RESUMO
Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are ß-sandwiches with a predominant ß-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross ß-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.
Assuntos
Conectina , Microscopia de Força Atômica , Músculo Esquelético , Agregados Proteicos , Conectina/química , Conectina/metabolismo , Conectina/genética , Coelhos , Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X , BenzotiazóisRESUMO
Extracellular matrix (ECM) proteins provide anchorage and structural strength to cells and tissues in the body and, thus, are fundamental molecular components for processes of cell proliferation, growth, and function. Atomic force microscopy (AFM) has increasingly become a valuable approach for studying biological molecules such as ECM proteins at the level of individual molecules. Operational modes of AFM can be used to acquire the measurements of the physical, electronic, and mechanical properties of samples, as well as for viewing the intricate details of the surface chemistry of samples. Investigations of the morphology and properties of biomolecules at the nanoscale can be useful for understanding the interactions between ECM proteins and biological molecules such as cells, DNA, and other proteins. Methods for preparing protein samples for AFM studies require only basic steps, such as the immersion of a substrate in a dilute solution or protein, or the deposition of liquid droplets of protein suspensions on a flat, clean surface. Protocols of nanolithography have been used to define the arrangement of proteins for AFM studies. Using AFM, mechanical and force measurements with tips that are coated with ECM proteins can be captured in ambient or aqueous environments. In this review, representative examples of AFM studies are described for molecular-level investigations of the structure, surface assembly, protein-cell interactions, and mechanical properties of ECM proteins (collagen, elastin, fibronectin, and laminin). Methods used for sample preparation as well as characterization with modes of AFM will be discussed.
Assuntos
Proteínas da Matriz Extracelular , Microscopia de Força Atômica , Microscopia de Força Atômica/métodos , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Humanos , AnimaisRESUMO
BACKGROUND: Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model. METHODS: Eroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics. RESULTS: After pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA). CONCLUSION: This study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions.
Assuntos
Amelogenina , Esmalte Dentário , Microscopia Eletrônica de Varredura , Erosão Dentária , Remineralização Dentária , Difração de Raios X , Humanos , Remineralização Dentária/métodos , Esmalte Dentário/efeitos dos fármacos , Erosão Dentária/patologia , Concentração de Íons de Hidrogênio , Amelogenina/uso terapêutico , Amelogenina/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Microscopia de Força Atômica , Fosfatos de Cálcio/farmacologia , Propriedades de Superfície , Dente Pré-Molar , CristalizaçãoRESUMO
Staphylococcus aureus infections are the primary causes of morbidity, and mortality, particularly in immuno-compromised individuals. S. aureus associated infections are acquired from community, as well as hospital settings, and difficult to treat because of the emerging resistance against available antibiotics. One of the key factors of its resistance is the biofilm formation, which can be targeted to treat S. aureus-induced infections. Currently, there is no drug available that function by targeting the biofilm. This unmet need demands the discovery of drug candidates against S. aureus biofilm. The present study was designed to evaluate coumarin derivatives 1-21 against S. aureus biofilm. The 96-well plate crystal violet assay was employed for the quantification of biofilm. Results showed that the coumarin derivatives 2-4, 10, and 17 possess potent antibiofilm activity, with MBIC values between 25-100 µg/mL. The results were further confirmed through atomic force microscopy (AFM), scanning electron (SEM), and fluorescence microscopic studies. The quantitative RT-PCR analysis revealed the downregulation of biofilm associated genes, icaA and icaD. These coumarin derivatives were also found to be non-cytotoxic to fibroblasts. This study, therefore, identifies the antibiofilm potential of coumarin derivatives that will pave the way for further research on these derivatives.
Assuntos
Antibacterianos , Biofilmes , Cumarínicos , Testes de Sensibilidade Microbiana , Staphylococcus aureus , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cumarínicos/farmacologia , Cumarínicos/química , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Microscopia de Força AtômicaRESUMO
HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.
Assuntos
Elasticidade , Infecções por HIV , HIV-1 , HIV-1/fisiologia , Humanos , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Internalização do Vírus , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Simulação de Dinâmica Molecular , Microscopia de Força Atômica , Poro Nuclear/metabolismo , Indóis , Fenilalanina/análogos & derivadosRESUMO
We isolated a stress-tolerance-related gene from a genome library of Synechococcus sp. NKBG15041c. The expression of the gene in E. coli confers resistance against various stresses. The gene encodes a MoxR AAA+ ATPase, which was designated SyMRP since it belongs to the MRP subfamily. The recombinant SyMRP showed weak ATPase activity and protected citrate synthase from thermal aggregation. Interestingly, the chaperone activity of SyMRP is ATP-dependent. SyMRP exists as a stable hexamer, and ATP-dependent conformation changes were not detected via analytical ultracentrifugation (AUC) or small-angle X-ray scattering (SAXS). Although the hexameric structure predicted by AlphaFold 3 was the canonical flat-ring structure, the structures observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) were not the canonical ring structure. In addition, the experimental SAXS profiles did not show a peak that should exist in the symmetric-ring structure. Therefore, SyMRP seems to form a hexameric structure different from the canonical hexameric structure of AAA+ ATPase.
Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Synechococcus , Synechococcus/enzimologia , Synechococcus/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Espalhamento a Baixo Ângulo , Difração de Raios X , Microscopia de Força Atômica , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Atomic force microscopy (AFM) imaging enables the visualization of protein molecules with high resolution, providing insights into their shape, size, and surface topography. Here, we use AFM to study the aggregation process of protein S100A9 in physiological conditions, in the presence of calcium at a molar ratio 4Ca2+:S100A9. We find that S100A9 readily assembles into a worm-like fibril, with a period dimension along the fibril axis of 11.5 nm. The fibril's chain length extends up to 136 periods after an incubation time of 144 h. At room temperature, the fibril's bending stiffness was found to be 2.95×10-28 Nm2, indicating that the fibrils are relatively flexible. Additionally, the values obtained for the Young's modulus (Ex=6.96×105 Pa and Ey=3.37×105 Pa) are four orders of magnitude lower than those typically reported for canonical amyloid fibrils. Our findings suggest that, under the investigated conditions, a distinct aggregation mechanism may be in place in the presence of calcium. Therefore, the findings reported here could have implications for the field of biomedicine, particularly with regard to Alzheimer's disease.
Assuntos
Amiloide , Cálcio , Calgranulina B , Microscopia de Força Atômica , Microscopia de Força Atômica/métodos , Amiloide/química , Amiloide/ultraestrutura , Calgranulina B/química , Calgranulina B/metabolismo , Cálcio/metabolismo , Cálcio/química , Módulo de Elasticidade , Humanos , Agregados ProteicosRESUMO
Precise monitoring of biomolecular radiation damage is crucial for understanding X-ray-induced cell injury and improving the accuracy of clinical radiotherapy. We present the design and performance of lanthanide-DNA-origami nanodosimeters for directly visualizing radiation damage at the single-particle level. Lanthanide ions (Tb3+ or Eu3+) coordinated with DNA origami nanosensors enhance the sensitivity of X-ray irradiation. Atomic force microscopy (AFM) revealed morphological changes in Eu3+-sensitized DNA origami upon X-ray irradiation, indicating damage caused by ionization-generated electrons and free radicals. We further demonstrated the practical applicability of Eu3+-DNA-origami integrated chips in precisely monitoring radiation-mediated cancer radiotherapy. Quantitative results showed consistent trends with flow cytometry and histological examination under comparable X-ray irradiation doses, providing an affordable and user-friendly visualization tool for preclinical applications. These findings provide new insights into the impact of heavy metals on radiation-induced biomolecular damage and pave the way for future research in developing nanoscale radiation sensors for precise clinical radiography.
Assuntos
DNA , Elementos da Série dos Lantanídeos , Microscopia de Força Atômica , DNA/química , DNA/análise , Humanos , Elementos da Série dos Lantanídeos/química , Raios X , Dano ao DNA , Európio/químicaRESUMO
The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.
Assuntos
Colesterol , Proteínas do Envelope de Coronavírus , Microscopia de Força Atômica , SARS-CoV-2 , Colesterol/metabolismo , Colesterol/química , SARS-CoV-2/metabolismo , Humanos , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Envelope de Coronavírus/química , Membrana Celular/metabolismo , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , COVID-19/metabolismo , COVID-19/virologia , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Proteínas Viroporinas/metabolismo , Técnicas de Patch-Clamp , Multimerização Proteica , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/químicaRESUMO
The biodegradation of therapeutic magnetic-oxide nanoparticles (MONPs) in the human body raises concerns about their lifespan, functionality, and health risks. Interactions between apoferritin proteins and MONPs in the spleen, liver, and inflammatory macrophages significantly accelerate nanoparticle degradation, releasing metal ions taken up by apoferritin. This can alter the protein's biological structure and properties, potentially causing health hazards. This study examines changes in apoferritin's shape, electrical surface potential (ESP), and protein-core composition after incubation with cobalt-ferrite (CoFe2O4) oxide nanoparticles. Using atomic force microscopy (AFM) and scanning Kelvin probe force microscopy (SKPFM), we observed changes in the topography and ESP distribution in apoferritin nanofilms over time. After 48 h, the characteristic apoferritin hole (â¼1.35 nm) vanished, and the protein's height increased from â¼3.5 to â¼7.5 nm due to hole filling. This resulted in a significant ESP increase on the filled-apoferritin surface, attributed to the formation of a heterogeneous chemical composition and crystal structure (γ-Fe2O3, Fe3O4, CoO, CoOOH, FeOOH, and Co3O4). These changes enhance electrostatic interactions and surface charge between the protein and the AFM tip. This approach aids in predicting and improving the MONP lifespan while reducing their toxicity and preventing apoferritin deformation and dysfunction.
Assuntos
Apoferritinas , Cobalto , Apoferritinas/química , Apoferritinas/metabolismo , Cobalto/química , Nanopartículas de Magnetita/química , Microscopia de Força Atômica , Compostos Férricos/química , Compostos Férricos/farmacologia , Humanos , Óxidos/químicaRESUMO
Radiotherapy is one of the most common approaches for cancer treatment, especially in the case of peripheral nervous system tumors. As it requires exposure to high doses of ionizing radiation, it is important to look for substances that support efficient reduction of the tumor volume with simultaneous prevention of the surrounding noncancerous cells. Cannabidiol (CBD), which exhibits both anticancer and neuroprotective properties, was applied as a potential modulator of radiological response; however, its influence on cells undergoing irradiation remains elusive. Here, we have applied high-resolution optical spectroscopy techniques to capture biomolecules associated with CBD shielding of normal and damaging cancerous cells upon X-ray exposure. Conventional Raman (RS) and Fourier transformed infrared (FT-IR) spectroscopies provided semiquantitative information mainly about changes in the concentration of total lipids, DNA, cholesteryl esters, and phospholipids in cells. A through assessment of the single cells by atomic force microscopy coupled with infrared spectroscopy (AFM-IR) allowed us to determine not only the alterations in DNA content but also in its conformation due to cell treatment. Pronounced nanoscale changes in cholesteryl ester metabolites, associated with CBD treatment and radiation, were also observed. AFM-IR chemoselective maps of the single cells indicate the modified distribution of cholesteryl esters with 40 nm spatial resolution. Based on the obtained results, we propose a label-free and fast analytical method engaging optical spectroscopy to assess the mechanism of normal and cancerous cell susceptibility to ionizing radiation when pretreated with CBD.
Assuntos
Canabidiol , DNA , Análise Espectral Raman , Humanos , Análise Espectral Raman/métodos , Canabidiol/química , Canabidiol/análise , DNA/química , DNA/efeitos da radiação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Ésteres do Colesterol/química , Ésteres do Colesterol/análise , Microscopia de Força Atômica , Fosfolipídeos/químicaRESUMO
This study evaluated push-out bond test (POBT), surface roughness, and antimicrobial properties against Enterococcus faecalis of bioceramic sealers supplemented with silver nanoparticles (AgNPs). The sealers tested were CeraSeal®, EndoSequence® BC SealerTM, and Bio-C® Sealer. The POBT was measured with a Universal Testing Machine, and the type of failure was evaluated with a stereomicroscope. The roughness average (Sa) and peak-valley height (Sy) values were evaluated by atomic force microscopy. The bacterial growth inhibition was evaluated using a disk diffusion test, and antimicrobial activity was determined with the plate microdilution method. The POBT showed no significant difference between sealers with and those without NPs in cervical and apical thirds (p > 0.05). In the middle third, the adhesion force was significant for Endosequence BC Sealer® (p < 0.05). The results showed that the Sa and Sy parameters, when AgNPs were added, did not show a statistically significant difference compared to the groups without nanoparticles (p > 0.05). All tested sealers showed bacterial growth inhibition, but no significant difference was found. Their efficacy, in descending order of antibacterial activity when AgNPs were added, is as follows: EndoSequence® BC SealerTM > Bio-C® Sealer > CeraSeal®. The incorporation of AgNPs into bioceramics improves antimicrobial activity without affecting mechanical properties.
Assuntos
Enterococcus faecalis , Nanopartículas Metálicas , Materiais Restauradores do Canal Radicular , Prata , Propriedades de Superfície , Prata/química , Prata/farmacologia , Nanopartículas Metálicas/química , Materiais Restauradores do Canal Radicular/química , Materiais Restauradores do Canal Radicular/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/química , Teste de Materiais , Humanos , Testes de Sensibilidade Microbiana , Cerâmica/química , Cerâmica/farmacologia , Microscopia de Força Atômica , Fosfatos de Cálcio , Combinação de Medicamentos , Óxidos , SilicatosRESUMO
Poly(N-isopropylacrylamide) (PNIPAM) offers a promising platform for non-invasive and gentle cell detachment. However, conventional PNIPAM-based substrates often suffer from limitations including limited stability and reduced reusability, which hinder their widespread adoption in biomedical applications. In this study, PNIPAM copolymer films were formed on the surfaces of glass slides or silicon wafers using a two-step film-forming method involving coating and grafting. Subsequently, a comprehensive analysis of the films' surface wettability, topography, and thickness was conducted using a variety of techniques, including contact angle analysis, atomic force microscopy (AFM), and ellipsometric measurements. Bone marrow mesenchymal stem cells (BMMSCs) were then seeded onto PNIPAM copolymer films prepared from different copolymer solution concentrations, ranging from 0.2 to 10 mg·mL-1, to select the optimal culture substrate that allowed for good cell growth at 37 °C and effective cell detachment through temperature reduction. Furthermore, the stability and reusability of the optimal copolymer films were assessed. Finally, AFM and X-ray photoelectron spectroscopy (XPS) were employed to examine the surface morphology and elemental composition of the copolymer films after two rounds of BMMSC adhesion and detachment. The findings revealed that the surface properties and overall characteristics of PNIPAM copolymer films varied significantly with the solution concentration. Based on the selection criteria, the copolymer films derived from 1 mg·mL-1 solution were identified as the optimal culture substrates for BMMSCs. After two rounds of cellular adhesion and detachment, some proteins remained on the film surfaces, acting as a foundation for subsequent cellular re-adhesion and growth, thereby implicitly corroborating the practicability and reusability of the copolymer films. This study not only introduces a stable and efficient platform for stem cell culture and harvesting but also represents a significant advance in the fabrication of smart materials tailored for biomedical applications.
Assuntos
Resinas Acrílicas , Adesão Celular , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Resinas Acrílicas/química , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Propriedades de Superfície , Proliferação de Células/efeitos dos fármacos , Temperatura , Animais , Microscopia de Força Atômica , Células Cultivadas , Células da Medula Óssea/citologiaRESUMO
The nucleosome is one of the hallmarks of eukaryotes, a dynamic platform that supports many critical functions in eukaryotic cells. Here, we engineer the in vivo assembly of the nucleosome core in the model bacterium Escherichia coli. We show that bacterial chromosome DNA and eukaryotic histones can assemble in vivo to form nucleosome complexes with many features resembling those found in eukaryotes. The formation of nucleosomes in E. coli was visualized with atomic force microscopy and using tripartite split green fluorescent protein. Under a condition that moderate histones expression was induced at 1 µM IPTG, the nucleosome-forming bacterium is viable and has sustained growth for at least 110 divisions in longer-term growth experiments. It exhibits stable nucleosome formation, a consistent transcriptome across passages, and reduced growth fitness under stress conditions. In particular, the nucleosome arrays in E. coli genic regions have profiles resembling those in eukaryotic cells. The observed compatibility between the eukaryotic nucleosome and the bacterial chromosome machinery may reflect a prerequisite for bacteria-archaea union, providing insight into eukaryogenesis and the origin of the nucleosome.
Assuntos
Escherichia coli , Histonas , Microscopia de Força Atômica , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Escherichia coli/metabolismo , Escherichia coli/genética , Histonas/metabolismo , Histonas/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Células Eucarióticas/metabolismoRESUMO
Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.
Assuntos
Aprendizado Profundo , Microscopia de Força Atômica , Análise de Célula Única , Microscopia de Força Atômica/métodos , Análise de Célula Única/métodos , Humanos , Nanotecnologia/métodos , Ensaios de Triagem em Larga Escala/métodosRESUMO
Antimicrobial potential of bioactive glass (BAG) makes it promising for implant applications, specifically overcoming the toxicity concerns associated with traditional antibacterial nanoparticles. The 58S composition of BAG (with high Ca and absence of Na) has been known to exhibit excellent bioactivity and antibacterial behaviour, but the mechanisms behind have not been investigated in detail. In this pioneering study, we are using Atomic Force Microscopy (AFM) to gain insights into 58S BAG's adhesive interactions with planktonic cells of both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria; along with the impact of crystallinity on antibacterial properties. We have recorded greater bacterial inhibition by amorphous BAG compared to semi-crystalline glass-ceramics and stronger effect against gram-negative bacteria via conventional long-term antibacterial tests. AFM force distance curves has illustrated substantial bonding between bacteria and BAG within the initial one second (observed at a gap of 250 ms) of contact, with multiple binding events. Further, stronger adhesion of BAG with E.coli (~ 6 nN) compared to S. aureus (~ 3 nN) has been found which can be attributed to more adhesive nano-domains (size effect) distributed uniformly on E.coli surface. This study has revealed direct evidence of impact of contact time and 58S BAG's crystalline phase on bacterial adhesion and antimicrobial behaviour. Current study has successfully demonstrated the mode and mechanisms of initial bacterial adhesion with 58S BAG. The outcome can pave the way towards improving the designing of implant surfaces for a range of biomedical applications.
Assuntos
Antibacterianos , Aderência Bacteriana , Cerâmica , Escherichia coli , Vidro , Microscopia de Força Atômica , Staphylococcus aureus , Microscopia de Força Atômica/métodos , Cerâmica/química , Aderência Bacteriana/efeitos dos fármacos , Vidro/química , Staphylococcus aureus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
Ultraviolet-C (UV-C) radiation and ozone gas are potential mechanisms employed to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), each exhibiting distinct molecular-level modalities of action. To elucidate these disparities and deepen our understanding, we delve into the intricacies of SARS-CoV-2 inactivation via UV-C and ozone gas treatments, exploring their distinct molecular-level impacts utilizing a suite of advanced techniques, including biological atomic force microscopy (Bio-AFM) and single virus force spectroscopy (SVFS). Whereas UV-C exhibited no perceivable alterations in virus size or surface topography, ozone gas treatment elucidated pronounced changes in both parameters, intensifying with prolonged exposure. Furthermore, a nuanced difference was observed in virus-host cell binding post-treatment: ozone gas distinctly reduced SARS-CoV-2 binding to host cells, while UV-C maintained the status quo. The results derived from these methodical explorations underscore the pivotal role of advanced Bio-AFM techniques and SVFS in enhancing our understanding of virus inactivation mechanisms, offering invaluable insights for future research and applications in viral contamination mitigation.
Assuntos
COVID-19 , Microscopia de Força Atômica , Ozônio , SARS-CoV-2 , Raios Ultravioleta , Inativação de Vírus , Ozônio/química , Ozônio/farmacologia , SARS-CoV-2/efeitos dos fármacos , Humanos , Inativação de Vírus/efeitos dos fármacos , Inativação de Vírus/efeitos da radiação , Células Vero , Esterilização/métodos , Chlorocebus aethiops , Animais , Gases em Plasma/química , Gases em Plasma/farmacologiaRESUMO
The review considers the possibility of using atomic force microscopy (AFM) as a basic method for protein detection in solutions with low protein concentrations. The demand for new bioanalytical approaches is determined by the problem of insufficient sensitivity of systems used in routine practice for protein detection. Special attention is paid to demonstration of the use in bioanalysis of a combination of AFM and fishing methods as an approach of concentrating biomolecules from a large volume of the analyzed solution on a small surface area.
Assuntos
Microscopia de Força Atômica , Proteínas , Microscopia de Força Atômica/métodos , Proteínas/análise , Proteínas/química , Humanos , Animais , SoluçõesRESUMO
Photobiomodulation therapy (PBMT) is a form of treatment commonly used for routine clinical applications, such as wound healing of the skin and reduction of inflammation. Additionally, PBMT has been explored for its potential in pain relief. In this work, we investigated the effect of PBMT on ion content within the 50B11 sensory neurons cell line in vitro using X-Ray fluorescence (XRF) and atomic force microscope (AFM) analysis. Two irradiation protocols were selected utilizing near-infrared laser lights at 800 and 970 nm, with cell fixation immediately following irradiation. Results showed a decrease in Calcium content after irradiation with both protocols, and with lidocaine, used as an analgesic control. Furthermore, a reduction in Potassium content was observed, particularly evident when normalized to cellular volume. These findings provide valuable insights into the molecular impact of PBMT within 50B11 sensory neurons under normal conditions. Such understanding may contribute to the wider adoption of PBMT as a therapeutic approach.
Assuntos
Cálcio , Raios Infravermelhos , Terapia com Luz de Baixa Intensidade , Células Receptoras Sensoriais , Animais , Células Receptoras Sensoriais/efeitos da radiação , Células Receptoras Sensoriais/metabolismo , Cálcio/metabolismo , Camundongos , Linhagem Celular , Espectrometria por Raios X , Microscopia de Força Atômica , Potássio/metabolismo , Potássio/química , Lidocaína/farmacologiaRESUMO
Polymer composite scaffolds hold promise in bone tissue engineering due to their biocompatibility, mechanical properties, and reproducibility. Among these materials, polylactic acid (PLA), a biodegradable plastics has gained attention for its processability characteristics. However, a deeper understanding of how PLA scaffold surface properties influence cell behavior is enssential for advancing its applications. In this study, 3D-printed PLA scaffolds containing hydroxyapatite (HA) were analyzed using atomic force microscopy and nanomechanical mapping. The addition of HA significantly increased key surface properties compared to unmodified PLA scaffols. Notably, the HA-modified scaffold demonstrated Gaussian distribution of stiffness and adhesive forces, in contrast to the bimodal properties observed in the unmodified PLA scaffolds. Human adipose-derived mesenchymal stem cell (hADMSC) seeded on the 3D-printed PLA scaffolds blended with 10% HA (P10) exhibited strong attachment. After four weeks, osteogenic differentiation of hADMSCs was detected, with calcium deposition reaching 6.76% ± 0.12. These results suggest that specific ranges of stiffness and adhesive forces of the composite scaffold can support cell attachement, and mineralization. The study highlights that tailoring suface properties of composite scaffolds is crucial for modulating cellular interactions, thus advancing the development of effective bone replacement materials.