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1.
Proc Natl Acad Sci U S A ; 121(22): e2322479121, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38771871

RESUMO

The significance of biochemical cues in the tumor immune microenvironment in affecting cancer metastasis is well established, but the role of physical factors in the microenvironment remains largely unexplored. In this article, we investigated how the mechanical interaction between cancer cells and immune cells, mediated by extracellular matrix (ECM), influences immune escape of cancer cells. We focus on the mechanical regulation of macrophages' targeting ability on two distinct types of colorectal carcinoma (CRC) cells with different metastatic potentials. Our results show that macrophages can effectively target CRC cells with low metastatic potential, due to the strong contraction exhibited by the cancer cells on the ECM, and that cancer cells with high metastatic potential demonstrated weakened contractions on the ECM and can thus evade macrophage attack to achieve immune escape. Our findings regarding the intricate mechanical interactions between immune cells and cancer cells can serve as a crucial reference for further exploration of cancer immunotherapy strategies.


Assuntos
Neoplasias Colorretais , Matriz Extracelular , Macrófagos , Evasão Tumoral , Microambiente Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Macrófagos/imunologia , Humanos , Microambiente Tumoral/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/imunologia , Linhagem Celular Tumoral , Metástase Neoplásica , Animais , Camundongos , Comunicação Celular/imunologia
3.
Sci Rep ; 14(1): 2908, 2024 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-38316885

RESUMO

Breast cancer is the most common diagnosed cancer, the HER2-positive subtype account for 15% of all breast cancer. HER2-targeted therapy is the mainstay treatment for HER2-positive breast cancer. Cuproptosis is a novel form of programmed cell death, and is caused by mitochondrial lipoylation and destabilization of iron-sulfur proteins triggered by copper, which was considered as a key player in various biological processes. However, the roles of cuproptosis-related genes in HER2-positive breast cancer remain largely unknown. In the present study, we constructed a prognostic prediction model of HER2-positive breast cancer patients using TCGA database. Dysregulated genes for cells resistant to HER2-targeted therapy were analyzed in the GEO dataset. KEGG pathway, GO enrichment and GSEA was performed respectively. The immune landscape of DLAT was analyzed by CIBERSORT algorithm and TIDE algorithm. HER2-positive breast cancer patients with high CRGs risk score showed shorter OS. DLAT was downregulated and correlated with better survival of HER2-positive breast cancer patients (HR = 3.30, p = 0.022). High expressed DLAT was associated with resistant to HER2-targeted therapy. Knocking down DLAT with siRNA increased sensitivity of breast cancer to trastuzumab. KEGG pathway and GO enrichment of DEGs indicated that DLAT participates in various pathways correlated with organelle fission, chromosome segregation, nuclear division, hormone-mediated signaling pathway, regulation of intracellular estrogen receptor signaling pathway, condensed chromosome and PPAR signaling pathway. There was a negative correlation between TIDE and DLAT expression (r = - 0.292, p < 0.001), which means high DLAT expression is an indicator of sensitivity to immunotherapy. In conclusion, our study constructed a four CRGs signature prognostic prediction model and identified DLAT as an independent prognostic factor and associated with resistant to HER2-targeted therapy for HER2-positive breast cancer patients.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Prognóstico , Algoritmos , Apoptose , Cobre
4.
Angew Chem Int Ed Engl ; 63(8): e202317972, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38116884

RESUMO

The pursuit of highly efficient and stable wide-band gap (WBG) perovskite solar cells (PSCs), especially for monolithic perovskite/silicon tandem devices, is a key focus in achieving the commercialization of perovskite photovoltaics. In this study, we initially designed poly(ionic liquid)s (PILs) with varying alkyl chain lengths based on density functional theory calculations. Results pinpoint that PILs with longer alkyl chain lengths tend to exhibit more robust binding energy with the perovskite structure. Then we synthesized the PILs to craft a hydrophobic hydrogen-bonded polymer network (HHPN) that passivates the WBG perovskite/electron transport layer interface, inhibits ion migration and serves as a barrier layer against water and oxygen ingression. Accordingly, the HHPN effectively curbs nonradiative recombination losses while facilitating efficient carrier transport, resulting in substantially enhanced open-circuit voltage (Voc ) and fill factor. As a result, the optimized single-junction WBG PSC achieves an impressive efficiency of 23.18 %, with Voc as high as 1.25 V, which is the highest reported for WBG (over 1.67 eV) PSCs. These devices also demonstrate outstanding thermostability and humidity resistance. Notably, this versatile strategy can be extended to textured perovskite/silicon tandem cells, reaching a remarkable efficiency of 28.24 % while maintaining exceptional operational stability.

5.
Int J Biol Sci ; 19(16): 5319-5336, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37928256

RESUMO

Transfer RNAs (tRNAs) impact the development and progression of various cancers, but how individual tRNAs are modulated during triple-negative breast cancer (TNBC) progression remains poorly understood. Here, we found that XPOT (Exportin-T), a nuclear export protein receptor of tRNAs, is associated with poor prognosis in breast cancer and directly orchestrates the nuclear export of a subset of tRNAs, subsequently promoting protein synthesis and proliferation of human TNBC cells. XPOT knockdown inhibited TNBC cell proliferation in vitro, and RNA-seq indicated that XPOT is involved in the completion of cytokinesis in TNBC cells. High-throughput sequencing of tRNA revealed that XPOT specifically influenced a subset of tRNA isodecoders involved in nucleocytoplasmic trafficking, including tRNA-Ala-AGC-10-1. Through codon preferential analysis and protein mass spectrometry, we found that XPOT preferentially transported nuclear tRNA-Ala-AGC-10-1 to the cytoplasm, driving the translation of TPR Repeat Protein 19 (TTC19). TTC19 is also indispensable for cytokinesis and proliferation of TNBC cells. Altogether, these findings provide a novel regulatory translation mechanism for preferential tRNA isodecoder nucleocytoplasmic transport through XPOT, which coordinates the spatial location of specific tRNA and the translation of mRNA to facilitate TNBC proliferation and progression. Targeting XPOT may be a novel therapeutic strategy for treating TNBC.


Assuntos
Citocinese , Neoplasias de Mama Triplo Negativas , Humanos , Citocinese/genética , Neoplasias de Mama Triplo Negativas/genética , Proliferação de Células/genética , Transporte Biológico , Citoplasma , RNA de Transferência/genética , Linhagem Celular Tumoral , Proteínas de Transporte Nucleocitoplasmático
6.
Risk Manag Healthc Policy ; 16: 2521-2529, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38024490

RESUMO

Purpose: The market price of adebrelimab, the first Chinese-developed anti-PD-L1 antibody used as a first-line treatment for extensive stage-small-cell lung cancer (ES-SCLC), has garnered significant public attention. This study sought to investigate the affordable price of adebrelimab for Chinese patients with untreated ES-SCLC through a cost-effectiveness analysis. Patients and Methods: We conducted a cost-effectiveness analysis using a Markov model, incorporating a what-if scenario of adding adebrelimab to first-line etoposide/platinum (EP) chemotherapy is cost-effective for ES-SCLC patients from the perspective of the Chinese healthcare system. The model included three mutually exclusive health states, with transition probabilities derived from the CAPSTONE-1 trial. Health state utilities and costs were acquired from a myriad of authoritative sources. We compared the incremental cost-effectiveness ratios (ICERs) for adebrelimab plus EP chemotherapy (AEP) versus EP with a willingness-to-pay threshold of $37,654 per quality-adjusted life-years (QALYs) to estimate the affordable price ceiling of the upcoming adebrelimab. Results: For the entire ES-SCLC population, the estimated price ceiling of adebrelimab/mg was $0.542 (95% CI, $0.542-$0.552). Subgroup analyses found that the highest price ceiling of adebrelimab/mg was observed in ES-SCLC patients with lactate dehydrogenase concentration ≤ upper normal limit [$0.824 (95% CI, $0.815-$0.830)]; and the lowest was found in ES-SCLC patients with liver metastasis [$0.252 (95% CI, $0.250- $0.256)]. Sensitivity analysis revealed a heightened probability of cost-effectiveness for the first-line AEP as the price of adebrelimab decreased, encompassing both the entire ES-SCLC population and its subgroups. Conclusion: The affordable price range for adebrelimab/mg Chinese patients with untreated ES-SCLC was estimated to be between $0.252 and $0.824, with variations observed across different subgroups. In the context of universal healthcare coverage, our study provides valuable evidence to inform the implementation of a value-based pricing strategy for cancer treatment.

7.
Cancer Lett ; 573: 216370, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37660883

RESUMO

Low-dose metronomic (LDM) chemotherapy, the frequent and continuous use of low doses of conventional chemotherapeutics, is emerging as a promising form of chemotherapy utilization. LDM chemotherapy exerts immunomodulatory effects. However, the underlying mechanism is not fully understood. Here we found that suppressing tumor growth by LDM chemotherapy was dependent on the activation of CD8+T cells. LDM chemotherapy potentiated the cytotoxic function of CD8+T cells by stimulating cancer-cell autonomous type I interferon (IFN) induction. Mechanistically, LDM chemotherapy evoked mitochondrial dysfunction and increased reactive oxygen species (ROS) production. ROS triggered the oxidation of cytosolic mtDNA, which was sensed by cGAS-STING, consequently inducing type I IFN production in the cancer cells. Moreover, the cGAS-STING-IFN axis increased PD-L1 expression and predicted favorable clinical responses to chemoimmunotherapy. Antioxidant N-acetylcysteine inhibited oxidized mtDNA-induced type I IFN production and attenuated the efficacy of combination therapy with LDM chemotherapy and PD-L1 blockade. This study elucidates the critical role of intratumoral oxidized mtDNA sensing in LDM chemotherapy-mediated activation of CD8+T cell immune response. These findings may provide new insights for designing combinatorial immunotherapy for cancer patients.


Assuntos
Antígeno B7-H1 , DNA Mitocondrial , Humanos , Espécies Reativas de Oxigênio , Mitocôndrias , Linfócitos T CD8-Positivos
8.
Breast Cancer Res ; 25(1): 90, 2023 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-37507768

RESUMO

The communication between tumor cells and tumor microenvironment plays a critical role in cancer development. Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment and take part in breast cancer formation and progression. Here, by comparing the gene expression patterns in CAFs and normal fibroblasts, we found SPRY2 expression was significantly decreased in CAFs and decreased SPRY2 expression was correlated with worse prognosis in breast cancer patients. SPRY2 knockdown in fibroblasts promoted tumor growth and distant metastasis of breast cancer in mice. Loss of stromal SPRY2 expression promoted CAF activation dependent on glycolytic metabolism. Mechanically, SPRY2 suppressed Y10 phosphorylation of LDHA and LDHA activity by interfering with the interaction between LDHA and SRC. Functionally, SPRY2 knockdown in fibroblasts enhanced the stemness of tumor cell dependent on glycolysis in fibroblasts. Collectively, this work identified SPRY2 as a negative regulator of CAF activation, and SPRY2 in CAFs may potentially be therapeutically targeted in breast cancer treatment.


Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Animais , Camundongos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Fibroblastos/metabolismo , Neoplasias/metabolismo , Fosforilação , Prognóstico , Microambiente Tumoral/genética , Humanos , Feminino
9.
J Transl Med ; 21(1): 400, 2023 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-37340461

RESUMO

BACKGROUND: Upregulation of the PD-L1 (CD274) immune checkpoint ligand on the tumor surface facilitates tumor immune escape and limits the application of immunotherapy in various cancers, including breast cancer. However, the mechanisms underlying high PD-L1 levels in cancers are still poorly understood. METHODS: Bioinformatics analyses and in vivo and in vitro experiments were carried out to assess the association between CD8+ T lymphocytes and TIMELESS (TIM) expression, and to discover the mechanisms of TIM, the transcription factor c-Myc, and PD-L1 in breast cancer cell lines. RESULTS: The circadian gene TIM enhanced PD-L1 transcription and facilitated the aggressiveness and progression of breast cancer through the intrinsic and extrinsic roles of PD-L1 overexpression. Bioinformatic analyses of our RNA sequencing data in TIM-knockdown breast cancer cells and public transcriptomic datasets showed that TIM might play an immunosuppressive role in breast cancer. We found that TIM expression was inversely associated with CD8+ T lymphocyte infiltration in human breast cancer samples and subcutaneous tumor tissues. In vivo and in vitro experiments demonstrated that TIM knockdown increased CD8+ T lymphocyte antitumor activity. Furthermore, our results showed that TIM interacts with c-Myc to enhance the transcriptional capability of PD-L1 and facilitates the aggressiveness and progression of breast cancer through the intrinsic and extrinsic roles of PD-L1 overexpression. Moreover, public database analysis suggested that high TIM levels were positively related to PD-L1 inhibitor therapeutic response. CONCLUSIONS: Mechanistically, we first found that TIM could upregulate PD-L1 by interacting with c-Myc to enhance the transcriptional capability of c-Myc to PD-L1. Altogether, our findings not only provide a novel therapeutic strategy to treat breast cancer by targeting the oncogenic effect of TIM but also indicate that TIM is a promising biomarker for predicting the benefit of anti-PD-L1 immunotherapy.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Antígeno B7-H1/metabolismo , Neoplasias da Mama/genética , Linfócitos T CD8-Positivos , Perfilação da Expressão Gênica , Imunoterapia , Células MCF-7 , Transcriptoma
10.
Apoptosis ; 28(9-10): 1484-1495, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37368176

RESUMO

Neutrophil and neutrophil extracellular traps (NETs) were reported to be associated with tumor development, but the exact role and concrete mechanisms are still poorly understood, especially in triple negative breast cancer (TNBC). In this study, our results exhibited that NETs formation in TNBC tissues was higher than that in non-TNBC tissues, and NETs formation was distinctly correlated with tumor size, ki67 level and lymph node metastasis in TNBC patients. Subsequent in vivo experiments demonstrated that NETs inhibition could suppress TNBC tumor growth and lung metastasis. Further in vitro experiments uncovered that oncogenic function of NETs on TNBC cells were possibly dependent on TLR9 expression. We also found that neutrophils from peripheral blood of TNBC patients with postoperative fever were prone to form NETs and could enhance the proliferation and invasion of TNBC cells. Mechanistically, we revealed that NETs could interact with TLR9 to decrease Merlin phosphorylation which contributed to TNBC cell ferroptosis resistance. Our work provides a novel insight into the mechanism of NETs promoting TNBC progression and blocking the key modulator of NETs might be a promising therapeutic strategy in TNBC.


Assuntos
Armadilhas Extracelulares , Ferroptose , Neoplasias de Mama Triplo Negativas , Humanos , Armadilhas Extracelulares/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Neurofibromina 2/metabolismo , Ferroptose/genética , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/patologia , Apoptose , Neutrófilos/patologia , Proliferação de Células
11.
Front Plant Sci ; 14: 1128399, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37223797

RESUMO

Precisely discerning disease types and vulnerable areas is crucial in implementing effective monitoring of crop production. This forms the basis for generating targeted plant protection recommendations and automatic, precise applications. In this study, we constructed a dataset comprising six types of field maize leaf images and developed a framework for classifying and localizing maize leaf diseases. Our approach involved integrating lightweight convolutional neural networks with interpretable AI algorithms, which resulted in high classification accuracy and fast detection speeds. To evaluate the performance of our framework, we tested the mean Intersection over Union (mIoU) of localized disease spot coverage and actual disease spot coverage when relying solely on image-level annotations. The results showed that our framework achieved a mIoU of up to 55.302%, indicating the feasibility of using weakly supervised semantic segmentation based on class activation mapping techniques for identifying disease spots in crop disease detection. This approach, which combines deep learning models with visualization techniques, improves the interpretability of the deep learning models and achieves successful localization of infected areas of maize leaves through weakly supervised learning. The framework allows for smart monitoring of crop diseases and plant protection operations using mobile phones, smart farm machines, and other devices. Furthermore, it offers a reference for deep learning research on crop diseases.

12.
Insects ; 14(3)2023 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-36975962

RESUMO

The frequent occurrence of crop pests and diseases is one of the important factors leading to the reduction of crop quality and yield. Since pests are characterized by high similarity and fast movement, this poses a challenge for artificial intelligence techniques to identify pests in a timely and accurate manner. Therefore, we propose a new high-precision and real-time method for maize pest detection, Maize-YOLO. The network is based on YOLOv7 with the insertion of the CSPResNeXt-50 module and VoVGSCSP module. It can improve network detection accuracy and detection speed while reducing the computational effort of the model. We evaluated the performance of Maize-YOLO in a typical large-scale pest dataset IP102. We trained and tested against those pest species that are more damaging to maize, including 4533 images and 13 classes. The experimental results show that our method outperforms the current state-of-the-art YOLO family of object detection algorithms and achieves suitable performance at 76.3% mAP and 77.3% recall. The method can provide accurate and real-time pest detection and identification for maize crops, enabling highly accurate end-to-end pest detection.

13.
Antioxidants (Basel) ; 11(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36421464

RESUMO

Controlled atmosphere (CA) has been demonstrated to maintain the shelf-life quality of fruits, but its effect on the antioxidant activities and phenolic content of yellow peach is not comprehensive. This study analyzed the role of CA on the quality of shelf period, phenolic content and antioxidant activity of "Jinxiu" yellow peach. Yellow peach was left under specific aeration conditions (3.5-4% CO2, 2-3% O2, 92-95.5% N2, 1 ± 0.5 °C) and the control (1 ± 0.5 °C) for 21 d, to observe changes in physiological parameters of the fruit during 10 d of the shelf life (25 ± 1 °C). The result showed that CA reduced the weight loss rate (WLR), decay rate (DR), and browning index (BI) of yellow peaches. Furthermore, the CA held a high level of total flavonoid content (TFC), total phenol content (TPC) and phenolic content in the fruit. Antioxidant analysis showed that polyphenol oxidase (PPO) enzyme activity was lower and free radical scavenging capacity (DPPH, ABTS, and FRAP) and antioxidant enzyme activities (POD and PAL) were higher in the CA group. Combining the results of significance analysis, correlation analysis, principal component analysis (PCA) and hierarchical cluster analysis (HCA) clearly identified the differences between the CA group and the control group. The results showed that the CA could maintain higher phenolic content and reduce the oxidation of yellow peach fruit and enhance fruit quality by affecting the antioxidant activities of yellow peach.

14.
J Immunol Res ; 2022: 9529114, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35935587

RESUMO

Objective: To identify trastuzumab-resistant genes predicting drug response and poor prognosis in human epidermal growth factor receptor 2 positive (HER2+) breast cancer. Methods: Gene expression profiles from the GEO (Gene Expression Omnibus) database were obtained and analyzed. Differentially expressed genes (DEGs) between the pathological complete response (pCR) group and non-pCR group in a trastuzumab neoadjuvant therapy cohort and DEGs between Herceptin-resistant and wild-type cell lines were detected and evaluated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were performed to select the functional hub genes. The hub genes' prognostic power was validated by another trastuzumab adjuvant treatment cohort. Results: Fifty upregulated overlapping DEGs were identified by analyzing two trastuzumab resistance-related GEO databases. Functional analysis picked out ten hub genes enriched in mitochondrial function and metabolism pathways: ASCL1, CPT2, DLD, ELVOL7, GAMT, NQO1, SLC23A1, SPR, UQCRB, and UQCRQ. These hub genes could distinguish patients with trastuzumab resistance from the sensitive ones. Further survival analysis of hub genes showed that DLD overexpression was significantly associated with an unfavorable prognosis in HER2+ breast cancer patients. Conclusion: Ten novel trastuzumab resistance-related genes were discovered, of which DLD could be used for trastuzumab response prediction and prognostic prediction in HER2+ breast cancer.


Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Receptor ErbB-2 , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico
15.
Chem Sci ; 12(35): 11762-11768, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34659713

RESUMO

The addition of sulfonyl radicals to alkenes and alkynes is a valuable method for constructing useful highly functionalized sulfonyl compounds. The underexplored alkoxy- and fluorosulfonyl radicals are easily accessed by CF3 radical addition to readily available allylsulfonic acid derivatives and then ß-fragmentation. These substituted sulfonyl radicals add to aryl alkyl alkynes to give vinyl radicals that are trapped by trifluoromethyl transfer to provide tetra-substituted alkenes bearing the privileged alkoxy- or fluorosulfonyl group on one carbon and a trifluoromethyl group on the other. This process exhibits broad functional group compatibility and allows for the late-stage functionalization of drug molecules, demonstrating its potential in drug discovery and chemical biology.

16.
J Vet Med Sci ; 80(1): 173-180, 2018 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187697

RESUMO

Valnemulin, successfully developed by Sandoz in 1984, is a new generation derivative of pleuromutilin related to tiamulin. Valnemulin has low water-solubility, a short half-life period, low bioavailability, and instability. The application of valnemulin was restricted. Therefore, finding a more moderate delivery system is necessary to improve the shortcomings of valnemulin. The purpose of the study was to improve the strong stability and the irritation caused by of valnemulin hydrochloride power through pegylated-valnemulin prodrug mode. The prepared pegylated-valnemulin prodrug was characterized and evaluated by in vitro release performance under buffer solutions with pH levels of 7.4 and 3.6. The loading rate of valnemulin in PEG-succinic-valnemulin prodrug was determined by ultraviolet spectrophotometer and high performance liquid chromatography (HPLC). HPLC with evaporative light scattering detector was applied to determine the amount of PEG-succinic acid. The loading rate of valnemulin in PEG-succinic-valnemulin prodrug was 6.46%. PEG-succinic-valnemulin prodrug demonstrated a satisfactory solubility of valnemulin with 523 mg·ml-1 and excellent stability verified by the stability experiment. The result of the in vitro release test showed that the prepared PEG-valnemulin prodrug has controlled release ability and the release rate of valnemulin from PEG-valnemulin prodrug with a pH of 7.4 was 64.98%, which was higher than that of pH3.6 with release rate of 31.90%. Therefore, the prepared PEG-succinic-valnemulin prodrug has great application potential.


Assuntos
Polietilenoglicóis/química , Pró-Fármacos/química , Diterpenos/síntese química , Diterpenos/química , Diterpenos/farmacocinética , Estabilidade de Medicamentos , Meia-Vida , Técnicas In Vitro , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacocinética , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética , Solubilidade
17.
Wei Sheng Wu Xue Bao ; 52(7): 850-6, 2012 Jul 04.
Artigo em Chinês | MEDLINE | ID: mdl-23115969

RESUMO

OBJECTIVE: Expression, purification and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei. METHODS: The strong promoter and terminator of cellobiohydrolase I (cbh1) gene from T. reesei were amplified by PCR and inserted into pBluescriptIISK(+) to form vector pSKCST. The laccase gene from Pleurotus ostreatus was de novo synthesized according to T. reesei condon bias and cloned into the vector pLacdt resulting in the expression vector pSKLDT. The linearized pSKLDT was introduced into T. reesei strain Tu6 by protoplast-mediated transformation. The screened laccase expression transformants were grown in shake flasks on minimal medium and the recombinant laccase was purified and characterized. RESULTS: Transformants were isolated in selective screening medium plate and identified by PCR. The enzyme activity of laccase in transformant LC-7 was 237.134 U/mL which was 28.6 -fold higher than that in P. ostreatus. The specific activity of the purified enzyme was 9852 IU/mg. Enzymatic assay revealed that the optimum temperature for its activity was 50 degrees C and pH was 3.0. The optimum substrate was ABTS and the K(m) and V(max) for ABTS were 7.58 x 10(-2) mmol/L and 9.752 x 10(-3) mmol/L/min. Metal ions like Cu2+, Zn2+, Fe3+, Mn2+, Ba2+, Mg2+ and Fe2+ had different inhibitory effect on purified laccase. CONCLUSIONS: Under the regulation of cbh1 promoter and cbh1 signal peptide, heterologous laccase was successfully overexpressed in T. reesei.


Assuntos
Engenharia Genética/métodos , Lacase/genética , Lacase/metabolismo , Pleurotus/enzimologia , Pleurotus/genética , Trichoderma/genética , Biocatálise/efeitos dos fármacos , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/isolamento & purificação , Metais/farmacologia , Plasmídeos/genética , Temperatura
18.
Bioresour Technol ; 109: 116-22, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22305540

RESUMO

A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference.


Assuntos
Aspergillus niger/enzimologia , Biotecnologia/métodos , Celulose 1,4-beta-Celobiosidase/genética , Regulação Fúngica da Expressão Gênica , Lipase/biossíntese , Interferência de RNA , Trichoderma/genética , Southern Blotting , Celulose 1,4-beta-Celobiosidase/metabolismo , DNA Fúngico/isolamento & purificação , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transformação Genética , Trichoderma/metabolismo
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