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1.
Nano Lett ; 24(8): 2544-2552, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38349341

RESUMO

Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.


Assuntos
Pontos Quânticos , Viroses , Vírus , Humanos , Envelope Viral/metabolismo , Proteínas do Envelope Viral
2.
Fungal Genet Biol ; 173: 103899, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38802054

RESUMO

Fusarium head blight is a devastating disease that causes severe yield loses and mycotoxin contamination in wheat grain. Additionally, balancing the trade-off between wheat production and disease resistance has proved challenging. This study aimed to expand the genetic tools of the endophyte Phomopsis liquidambaris against Fusarium graminearum. Specifically, we engineered a UDP-glucosyltransferase-expressing P. liquidambaris strain (PL-UGT) using ADE1 as a selection marker and obtained a deletion mutant using an inducible promoter that drives Cas9 expression. Our PL-UGT strain converted deoxynivalenol (DON) into DON-3-G in vitro at a rate of 71.4 % after 36 h. DON inactivation can be used to confer tolerance in planta. Wheat seedlings inoculated with endophytic strain PL-UGT showed improved growth compared with those inoculated with wildtype P. liquidambaris. Strain PL-UGT inhibited the growth of Fusarium graminearum and reduced infection rate to 15.7 %. Consistent with this finding, DON levels in wheat grains decreased from 14.25 to 0.56 µg/g when the flowers were pre-inoculated with PL-UGT and then infected with F. graminearum. The expression of UGT in P. liquidambaris was nontoxic and did not inhibit plant growth. Endophytes do not enter the seeds nor induce plant disease, thereby representing a novel approach to fungal disease control.


Assuntos
Ascomicetos , Endófitos , Fusarium , Glucosiltransferases , Doenças das Plantas , Tricotecenos , Triticum , Triticum/microbiologia , Triticum/genética , Tricotecenos/metabolismo , Fusarium/genética , Fusarium/efeitos dos fármacos , Fusarium/enzimologia , Endófitos/genética , Endófitos/enzimologia , Endófitos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Ascomicetos/genética , Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Resistência à Doença/genética , Micotoxinas/metabolismo
3.
Angew Chem Int Ed Engl ; 63(31): e202402880, 2024 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-38758629

RESUMO

Lysine-specific peptide and protein modification strategies are widely used to study charge-related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge-related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge-related protein properties, such as liquid-liquid phase separation behaviour of α-synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine-containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.


Assuntos
Hidroxilamina , Lisina , Lisina/química , Hidroxilamina/química , Proteínas/química , Amidinas/química , alfa-Sinucleína/química , Peptídeos/química
4.
Bioconjug Chem ; 34(6): 1037-1044, 2023 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-37204067

RESUMO

Sphingomyelinase (SMase), a hydrolase of sphingomyelin (SM) enriched in the outer leaflet of the plasma membrane of mammalian cells, is closely associated with the onset and development of many diseases, but the specific mechanisms of SMase on the cell structure, function, and behavior are not yet fully understood due to the complexity of the cell structure. Artificial cells are minimal biological systems constructed from various molecular components designed to mimic cellular processes, behaviors, and structures, which are excellent models for studying biochemical reactions and dynamic changes in cell membranes. In this work, we presented an artificial cell model that mimics the lipid composition and content of the outer leaflet of mammalian plasma membranes for studying the effect of SMase on cell behavior. The results confirmed that the artificial cells can respond to SM degradation by producing ceramides that enrich and alter the membrane charge and permeability, thus inducing the budding and fission of the artificial cells. Thus, the artificial cells developed here provide a powerful tool to study the mechanism of action of cell membrane lipids on cell biological behavior, paving the way for further molecular mechanism studies.


Assuntos
Células Artificiais , Esfingomielinas , Animais , Esfingomielinas/análise , Esfingomielinas/metabolismo , Esfingomielinas/farmacologia , Ceramidas/química , Ceramidas/metabolismo , Ceramidas/farmacologia , Membrana Celular/metabolismo , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Mamíferos/metabolismo
5.
J Pept Sci ; 29(3): e3454, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36181422

RESUMO

Considering the fact that site-selective late-stage diversification of peptides and proteins remains a challenge for biochemistry, strategies targeting low-abundance natural amino acids need to be further developed. As an extremely oxidation-sensitive and low-abundance amino acid, methionine emerges as a promising target for chemo- and site-selective modification. Herein we report an efficient and highly selective modification on methionine residues by one-pot O- and N-transfer reaction, generating sulfoximine-modified peptides with near-perfect conversion within 10 min. Moreover, the great tolerance to other natural amino acids has been demonstrated in reactions with various peptide substrates. To demonstrate the generality of this protocol, we have modified natural peptides and obtained sulfoximination products with high conversion rates. This methodology provides a novel strategy as the expansion of the methionine-based peptide functionalization toolbox.


Assuntos
Metionina , Proteínas , Metionina/química , Metionina/metabolismo , Proteínas/química , Peptídeos/química , Racemetionina/metabolismo , Estresse Oxidativo
6.
Anal Chem ; 94(21): 7567-7575, 2022 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-35581735

RESUMO

Mammalian cell display technology uses eukaryotic protein expression system to display proteins on cell surfaces and has become an important method in biological research. Although mammalian cell display technology has many advantages and development potential, certain attributes of the displayed protein remain uncharacterized, such as whether the displayed proteins re-enter the cell and how displayed proteins move into the cell. Here, we present the endocytosis mechanism, motility behavior, and transport kinetics of displayed proteins determined using HaloTag as the displayed protein and quantum dot-based single-particle tracking. The displayed protein enters the cell through clathrin-mediated endocytosis and is transported through the cell in three stages, which is dependent on microfilaments and microtubules. The dynamic information obtained in this study provides answers to questions about endocytosis and postendocytosis transport of displayed proteins and, therefore, is expected to facilitate the development of surface display technology.


Assuntos
Pontos Quânticos , Citoesqueleto de Actina , Animais , Membrana Celular , Endocitose , Mamíferos
7.
Fungal Genet Biol ; 158: 103650, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34923123

RESUMO

Fusarium head blight (FHB) is a disease that affects wheat crops worldwide and is caused by Fusarium graminearum. Effective and safe strategies for the prevention and treatment of the disease are very limited. Phomopsis liquidambaris, a universal endophyte, can colonize wheat. Two engineered strains, Phomopsis liquidambaris OE-Chi and IN-Chi, were constructed by transformation with a plasmid and integration of a chitinase into the genome, respectively. The OE-Chi and IN-Chi strains could inhibit the expansion of Fusarium sp. in plate confrontation assays in vitro. Colonization of the OE-Chi strain in wheat showed better effects than colonization of the IN-Chi strain and alleviated the inhibition of wheat growth caused by F. graminearum. The shoot length, root length and fresh weight of infected wheat increased by 164.9%, 115.4%, and 190.7%, respectively, when the plants were inoculated with the OE-Chi strain. The peroxidase (POD) activity in the wheat root increased by 38.0%, and it was maintained at a high level in the shoot, which suggested that the OE-Chi strain could enhance the resistance of wheat to F. graminearum. The root and shoot superoxide dismutase (SOD) activities were decreased by 11.8% and 19.0%, respectively, which may be helpful for colonization by the OE-Chi strain. These results suggested that the Phomopsis liquidambaris OE-Chi strain may be a potential endophyte in the biocontrol of FHB.


Assuntos
Quitinases , Fusarium , Ascomicetos , Quitinases/genética , Endófitos/genética , Fusarium/genética , Doenças das Plantas , Triticum
8.
J Assist Reprod Genet ; 37(4): 963-972, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32318905

RESUMO

PURPOSE: To establish a mathematical model for assessing the true ovarian reserve based on the predicted probability of poor ovarian response (POR). METHODS: In this retrospective cohort study, a total of 1523 GnRH-antagonist cycles in 2017 were firstly analyzed. The ovarian responses were calculated based on the number of retrieved oocytes. The continuous variables were converted into categorical variables according to cutoff values generated by the decision tree method. The optimal model was identified using forward stepwise multiple logistic regression with 5-fold cross-validation and further verified its performances using outer validation data. RESULTS: The predictors in our model were anti-Müllerian hormone (AMH), antral follicle counts (AFC), basal follicle-stimulating hormone (FSH), and age, in order of their significance, named AAFA model. The AUC, sensitivity, specificity, positive predictive value, and negative predictive value of AAFA model in inner validation and outer validation data were 0.861 and 0.850, 0.603 and 0.519, 0.917 and 0.930, 0.655 and 0.570, and 0.899 and 0.915. Ovarian reserve of 16 subgroups was further ranked according to the predicted probability of POR and further divided into 4 groups of A-D using clustering analysis. The incidence of POR in the four groups was 0.038 (0.030-0.046), 0.139 (0.101-0.177), 0.362 (0.308-0.415), and 0.571 (0.525-0.616), respectively. The order of ovarian reserve from adequate to poor followed the order of A to D. CONCLUSION: We have established an easy applicable AAFA model for assessing true ovarian reserve and may have important implications in both infertile women and general reproductive women in Chinese or Asian population.


Assuntos
Fertilização in vitro , Folículo Ovariano/crescimento & desenvolvimento , Reserva Ovariana/fisiologia , Ovário/crescimento & desenvolvimento , Hormônio Antimülleriano/genética , Feminino , Hormônio Foliculoestimulante , Humanos , Infertilidade Feminina/patologia , Infertilidade Feminina/prevenção & controle , Modelos Teóricos , Recuperação de Oócitos/métodos , Folículo Ovariano/transplante , Ovário/transplante , Indução da Ovulação/métodos , Probabilidade
9.
Adv Mater ; 36(2): e2306808, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37732588

RESUMO

The plasma membranes (PM) of mammalian cells contain diverse lipids, proteins, and carbohydrates that are important for systemic recognition and communication in health and disease. Cell membrane coating technology that imparts unique properties of natural plasma membranes to the surface of encapsulated nanoparticles is thus becoming a powerful platform for drug delivery, immunomodulation, and vaccination. However, current coating methods fail to take full advantage of the natural systems because they disrupt the complex and functionally essential features of PMs, most notably the chemical diversity and compositional differences of lipids in two leaflets of the PM. Herein, a new lipid coating approach is reported in which the lipid composition is optimized through a combination of biomimetic and systematic variation approaches for the custom design of nanocarrier systems for precision drug delivery. Nanocarriers coated with the optimized lipids offer unique advantages in terms of bioavailability and efficiency in tumor targeting, tumor penetration, cellular uptake, and drug release. This pilot study provides new insight into the rational design and optimization of nanocarriers for cancer chemotherapeutic drugs and lays the foundation for further customization of cell membrane-mimicking nanocarriers through systematic incorporation of other components.


Assuntos
Nanopartículas , Neoplasias , Animais , Membrana Celular/metabolismo , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Lipídeos/química , Mamíferos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Projetos Piloto , Humanos
10.
J Agric Food Chem ; 71(3): 1391-1404, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36625777

RESUMO

Fusarium head blight is one of the most serious diseases caused by Fusarium graminearum in wheat. Here, we developed a new way to prevent and control Fusarium head blight by introducing the resistance genes Fhb1 and Fhb7 into the endophytic fungus Phomopsis liquidambaris, named PL-Fhb1 and PL-Fhb7, respectively, which could colonize wheat. The wheat seedlings were preinoculated with PL-Fhb1 and PL-Fhb7 to enhance the resistance against deoxynivalenol (DON) and PL-Fhb1 and PL-Fhb7 inhibited the growth of F. graminearum by 73% and 49%, respectively. The incidence rate of diseased spikes decreased to 35.2% and 45.4%, and the corresponding DON levels for wheat grains decreased from 13.2 to 1.79 µg/g and from 13.2 µg/g to 0.39 µg/g when the leaves were preinoculated with PL-Fhb1 and PL-Fhb7 after overwintering, respectively. The incidence rates of diseased spikes decreased to 25.7% and 34.7%, and the DON levels for wheat grains decreased from 17.48 µg/g to 1.23 µg/g and from 17.48 µg/g to 0 µg/g when the wheat flowers were inoculated with PL-Fhb1 and PL-Fhb7, and the wheat flowers were subsequently infected with F. graminearum, respectively. It was confirmed that DON was transformed into DON-glutathione (GSH) by PL-Fhb7 using high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, PL-Fhb1 may have increased plant immunity and enhanced the resistance to F. graminearum. This study indicates that engineered endophytes can improve the resistance to Fusarium head blight and presents a new method for the biological control of Fusarium head blight.


Assuntos
Ascomicetos , Fusarium , Triticum/microbiologia , Doenças das Plantas/microbiologia
11.
Med Biol Eng Comput ; 60(11): 3113-3124, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36097237

RESUMO

In medicine, identifying the indirect immunofluorescence of human epithelial type 2 (HEp-2) cells plays a decisive role in the diagnosis of autoimmune diseases. The manual interpretation of Hep-2 cell images may lead to some limitations, such as subjectivity, inconsistency and low efficiency. Therefore, it is very important to automatically identify HEp-2 images. Inspired by the outstanding performance of neural networks in image classification tasks, we propose a multi-class and multiple-binary classifier (MCMBC) for the classification of HEp-2 cells. MCMBC is an ensemble learner that contains two kinds of sub-classifiers: multi-class (MC) and multiple-binary (MB). The MC sub-classifier adopts a multi-scale convolutional neural network (MSCNN) that increases the efficiency of information transmission between layers. On the basis of classification results of the MC sub-classifier on validation sets, we can find easy-to-confuse class pairs. An easy-to-confuse class pair is two classes that are not easy to be identified from each other. The MB sub-classifiers adopt multiple-binary pre-trained VGG16 networks that are used to deal with these class pairs. The final prediction for a sample possibly belonging to an easy-to-confuse class is decided by the assembled features extracted from the last fully connected layer of MC and the output of MB sub-classifiers. To evaluate the proposed model, experiments were conducted on the ICPR 2014 Task-2 dataset. Experimental results show that MCMBC performs better than the state-of-the-art method (84.68% vs. 83.35% on the criterion of average classification accuracy (ACA) and 82.89% vs. 82.67% on the criterion of mean classification accuracy (MCA)).


Assuntos
Redes Neurais de Computação , Humanos
12.
ACS Synth Biol ; 10(8): 2030-2039, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34251173

RESUMO

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.


Assuntos
Ascomicetos , Endófitos , Flavanonas/biossíntese , Quempferóis/biossíntese , Ascomicetos/genética , Ascomicetos/metabolismo , Endófitos/genética , Endófitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavanonas/genética , Quempferóis/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
13.
ACS Appl Mater Interfaces ; 13(36): 42522-42532, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34463488

RESUMO

Tumor-targeted delivery and controlled release of antitumor drugs are promising strategies for increasing chemotherapeutic efficacy and reducing adverse effects. Although mesoporous silica nanoparticles (MSNs) have been known as a potential delivery system for doxorubicin (DOX), they have restricted applications due to their uncontrolled leakage and burst release from their large open pores. Herein, we engineered a smart drug-delivery system (smart MSN-drug) based on MSN-drug loading, cell membrane mimetic coating, on-demand pore blocking/opening, and tumor cell targeting strategies. The pore size of DOX-loaded MSNs was narrowed by polydopamine coating, and the pores/channels were blocked with tumor-targeting ligands anchored by tumor environment-rupturable -SS- chains. Furthermore, a cell membrane mimetic surface was constructed to enhance biocompatibility of the smart MSN-drug. Confocal microscopy results demonstrate highly selective uptake (12-fold in comparison with L929 cell) of the smart MSN-drug by HeLa cells and delivery into the HeLa cellular nuclei. Further in vitro IC50 studies showed that the toxicity of the smart MSN-drug to HeLa cells was 4000-fold higher than to the normal fibroblast cells. These exciting results demonstrate the utility of the smart MSN-drug capable of selectively killing tumor cells and saving the normal cells.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Animais , Antineoplásicos/química , Doxorrubicina/química , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Células HeLa , Humanos , Indóis/química , Indóis/toxicidade , Camundongos , Nanopartículas/toxicidade , Fosforilcolina/análogos & derivados , Fosforilcolina/toxicidade , Polímeros/química , Polímeros/toxicidade , Porosidade , Dióxido de Silício/química , Dióxido de Silício/toxicidade , Microambiente Tumoral/fisiologia
14.
ACS Appl Bio Mater ; 3(11): 7382-7387, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-35019481

RESUMO

A facile strategy to construct dual-drug delivery nanoparticles (TL-CPT NPs), which possessed higher loading content of CPT and TPP-LND. Notably, TL-CPT NPs showed promising ultrarapid pH/GSH response to release more than 86% of loaded TPP-LND or 93% of loaded CPT in just 2 h. The results showed that the nanoparticles hierarchically delivered CPT and TPP-LND to targeted different organelles without mutual influence benefiting the ultrarapid pH/GSH response to drug release, and further significantly and synergistically induced cell apoptosis and improved chemotherapeutic efficiency in cancer cells.

15.
ACS Appl Mater Interfaces ; 12(29): 32432-32445, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32573194

RESUMO

To maximize the utilization and response to the high oxidative stress environment of tumor sites while avoiding the dilemma of enhancing reactive oxygen species (ROS) response in a single way, mitochondrial targeting combined with fluorescent self-reporting polymeric nanocarriers (1K-TPP and 2K-TPP) with grafted structures were synthesized via a chemoenzymatic method in a high yield to simultaneously enhance the drug delivery of endogenous ROS responses. 1K-TPP and 2K-TPP loaded doxorubicin (DOX) at a high content over 12% and formed homogeneous spherical micelles. In vitro, both of them showed promising high sensitivity (detection limit below 200 nM H2O2), fast response, and ratiometric fluorescent self-reporting properties (fluorescent enhancement more than 200 times) to ROS and excellent stability under physiological conditions, while achieving a rapid release of the DOX in response to 1 mM H2O2. Cell co-localization experiments exhibited that they had favorable mitochondrial targeting, and mitochondrial isolation experiments also confirmed that the TPP-modified 1K-TPP selectively accumulated nearly three times in mitochondria than that in total cells. The internalization of 1K-TPP and 2K-TPP into cancer cells was greatly improved by nearly 200% compared to that of unmodified control (1K-OH and 2K-OH) and also explored a unique energy-dependent endocytosis. Furthermore, stimulation of endogenous ROS enhanced the green fluorescence intensity (up to 51.4%) of the linked probe so as to destroy the internal structure of the nanocarriers, achieving self-reporting of the drug's intracellular release and tracking of the intracellular location of nanocarriers. The cytotoxicity of DOX-loaded 1K-TPP and 2K-TPP in tumor cells with a higher ROS content showed statistical superiority to that of 1K-OH and 2K-OH, benefiting from the extremely good endogenous ROS response sensitivity leading to the differential selective release of drugs. These results demonstrate the potential of 1K-TPP and 2K-TPP, especially for 1K-TPP, as mitochondria-targeted, fluorescent self-reporting nanocarriers for combined enhancement of endogenous ROS responsiveness.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Fluorescência , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antibióticos Antineoplásicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Mitocôndrias/metabolismo , Estrutura Molecular , Nanopartículas/química , Tamanho da Partícula , Polímeros/síntese química , Polímeros/química , Propriedades de Superfície
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