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1.
Anal Chem ; 92(16): 11297-11304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32683857

RESUMO

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.


Assuntos
Microscopia de Força Atômica/métodos , Nanoestruturas/química , Análise Espectral Raman/métodos , Vírion/química , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Capsídeo/química , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Análise Discriminante , Herpesvirus Humano 1/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Análise dos Mínimos Quadrados , Levivirus/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Células Vero
2.
PLoS One ; 15(7): e0227395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628681

RESUMO

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.


Assuntos
Aderência Bacteriana , Lactococcus lactis/fisiologia , Microscopia de Força Atômica/métodos , Paracoccus/fisiologia , Células Imobilizadas/fisiologia , Vidro/química , Indóis/química , Polímeros/química , Água do Mar/microbiologia , Análise de Célula Única , Propriedades de Superfície , Titânio/química
3.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
4.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510478

RESUMO

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.


Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Matriz Extracelular/patologia , Microscopia de Força Atômica/métodos , Osteoartrite/patologia , Elasticidade , Humanos
5.
AAPS PharmSciTech ; 21(5): 142, 2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32419061

RESUMO

Mucoadhesion-based drug delivery systems have recently gained interest because of their bio-adhesion capability, which results in enhanced residence time leading to prolonged duration of action with the mucosal surface, potentially improving compliance and convenience. Mucoadhesion testing of these formulations is widely reported; however, this is technically challenging due to the absence of any standard methods and difficulty in conducting mucoadhesion, formulation-mucosal surface interaction, mucosal surface topography and drug release in a single experiment. As these measurements are currently conducted separately, on replicate formulations, results can often be subjective and difficult to correlate. Hence, the aim of the present study was to develop a new AFM-based single-entity ex vivo muco-dissolution (MUCO-DIS) technique to simultaneously evaluate mucoadhesion force, 3D surface topography, polymer dissolution and drug release characteristics. To demonstrate the potential of the current technique, the interactions between model pectin microparticles containing metformin HCl and a range of gastrointestinal mucosal surfaces (gastric, small intestine, large intestine and buccal) were studied. This novel system has not only successfully determined the mucoadhesion force, polymer dissolution and drug release information but has also highlighted the difference in microparticle performance with different mucosal targets. The current work has highlighted the potential of this newly developed MUCO-DIS system and we believe this will be a valuable tool for characterising these popular pharmaceutical formulations. This technique could also provide an opportunity to other scientific fields to evaluate materials, substrate behaviour and their interactions in their hydrated state at nanoscale with real-time chemical and surface mapping.


Assuntos
Absorção Intestinal , Microscopia de Força Atômica/métodos , Membrana Mucosa , Nanotecnologia/métodos , Adesividade , Animais , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Excipientes , Técnicas In Vitro , Metformina/administração & dosagem , Metformina/química , Nanopartículas , Solubilidade , Suínos
6.
Nat Protoc ; 15(6): 2107-2139, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32451442

RESUMO

Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3-4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.


Assuntos
Hidrodinâmica , Microscopia de Força Atômica/métodos , RNA Longo não Codificante/química , Processamento de Imagem Assistida por Computador
7.
Nat Commun ; 11(1): 2185, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366822

RESUMO

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.


Assuntos
DNA/química , Fluorescência , Fractais , Oligonucleotídeos/química , Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Nanoestruturas/química
8.
AAPS PharmSciTech ; 21(4): 125, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350635

RESUMO

Sunlight is important to health, but higher exposure to radiation causes early aging of the skin and skin damage that can lead to skin cancers. This study aimed at producing a stable octyl p-methoxycinnamate (OMC)-loaded nanostructured lipid carrier (NLC) sunscreen, which can help in the photoprotective effect. NLC was produced by emulsification-sonication method and these systems were composed of myristyl myristate (MM), caprylic capric triglyceride (CCT), Tween® 80 (TW), and soybean phosphatidylcholine (SP) and characterized by dynamic light scattering (DLS), zeta potential (ZP) measurement, atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and in vitro release studies. Pre-formulation studies were performed changing TW concentrations and no differences were found at concentrations of 1.0 and 2.0%. Two selected formulations were designed and showed an average size of 91.5-131.7, polydispersity index > 0.2, and a negative value of ZP. AFM presented a sphere-like morphology and SEM showed ability to form a thin film. DSC exhibited that the incorporation of OMC promoted reduction of enthalpy due to formation of a more amorphous structure. Drug release shows up to 55.74% and 30.57%, and this difference could be related to the presence of SP in this formulation that promoted a more amorphous structure; the release mechanism study indicated Fickian diffusion and relaxation. Sun protection factor (SPF) evaluation was performed using NLC and presented values around 40, considerably higher than those observed in the literature. The developed formulations provide a beneficial alternative to conventional sunscreen formulations.


Assuntos
Cinamatos/síntese química , Portadores de Fármacos/síntese química , Lipídeos/síntese química , Nanoestruturas/química , Fator de Proteção Solar/métodos , Protetores Solares/síntese química , Varredura Diferencial de Calorimetria/métodos , Cinamatos/farmacocinética , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Lipídeos/farmacocinética , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Varredura/métodos , Tamanho da Partícula , Protetores Solares/farmacocinética
9.
J Vis Exp ; (157)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32225151

RESUMO

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.


Assuntos
Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Propriedades de Superfície
10.
Phys Rev Lett ; 124(11): 118102, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32242730

RESUMO

The fine interplay between the simultaneous stretching and confinement of amyloid fibrils is probed by combining a microcapillary setup with atomic force microscopy. Single-molecule statistics reveal how the stretching of fibrils changed from force to confinement dominated at different length scales. System order, however, is solely ruled by confinement. Coarse-grained simulations support the results and display the potential to tailor system properties by tuning the two effects. These findings may further help shed light on in vivo amyloid fibril growth and transport in highly confined environments such as blood vessels.


Assuntos
Amiloide/química , Modelos Químicos , Amiloide/metabolismo , Simulação por Computador , Microscopia de Força Atômica/métodos
11.
Proc Natl Acad Sci U S A ; 117(17): 9318-9328, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32273391

RESUMO

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.


Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças Ópticas
12.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098817

RESUMO

The bacterial cell envelope is essential for viability, the environmental gatekeeper and first line of defense against external stresses. For most bacteria, the envelope biosynthesis is also the site of action of some of the most important groups of antibiotics. It is a complex, often multicomponent structure, able to withstand the internally generated turgor pressure. Thus, elucidating the architecture and dynamics of the cell envelope is important, to unravel not only the complexities of cell morphology and maintenance of integrity but also how interventions such as antibiotics lead to death. To address these questions requires the capacity to visualize the cell envelope in situ via high-spatial resolution approaches. In recent years, atomic force microscopy (AFM) has brought novel molecular insights into the assembly, dynamics, and functions of bacterial cell envelopes. The ultrafine resolution and physical sensitivity of the technique have revealed a wealth of ultrastructural features that are invisible to traditional optical microscopy techniques or imperceptible in their true physiological state by electron microscopy. Here, we discuss recent progress in our use of AFM imaging for understanding the architecture and dynamics of the bacterial envelope. We survey recent studies that demonstrate the power of the technique to observe isolated membranes and live cells at (sub)nanometer resolution and under physiological conditions and to track in vitro structural dynamics in response to growth or to drugs.


Assuntos
Bactérias/ultraestrutura , Membrana Celular/ultraestrutura , Parede Celular/ultraestrutura , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Proteínas de Membrana , Microscopia de Força Atômica/métodos
13.
J Phys Chem Lett ; 11(5): 1697-1701, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32039604

RESUMO

Here we demonstrate sub-10 nm spatial resolution sampling of a volume of ∼360 molecules with a strong field enhancement at the sample-tip junction by implementing noble metal substrates (Au, Ag, Pt) in photoinduced force microscopy (PiFM). This technique shows the versatility and robustness of PiFM and is promising for application in interfacial studies with hypersensitivity and super spatial resolution.


Assuntos
Metais/química , Microscopia de Força Atômica/métodos , Animais , Bovinos , Ouro/química , Platina/química , Soroalbumina Bovina/química , Dióxido de Silício/química , Prata/química
14.
Micron ; 131: 102827, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31951938

RESUMO

One of the main obstacles to studying the surface ultrastructure of microbial cells by atomic force microscopy (AFM) is determining how to immobilize live cells on the AFM substrates. Each method has its own advantages and disadvantages. The aim of this study was to characterize a new simple and inexpensive method using two types of polyethersulfone (PES) membrane filters that differ in pore size (micropore and nanopore) to immobilize live and dead Brevibacillus laterosporus for AFM imaging. B. laterosporus was easily trapped by the microporous PES membrane, facilitating the successful AFM scanning of the bacterial surface ultrastructure. In addition, B. laterosporus strongly attached to the nanoporous membranes and withstood the pulling forces exerted by the AFM tip during scanning. These methods of immobilization did not affect the cell viability. The nanostructure and roughness of the bacterial surface were also observed for live, fixed, and air-dried cells. Live and dead bacteria displayed similar morphologies at low resolution, while at high resolution, live bacteria displayed a more convoluted surface ("brain-like structure").


Assuntos
Brevibacillus/ultraestrutura , Microscopia de Força Atômica/métodos
15.
Biochim Biophys Acta Biomembr ; 1862(5): 183188, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31930963

RESUMO

Langmuir films prepared from bovine erythrocyte membranes (LFBEM) were studied and transferred to alkylated glasses (Langmuir-Blodgett films, LBBEM) in order to assess the effects of membrane molecular packing on Bovine Erythrocyte Acetylcholinesterase (BEA) catalytic activity. Surface pressure (π) vs Area isotherms showed three 2D-transitions at ~7, ~18 and ~44 mN/m and a collapse pressure at πc = 49 mN/m. The 0-12-0 mN/m compression-decompression cycles resulted reversible while those 0-40-0 mN/m exhibited a significant hysteresis. Taken together, EFM, BAM and AFM images and the stability of the film after 3C-D cycles, we can suggest that over the air-water interface as well as over the silanized glass substrate the surface is mostly covered by a monolayer with a few particles dispersed. Acetylthiocholine hydrolysis was assayed with BEA in bovine erythrocyte membrane suspensions (SBEM) and in LBBEM packed at 10 (LBBEM,10) and 35 mN/m (LBBEM,35), which gave the following kinetic parameters: Vmax = 3.41 ± 0.15, 0.021 ± 0.002 and 0.030 ± 0.003 nmol.min-1·µg prot-1 and KM = 0.11 ± 0.02, 0.047 ± 0.017 and 0.026 ± 0.017 mM, respectively. Although from SBEM to LBBEM we lost active enzyme, the catalytic efficiency (Vmax/KM) increased ~750 times. Eugenol and 1,8-cineol inhibited BEA catalytic activity in LBBEM,35. Our results demonstrate the transmission of information between the membrane and the environment within the subphase immediately below the membrane, where anchored proteins are hosted. This was reflected by the membrane packing-induced modulation of BEA catalytic activity. Furthermore, LBBEM provides a proof of concept for the development of biosensors to screen new green pesticides acting through BEA interaction.


Assuntos
Acetilcolinesterase/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/fisiologia , Adsorção/fisiologia , Animais , Catálise , Bovinos , Membrana Eritrocítica/fisiologia , Hidrólise , Cinética , Microscopia de Força Atômica/métodos , Estudo de Prova de Conceito , Propriedades de Superfície , Água/química
16.
Biochim Biophys Acta Biomembr ; 1862(5): 183198, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958436

RESUMO

Binding of integrin alphaIIbbeta3 (αiibß3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibß3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibß3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210-450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibß3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions.


Assuntos
Estresse Nitrosativo/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Integrinas/química , Integrinas/metabolismo , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica/métodos , Nitratos/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oligopeptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Ligação Proteica , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/metabolismo
17.
Chemphyschem ; 21(3): 188-193, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31912640

RESUMO

Tip-enhanced Raman spectroscopy (TERS) is a promising technique for structural studies of biological systems and biomolecules, owing to its ability to provide a chemical fingerprint with sub-diffraction-limit spatial resolution. This application of TERS has thus far been limited, due to difficulties in generating high field enhancements while maintaining biocompatibility. The high sensitivity achievable through TERS arises from the excitation of a localized surface plasmon resonance in a noble metal atomic force microscope (AFM) tip, which in combination with a metallic surface can produce huge enhancements in the local optical field. However, metals have poor biocompatibility, potentially introducing difficulties in characterizing native structure and conformation in biomolecules, whereas biocompatible surfaces have weak optical field enhancements. Herein, a novel, biocompatible, highly enhancing surface is designed and fabricated based on few-monolayer mica flakes, mechanically exfoliated on a metal surface. These surfaces allow the formation of coupled plasmon enhancements for TERS imaging, while maintaining the biocompatibility and atomic flatness of the mica surface for high resolution AFM. The capability of these substrates for TERS is confirmed numerically and experimentally. We demonstrate up to five orders of magnitude improvement in TERS signals over conventional mica surfaces, expanding the sensitivity of TERS to a wide range of non-resonant biomolecules with weak Raman cross-sections. The increase in sensitivity obtained through this approach also enables the collection of nanoscale spectra with short integration times, improving hyperspectral mapping for these applications. These mica/metal surfaces therefore have the potential to revolutionize spectromicroscopy of complex, heterogeneous biological systems such as DNA and protein complexes.


Assuntos
Silicatos de Alumínio/química , Materiais Biocompatíveis/química , Ouro/química , Microscopia de Força Atômica/instrumentação , DNA/análise , Microscopia de Força Atômica/métodos , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície/métodos
18.
BMB Rep ; 53(2): 74-81, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31964473

RESUMO

Under physiological and pathological conditions, mechanical forces generated from cells themselves or transmitted from extracellular matrix (ECM) through focal adhesions (FAs) and adherens junctions (AJs) are known to play a significant role in regulating various cell behaviors. Substantial progresses have been made in the field of mechanobiology towards novel methods to understand how cells are able to sense and adapt to these mechanical forces over the years. To address these issues, this review will discuss recent advancements of traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) to measure multiple aspects of cellular forces exerted by cells at cell-ECM and cell-cell junctional intracellular interfaces. We will also highlight how these methods can elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in regulating various cellular functions. [BMB Reports 2020; 53(2): 74-81].


Assuntos
Matriz Extracelular/fisiologia , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodos , Biopolímeros , Adesão Celular/fisiologia , Matriz Extracelular/química , Adesões Focais/química , Adesões Focais/fisiologia , Hidrogéis , Junções Intercelulares/química , Junções Intercelulares/fisiologia , Estresse Mecânico , Tração
19.
Micron ; 130: 102814, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31931325

RESUMO

Undersampling is a simple but efficient way to increase the imaging rate of atomic force microscopy (AFM). One major challenge in this approach is that of accurate image reconstruction from a limited number of measurements. In this work, we present a deep neural network (DNN) approach to reconstruct µ-path sub-sampled AFM images. Our network consists of two sub-networks, namely a RED-net and a U-net, in series, and is trained end-to-end from random images masked according to µ-path sub-sampling patterns. Using both simulation and experiments, the DNN is shown to yield better image quality than three existing optimization-based methods for reconstruction: basis pursuit, a variant of total variation minimization, and inpainting.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Força Atômica/métodos , Redes Neurais de Computação , Algoritmos
20.
Nucleic Acids Res ; 48(3): e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31840169

RESUMO

Ionizing radiation produces clustered DNA damage that contains two or more lesions in 10-20 bp. It is believed that the complexity of clustered damage (i.e., the number of lesions per damage site) is related to the biological severity of ionizing radiation. However, only simple clustered damage containing two vicinal lesions has been demonstrated experimentally. Here we developed a novel method to analyze the complexity of clustered DNA damage. Plasmid DNA was irradiated with densely and sparsely ionizing Fe-ion beams and X-rays, respectively. Then, the resulting DNA lesions were labeled with biotin/streptavidin and observed with atomic force microscopy. Fe-ion beams produced complex clustered damage containing 2-4 lesions. Furthermore, they generated two or three clustered damage sites in a single plasmid molecule that resulted from the hit of a single track of Fe-ion beams. Conversely, X-rays produced relatively simple clustered damage. The present results provide the first experimental evidence for complex cluster damage.


Assuntos
Dano ao DNA , Microscopia de Força Atômica/métodos , DNA/efeitos da radiação , DNA/ultraestrutura , Ferro , Raios X
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