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1.
Nat Commun ; 11(1): 4502, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908136

RESUMO

Biological tissues, such as muscle, can increase their mechanical strength after swelling due to the existence of many biological membrane barriers that can regulate the transmembrane transport of water molecules and ions. Oppositely, typical synthetic materials show a swelling-weakening behavior, which always suffers from a sharp decline in mechanical strength after swelling, because of the dilution of the network. Here, we describe a swelling-strengthening phenomenon of polymer materials achieved by a bioinspired strategy. Liposomal membrane nanobarriers are covalently embedded in a crosslinked network to regulate transmembrane transport. After swelling, the stretched network deforms the liposomes and subsequently initiates the transmembrane diffusion of the encapsulated molecules that can trigger the formation of a new network from the preloaded precursor. Thanks to the tough nature of the double-network structure, the swelling-strengthening phenomenon is achieved to polymer hydrogels successfully. Swelling-triggered self-strengthening enables the development of various dynamic materials.


Assuntos
Materiais Biomiméticos/química , Hidrogéis/química , Lipossomos/química , Nanoestruturas/química , Força Compressiva , Reagentes para Ligações Cruzadas/química , Lipossomos/ultraestrutura , Teste de Materiais , Microscopia Eletrônica de Transmissão , Nanoestruturas/ultraestrutura , Resistência à Tração
2.
Nat Commun ; 11(1): 4535, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913217

RESUMO

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.


Assuntos
Nanopartículas/química , Coroa de Proteína/química , Química Click , Reagentes para Ligações Cruzadas/química , Células Endoteliais , Humanos , Poliestirenos/química , Ligação Proteica , Coroa de Proteína/análise , Dióxido de Silício/química , Células THP-1
3.
Int J Nanomedicine ; 15: 4825-4845, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32753868

RESUMO

Background: Nanosized drug delivery systems (NDDSs) have shown excellent prospects in tumor therapy. However, insufficient penetration of NDDSs has significantly impeded their development due to physiological instability and low passive penetration efficiency. Methods: Herein, we prepared a core cross-linked pullulan-modified nanosized system, fabricated by visible-light-induced diselenide bond cross-linked method for transporting ß-Lapachone and doxorubicin prodrug (boronate-DOX, BDOX), to improve the physiological stability of the NDDSs for efficient passive accumulation in tumor blood vessels (ß-Lapachone/BDOX-CCS). Additionally, ultrasound (US) was utilized to transfer ß-Lapachone/BDOX-CCS around the tumor vessel in a relay style to penetrate the tumor interstitium. Subsequently, ß-Lapachone enhanced ROS levels by overexpressing NQO1, resulting in the transformation of BDOX into DOX. DOX, together with abundant levels of ROS, achieved synergistic tumor therapy. Results: In vivo experiments demonstrated that ultrasound (US) + cross-linked nanosized drug delivery systems (ß-Lapachone/BDOX-CCS) group showed ten times higher DOX accumulation in the tumor interstitium than the non-cross-linked (ß-Lapachone/BDOX-NCS) group. Conclusion: Thus, this strategy could be a promising method to achieve deep penetration of NDDSs into the tumor.


Assuntos
Doxorrubicina/uso terapêutico , Nanopartículas/química , Naftoquinonas/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Ultrassonografia , Animais , Ácidos Borônicos/química , Permeabilidade Capilar/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Feminino , Glucanos/química , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/farmacocinética , Tamanho da Partícula , Pró-Fármacos/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Distribuição Tecidual/efeitos dos fármacos
4.
Nat Commun ; 11(1): 3850, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737322

RESUMO

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Proteoma/análise , Coloração e Rotulagem/métodos , Acrilamidas/química , Reagentes para Ligações Cruzadas/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Hidrogéis/química , Espaço Intracelular/química , Succinimidas/química , Inclusão do Tecido/métodos
5.
Nat Commun ; 11(1): 4032, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788575

RESUMO

Hydrogel-based devices are widely used as flexible electronics, biosensors, soft robots, and intelligent human-machine interfaces. In these applications, high stretchability, low hysteresis, and anti-fatigue fracture are essential but can be rarely met in the same hydrogels simultaneously. Here, we demonstrate a hydrogel design using tandem-repeat proteins as the cross-linkers and random coiled polymers as the percolating network. Such a design allows the polyprotein cross-linkers only to experience considerable forces at the fracture zone and unfold to prevent crack propagation. Thus, we are able to decouple the hysteresis-toughness correlation and create hydrogels of high stretchability (~1100%), low hysteresis (< 5%), and high fracture toughness (~900 J m-2). Moreover, the hydrogels show a high fatigue threshold of ~126 J m-2 and can undergo 5000 load-unload cycles up to 500% strain without noticeable mechanical changes. Our study provides a general route to decouple network elasticity and local mechanical response in synthetic hydrogels.


Assuntos
Reagentes para Ligações Cruzadas/química , Hidrogéis/química , Poliproteínas/química , Estresse Mecânico , Resinas Acrílicas/química , Fluorescência , Fenômenos Mecânicos
6.
Int J Nanomedicine ; 15: 4991-5004, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764931

RESUMO

Introduction: Various materials and approaches have been used to reduce the mesh-induced inflammatory response and modify the mesh with tissue-matched mechanical properties, aiming to improve the repair of abdominal wall defects. Materials and Methods: In this study, we fabricated a polycaprolactone (PCL)/silk fibroin (SF) mesh integrated with amoxicillin (AMX)-incorporating multiwalled carbon nanotubes (MWCNTs) via electrospinning, grafting and crosslinking, developing a sustainable antibiotic and flexible mesh. AMX was loaded into the hollow tubular MWCNTs by physical adsorption, and a nanofibrous structure was constructed by electrospinning PCL and SF (40:60 w/w). The AMX@MWCNTs were then chemically grafted onto the surfaces of the PCL/SF nanofibers by treating with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution for simultaneous crosslinking and coating. The incorporation of AMX into the MWCNTs (AMX@MWCNTs) and the integration of the AMX@MWCNTs with the PCL/SF nanofibers were characterized. Then, the functional mesh was fabricated and fully evaluated in terms of antibacterial activity, mechanical properties and host response. Results: Our results demonstrated that the PCL/SF nanofibrous structure was fabricated successfully by electrospinning. After integrating with AMX@MWCNT by grafting and crosslinking, the functional mesh showed undeformed structure, modified surface hydrophilicity and biocompatible interfaces, abdominal wall-matched mechanical properties, and a sustained-release antibiotic profile in E. coli growth inhibition compared to those of PCL/SF mesh in vitro. In a rat model with subcutaneous implantation, the functional mesh incited less mesh-induced inflammatory and foreign body responses than PCL/SF mesh within 14 days. The histological analysis revealed less infiltration of granulocytes and macrophages during this period, resulting in the loosely packed collagen deposition on the functional mesh and prominent collagen incorporation. Discussion: Therefore, this designed PCL/SF-AMX@MWCNT nanofibrous mesh, functionalized with antibacterial and tissue-matched mechanical properties, provides a promising alternative for the repair of abdominal wall defects.


Assuntos
Amoxicilina/química , Antibacterianos/química , Nanofibras/química , Nanotecnologia/métodos , Telas Cirúrgicas , Amoxicilina/farmacocinética , Amoxicilina/farmacologia , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroínas/química , Inflamação/etiologia , Masculino , Teste de Materiais , Camundongos , Nanotubos de Carbono/química , Poliésteres/química , Ratos Sprague-Dawley , Telas Cirúrgicas/efeitos adversos
7.
Arch Biochem Biophys ; 690: 108468, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679196

RESUMO

Hsp90 is a ubiquitous, homodimer and modular molecular chaperone. Each Hsp90 protomer has three different domains, named the N-terminal domain (NTD), middle domain (MD) and C-terminal domain (CTD). The Hsp90 molecular cycle involves ATP binding and hydrolysis, which drive conformational changes. Hsp90 is critical for the viability of eukaryotic organisms, including the protozoan that causes the severe form of malaria, Plasmodium falciparum, the growth and differentiation of which are compromised when Hsp90 is inhibited. Here, we characterize the structure of a recombinant P. falciparum Hsp90 (PfHsp90) protein, as well as its MD (PfHsp90MD) and NTD plus MD (PfHsp90NMD) constructs. All the proteins were obtained with high purity and in the folded state. PfHsp90 and PfHsp90NMD interacted with adenosine nucleotides via the NTD, and Mg2+ was critical for strong binding. PfHsp90 behaved mostly as elongated and flexible dimers in solution, which dissociate with a sub-micromolar dissociation constant. The PfHsp90MD and PfHsp90NMD constructs behaved as globular and elongated monomers, respectively, confirming the importance of the CTD for dimerization. Small angle X-ray scattering data were obtained for all the constructs, and ab initio models were constructed, revealing PfHsp90 in an open conformation and as a greatly elongated and flexible protein.


Assuntos
Proteínas de Choque Térmico HSP90/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Adenosina/química , Trifosfato de Adenosina/química , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Hidrólise , Magnésio/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica
8.
Nat Commun ; 11(1): 3128, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561732

RESUMO

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein-protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.


Assuntos
Reagentes para Ligações Cruzadas/química , Formaldeído/química , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Aminoácidos/química , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , Proteínas/metabolismo
9.
J Chromatogr A ; 1624: 461155, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32540056

RESUMO

The topic in the present paper is to prepare molecularly imprinted polymer (MIP) using the template molecule with low purity. For the first time, a surrounding of macromolecular crowding was established to promote the formation of the complex of the template with functional monomer efficiently thus highly pure template molecule was unnecessary. In this study, the MIP monolith was synthesized using low purity lactucopicrin as template in place of highly pure one, and polystyrene was used as macromolecular crowding agent. 4-Vinylpyridine and ethyleneglycol dimethacrylate were used as functional monomer and crosslinker, respectively. Polymerization parameters, including the ratio of functional monomer/template, various template concentrations, and PS concentration on the affinity of the resulting MIP were systematically investigated. For the lactucopicrin MIP made with the purity of lactucopicrin of 92%, the imprinting factor can be up to 2.2. The resulting MIP was filled in solid phase extraction (SPE) cartridge to purify lactucopicrin from the crude extract of Cichorium glandulosum Boiss. et Huet. After two cycles of MIP SPE for the crude extract, the highest recovery and purity of lactucopicrin was 64.8% and 97.8%, respectively. The results indicated that the use of macromolecular crowding agent is an effective method for improving the performance of the MIP prepared with the template of low purity, particularly valuable to the cases in which the highly pure target molecule is hard to be obtained.


Assuntos
Impressão Molecular , Polímeros/química , Asteraceae/química , Reagentes para Ligações Cruzadas/química , Lactonas/química , Lactonas/isolamento & purificação , Metacrilatos/química , Polimerização , Poliestirenos/química , Piridinas/química , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Extração em Fase Sólida
10.
Int J Nanomedicine ; 15: 3669-3680, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547021

RESUMO

Introduction: The exhaustion and poor homing of activated lymphocytes are critical obstacles in adoptive cell immunotherapy for solid tumors. In order to effectively deliver immune cells into tumors, we encapsulated interferon-α2b (IFN-α2b) into macroporous hydrogels as an enhancement factor and utilized low-dose irradiation (LDI) as a tumoral attractor of T cells. Methods: Hydroxypropyl cellulose hydrogels were prepared by irradiation techniques, and the cross-sectional microstructure was characterized by scanning electron microscopy. The synergistic antitumor mechanism of combination of IFN-α2b and CIK cells was evaluated by detecting the expression of activation marker CD69 on CIK cell surface and IFN-γ production by CIK cells. The in vivo antitumor activity of IFN-α2b-incorporated hydroxypropyl cellulose hydrogels combined with CIK and radiation was evaluated in an MKN-45 xenografted nude mice model. Results: The bioactivity of IFN-α2b was well maintained in ultraviolet-reactive, rapidly cross-linkable hydroxypropyl cellulose hydrogels. In vitro studies demonstrated IFN-α2b-activated T cells, as evidenced by upregulating early activation marker CD69 and secretion inflammatory cytokine IFN-γ. In vivo real-time image showed our hydrogels kept a higher amount of drug delivery at the tumor site for a long time compared with free drug injection. Low-dose irradiation promoted T cell accumulation and infiltration in subcutaneous tumors. Combination of IFN-α2b-loaded hydrogels (Gel-IFN) with T cells and LDI exhibited higher efficacy to eradicate human gastric cancer xenograted tumors with less proliferating cells and more necrotic regions compared with IFN-α2b or T cells alone. Discussion: HPC hydrogels kept the activity of IFN-α2b and stably release of IFN-α2b to stimulate T cells for a long time. At the same time, low-dose radiation recruits T cells into tumors. This innovative integration mode of IFN-α2b-loaded hydrogels and radiotherapy offers a potent strategy to improve the therapeutic outcome of T cell therapy.


Assuntos
Antineoplásicos/uso terapêutico , Reagentes para Ligações Cruzadas/química , Hidrogéis/química , Interferon-alfa/uso terapêutico , Luz , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/radioterapia , Linfócitos T/imunologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Celulose/análogos & derivados , Celulose/química , Relação Dose-Resposta à Radiação , Elétrons , Humanos , Interferon-alfa/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Proc Natl Acad Sci U S A ; 117(27): 15497-15503, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576692

RESUMO

Bioadhesives such as tissue adhesives, hemostatic agents, and tissue sealants have potential advantages over sutures and staples for wound closure, hemostasis, and integration of implantable devices onto wet tissues. However, existing bioadhesives display several limitations including slow adhesion formation, weak bonding, low biocompatibility, poor mechanical match with tissues, and/or lack of triggerable benign detachment. Here, we report a bioadhesive that can form instant tough adhesion on various wet dynamic tissues and can be benignly detached from the adhered tissues on demand with a biocompatible triggering solution. The adhesion of the bioadhesive relies on the removal of interfacial water from the tissue surface, followed by physical and covalent cross-linking with the tissue surface. The triggerable detachment of the bioadhesive results from the cleavage of bioadhesive's cross-links with the tissue surface by the triggering solution. After it is adhered to wet tissues, the bioadhesive becomes a tough hydrogel with mechanical compliance and stretchability comparable with those of soft tissues. We validate in vivo biocompatibility of the bioadhesive and the triggering solution in a rat model and demonstrate potential applications of the bioadhesive with triggerable benign detachment in ex vivo porcine models.


Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Ferida Cirúrgica/terapia , Adesivos Teciduais/química , Adesividade , Animais , Reagentes para Ligações Cruzadas/química , Modelos Animais de Doenças , Feminino , Teste de Materiais , Ratos , Bicarbonato de Sódio/química , Soluções , Succinimidas/química , Suínos , Técnicas de Fechamento de Ferimentos/instrumentação
12.
Nat Struct Mol Biol ; 27(7): 678-682, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514175

RESUMO

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleoside adducts offer a confident and quantitative measure of direct RNA-protein interaction. Using RBS-ID, we profiled ~2,000 human RBSs and probed Streptococcus pyogenes Cas9 to discover residues important for genome editing.


Assuntos
Biologia Molecular/métodos , RNA/química , RNA/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Proteína 9 Associada à CRISPR/metabolismo , Cromatografia Líquida , Reagentes para Ligações Cruzadas/química , Células HeLa , Humanos , Ácido Fluorídrico/química , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/metabolismo , Espectrometria de Massas em Tandem
13.
AAPS PharmSciTech ; 21(5): 173, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32548717

RESUMO

Innovative strategies for periodontal regeneration have been the focus of research clusters across the globe for decades. In order to overcome the drawbacks of currently available options, investigators have suggested a novel concept of functionally graded membrane (FGM) templates with different structural and morphological gradients. Chitosan (CH) has been used in the past for similar purpose. However, the composite formulation of composite and tetracycline when cross-linked with glutaraldehyde have received little attention. Therefore, the purpose of the study was to investigate the drug loading and release characteristics of novel freeze gelated chitosan templates at different percentages of glutaraldehyde. These were cross-linked with 0.1 and 1% glutaraldehyde and loaded with doxycycline hyclate. The electron micrographs depicted porous morphology of neat templates. After cross-linking, these templates showed compressed ultrastructures. Computerized tomography analysis showed that the templates had 88 to 92% porosity with average pore diameter decreased from 78 to 44.9 µm with increasing concentration. Fourier transform infrared spectroscopy showed alterations in the glycosidic segment of chitosan fingerprint region which after drug loading showed a dominant doxycycline spectral composite profile. Interestingly, swelling profile was not affected by cross-linking either at 0.1 and 1% glutaraldehyde and template showed a swelling ratio of 80%, which gained equilibrium after 15 min. The drug release pattern also showed a 40 µg/mL of release after 24 h. These doxycycline-loaded templates show their tendency to be used in a functionally graded membrane facing the defect site.


Assuntos
Materiais Biocompatíveis/química , Quitosana/química , Reagentes para Ligações Cruzadas/química , Congelamento , Regeneração Tecidual Guiada Periodontal/métodos , Materiais Biocompatíveis/farmacocinética , Quitosana/farmacocinética , Reagentes para Ligações Cruzadas/farmacocinética , Liberação Controlada de Fármacos , Géis , Glutaral/química , Glutaral/farmacocinética , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
14.
PLoS Comput Biol ; 16(5): e1007890, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453720

RESUMO

The biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.


Assuntos
Deformação Eritrocítica , Membrana Eritrocítica/fisiologia , Eritrócitos/citologia , Miosinas/química , Citoesqueleto de Actina/química , Reagentes para Ligações Cruzadas/química , Glicoforinas/química , Humanos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/química , Faloidina/química , Rodaminas/química , Estresse Mecânico
15.
Nat Commun ; 11(1): 2470, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424320

RESUMO

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Pele/patologia , Cicatrização , Animais , Proliferação de Células , Sobrevivência Celular , Reagentes para Ligações Cruzadas/química , Dependovirus , Edição de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hidrogéis/química , Cinética , Camundongos , Proteínas Proto-Oncogênicas c-sis , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Proc Natl Acad Sci U S A ; 117(23): 12817-12825, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444491

RESUMO

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.


Assuntos
Citoesqueleto/metabolismo , Módulo de Elasticidade , Mecanotransdução Celular , Tecidos Suporte/química , Actinina/metabolismo , Polaridade Celular , Reagentes para Ligações Cruzadas/química , Citoesqueleto/ultraestrutura , Fibroblastos/metabolismo , Humanos , Modelos Teóricos , Miosinas/metabolismo
17.
Nat Methods ; 17(6): 609-613, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424271

RESUMO

We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.


Assuntos
Hidrogéis/química , Coloração e Rotulagem/métodos , Engenharia Tecidual/métodos , Acrilamida/química , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Bioimpressão , Córtex Cerebral/química , Reagentes para Ligações Cruzadas/química , Módulo de Elasticidade , Hipocampo/química , Humanos , Teste de Materiais , Camundongos , Estresse Mecânico , Resistência à Tração
18.
Mol Cell Biol ; 40(13)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32253346

RESUMO

Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.


Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerase I/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Reagentes para Ligações Cruzadas/química , Proteínas de Ligação a DNA/química , Espectrometria de Massas , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Ativação Transcricional
19.
Food Chem ; 318: 126404, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32135426

RESUMO

The physicochemical properties of collagen casings were successfully improved by glutaraldehyde (GA) cross-linking, where the properties could be further regulated by drying temperature. Transverse direction (TD) showed a lower heat shrinkage rate than that in machine direction (MD). GA cross-linking significantly improved the mechanical properties of films under wet and boiled state. The mechanical properties of films in MD were more susceptible to wet and boiling water. The chemical composition was unchanged after GA cross-linking, but higher drying temperatures led to higher triple helix contents. The GA cross-linking mainly promoted the low temperature thermostability of collagen casings. All film samples had a rough fibrous morphology and a majority of collagen fibers was oriented under the lower drying temperature (55 â„ƒ). These results reported in this study can be used to better guide the preparation of collagen casings.


Assuntos
Colágeno/química , Reagentes para Ligações Cruzadas/química , Glutaral/química , Animais , Dessecação , Módulo de Elasticidade , Ligação de Hidrogênio , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Temperatura
20.
Nature ; 579(7800): 603-608, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132710

RESUMO

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption1. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.


Assuntos
Acetaldeído/química , Reagentes para Ligações Cruzadas/química , Dano ao DNA , Reparo do DNA , Replicação do DNA/fisiologia , DNA/química , Etanol/química , Anemia de Fanconi/metabolismo , Animais , Cisplatino/química , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/farmacologia , Mutagênese/efeitos dos fármacos , Nucleotidiltransferases/metabolismo , Mutação Puntual/efeitos dos fármacos , Mutação Puntual/genética , Xenopus , Proteínas de Xenopus/metabolismo
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