.8
Yu Ji,Suchen Qu,Gaoyu Shi,Liansheng Fan,Jing Qian,Zhaorui Sun,Feng Lu,Xin Han
Hypoxia is one of the most typical features among various types of solid tumors, which creates an immunosuppressive tumor microenvironment (TME) and limits the efficacy of cancer treatment. Alleviating hypoxia becomes a key strategy to reshape hypoxic TME which improves cancer immunotherapy. However, it remains challenging to perform tumor precision therapy with controllable switches through hypoxia-activated gene editing and prodrugs to alleviate hypoxia. In this study, silica-coated second near-infrared window (NIR-II) emitting silver sulfide quantum dots are used as the carrier to load the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) system to target hypoxia-inducible factor-1 (HIF-1α) and guide tumor-targeted imaging. To reduce the off-target effects in nontumor cells and better control safety risks, a TME-triggered cascade-activation nanodiagnostic and therapeutic platform (AA@Cas-H@HTS) is designed, which achieves the hypoxia activation of prodrug tirapazamine (TPZ) and spatiotemporal release of CRISPR/Cas9 ribonucleoprotein. Tumor hypoxia is greatly alleviated by the synergistic function of HIF-1α depletion by gene editing and TPZ activation. Importantly, targeting HIF-1α disrupts the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) signaling pathway, which effectively reshapes the immune-suppressive TME and activates T cell-mediated antitumor immunity. Taken together, we have provided a TME-triggered cascade-activation nanoplatform to alleviate hypoxia for improved cancer immunotherapy.
研究领域:
癌症生物学
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免疫学
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纳米技术
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分子生物学
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诊疗学
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产品关联
DOI
PubMed
高影响因子
Cell Reports Medicine
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2025
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IF 10.6
Kaiyu Xu,Ying Zhang,Yongxuan Chen,Xiaojia Zhu,Yu Li,Longbao Lv,Xiechao He,Zhengfei Hu,Yifan Li,Maosen Ye,Dewei Jiang,Zhanlong He,Weihua Jin,Yanyan Li,Xiaomei Yu,Deng-Feng Zhang,Karl Herrup,Ping Zheng,Yong-Gang Yao,Dong-Dong Wu,Jiali Li
Ataxia-telangiectasia (A-T) is a hereditary neurodegenerative disorder caused by mutations in the ATM (ataxia-telangiectasia mutated) gene. Although existing rodent models reproduce some of the multi-systemic features of A-T, they notably fail to recapitulate the severe neurological manifestations, particularly the profound cerebellar atrophy and associated ataxia. To address this limitation, we have generated ATM-deficient rhesus macaques using CRISPR-Cas9. These macaques exhibit hallmark features of A-T, including growth retardation, lymphopenia, elevated a-fetoprotein levels, oculocutaneous telangiectasias, heightened sensitivity to ionizing radiation, and most critically, cerebellar atrophy, Purkinje cell loss, and early-stage cerebellar neurodegeneration leading to significant motor impairments. Single-nucleus transcriptomic profiling of the cerebellum revealed pronounced gene expression changes associated with ATM deficiency, particularly in molecular layer interneurons (MLIs), which are implicated in Purkinje cell loss. This non-human primate model provides deeper insights into the pathogenesis of A-T and represents a promising and valuable platform for developing therapeutic strategies.
研究领域:
神经生物学
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遗传学
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分子医学
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转化医学
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发育生物学
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基因编辑
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神经退行性疾病
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CONTROLLED RELEASE
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2025
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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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产品关联
DOI
PubMed
高影响因子
PLoS Genetics
·
2022
·Vol. 18
·期 3
·e1009841
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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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产品关联
DOI
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
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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高影响因子
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