RESUMEN
Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.
Asunto(s)
Esclerosis Amiotrófica Lateral , Axones , Demencia Frontotemporal , Mutación , Proteína FUS de Unión a ARN , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Axones/patología , Axones/metabolismo , Animales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Demencia Frontotemporal/metabolismo , Femenino , Masculino , Xenopus laevis , Conos de Crecimiento/metabolismo , Humanos , Modelos Animales de EnfermedadRESUMEN
Although fusogenic liposomes offer a promising approach for the delivery of antibiotic payloads across the cell envelope of Gram-negative bacteria, there is still a limited understanding of the individual nanocarrier interactions with the bacterial target. Using super-resolution microscopy, we characterize the interaction dynamics of positively charged fusogenic liposomes with Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The liposomes merge with the outer membrane (OM) of Gram-negative bacteria, while attachment or lipid internalization is observed in Gram-positive cells. Employing total internal reflection fluorescence microscopy, we demonstrated liposome fusion with model supported lipid bilayers. For whole E. coli cells, however, we observed heterogeneous membrane integrations, primarily involving liposome attachment and hemifusion events. With increasing lipopolysaccharide length, the likelihood of full-fusion events was reduced. The integration of artificial lipids into the OM of Gram-negative cells led to membrane destabilization, resulting in decreased bacterial vitality, membrane detachment, and improved codelivery of vancomycinâan effective antibiotic against Gram-positive cells. These findings provide significant insights into the interactions of individual nanocarriers with bacterial envelopes at the single-cell level, uncovering effects that would be missed in bulk measurements. This highlights the importance of conducting single-particle and single-cell investigations to assess the performance of next-generation drug delivery platforms.
Asunto(s)
Escherichia coli , Liposomas , Liposomas/metabolismo , Escherichia coli/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Sistemas de Liberación de Medicamentos , Membrana Celular/metabolismo , Bacterias GramnegativasRESUMEN
Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson's disease patient's brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.
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Células Madre Pluripotentes Inducidas , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , Vesículas Sinápticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Roedores/metabolismo , LípidosRESUMEN
The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phages of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we employed Escherichia coli OMV derived SLBs and use both fluorescent imaging and mechanical sensing techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalized with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phages with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with the phage. The work presented here shows how interactions occurring between the phages and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phages that work against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.
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Bacteriófagos , Membrana Dobles de Lípidos , Membrana Dobles de Lípidos/química , Escherichia coli , Antibacterianos/farmacologíaRESUMEN
As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.
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Transportadoras de Casetes de Unión a ATP , Técnicas Biosensibles , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.
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Membrana Externa Bacteriana , Membrana Dobles de Lípidos , Antibacterianos , Escherichia coli , Proteínas Fluorescentes Verdes , Humanos , Membrana Dobles de Lípidos/química , Microscopía de Fuerza AtómicaRESUMEN
The development of effective pathogen reduction strategies is required due to the rise in antibiotic-resistant bacteria and zoonotic viral pandemics. Photodynamic inactivation (PDI) of bacteria and viruses is a potent reduction strategy that bypasses typical resistance mechanisms. Naturally occurring riboflavin has been widely used in PDI applications due to efficient light-induced reactive oxygen species (ROS) release. By rational design of its core structure to alter (photo)physical properties, we obtained derivatives capable of outperforming riboflavin's visible light-induced PDI against E. coli and a SARS-CoV-2 surrogate, revealing functional group dependency for each pathogen. Bacterial PDI was influenced mainly by guanidino substitution, whereas viral PDI increased through bromination of the flavin. These observations were related to enhanced uptake and ROS-specific nucleic acid cleavage mechanisms. Trends in the derivatives' toxicity towards human fibroblast cells were also investigated to assess viable therapeutic derivatives and help guide further design of PDI agents to combat pathogenic organisms.
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COVID-19 , Fotoquimioterapia , Bacterias , Escherichia coli , Humanos , Luz , Fármacos Fotosensibilizantes/química , Especies Reactivas de Oxígeno/farmacología , Riboflavina/farmacología , SARS-CoV-2RESUMEN
Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.
RESUMEN
Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.
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Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor-based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point-of-care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound-induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.
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Infección de Heridas , Antibacterianos/farmacología , Biopelículas , Humanos , Cicatrización de Heridas , Infección de Heridas/tratamiento farmacológicoRESUMEN
Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor-based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point-of-care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound-induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.
RESUMEN
Fibrillar amyloids exhibit a fascinating range of mechanical, optical, and electronic properties originating from their characteristic ß-sheet-rich structure. Harnessing these functionalities in practical applications has so far been hampered by a limited ability to control the amyloid self-assembly process at the macroscopic scale. Here, we use core-shell electrospinning with microconfinement to assemble amyloid-hybrid fibers, consisting of densely aggregated fibrillar amyloids stabilized by a polymer shell. Up to centimeter-long hybrid fibers with micrometer diameter can be arranged into aligned and ordered arrays and deposited onto substrates or produced as free-standing networks. Properties that are characteristic of amyloids, including their high elastic moduli and intrinsic fluorescence signature, are retained in the hybrid fiber cores, and we show that they fully persist through the macroscopic fiber patterns. Our findings suggest that microlevel confinement is key for the guided assembly of amyloids from monomeric proteins.
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Amiloide , PolímerosRESUMEN
Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9-12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.
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Diseño de Fármacos , Ingeniería de Proteínas , Proteínas/síntesis química , Proteínas/metabolismo , Células 3T3 , Animales , Biología Celular , Supervivencia Celular , Biología Computacional , Endocitosis , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas In Vitro , Cinética , Ligandos , Ratones , Microscopía de Fuerza Atómica , Modelos Moleculares , Biología SintéticaRESUMEN
The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.
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Proteínas Bacterianas/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Desoxirribonucleasas/química , Proteínas de Escherichia coli/química , Exonucleasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo Celular , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Humanos , Hidrólisis , Proteína Homóloga de MRE11/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Zinc/químicaRESUMEN
Antibiotic resistance has become one of the greatest challenges for modern medicine, and new approaches for the treatment of bacterial infections are urgently needed to avoid widespread vulnerability again to infections that have so far been easily treatable with existing drugs. Among the many approaches investigated to overcome this challenge is the use of engineered nanostructures for the precise and targeted delivery of existing antimicrobial agents in a fashion that will potentiate their effect. This idea leans on lessons learned from pioneering research in cancer, where the targeted delivery of anti-cancer drugs to mammalian cells has been a topic for some time. In particular, new research has demonstrated that nanomaterials can be functionalised with active antimicrobials and, in some cases, with targeting molecules that potentiate the efficiency of the antimicrobials. In this mini-review, we summarise results that demonstrate the potential for nanoparticles, dendrimers and DNA nanostructures for use in antimicrobial delivery. We consider material aspects of the delivery vehicles and ways in which they can be functionalised with antibiotics and antimicrobial peptides, and we review evidence for their efficacy to kill bacteria both in vitro and in vivo. We also discuss the advantages and limitations of these materials and highlight the benefits of DNA nanostructures specifically for their versatile potential in the present context.
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Antiinfecciosos , Infecciones Bacterianas , Nanopartículas , Nanoestructuras , Animales , Antibacterianos/farmacología , Antiinfecciosos/farmacologíaRESUMEN
Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.
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Proteínas Arqueales/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Sulfolobus acidocaldarius/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Proteína Homóloga de MRE11/química , Proteína Homóloga de MRE11/genética , Microscopía de Fuerza Atómica , Unión Proteica , Sulfolobus acidocaldarius/química , Sulfolobus acidocaldarius/enzimología , Sulfolobus acidocaldarius/metabolismoRESUMEN
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
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Agregado de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Benzotiazoles/metabolismo , Calcio/metabolismo , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación , Conformación Proteica , Espectroscopía de Protones por Resonancia Magnética , Relación Estructura-ActividadRESUMEN
We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.
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Antibacterianos/farmacología , ADN/química , Portadores de Fármacos/química , Muramidasa/farmacología , Nanoestructuras/química , Animales , Antibacterianos/química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/toxicidad , Bacillus subtilis/química , Bacillus subtilis/efectos de los fármacos , Células COS , Chlorocebus aethiops , ADN/toxicidad , Portadores de Fármacos/toxicidad , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Muramidasa/química , Nanoestructuras/toxicidad , Conformación de Ácido NucleicoRESUMEN
In this review, we discuss methods and advancements in fluorescence lifetime imaging microscopy that permit measurements to be performed at faster speed and higher resolution than previously possible. We review fast single-photon timing technologies and the use of parallelized detection schemes to enable high-throughput and high content imaging applications. We appraise different technological implementations of fluorescence lifetime imaging, primarily in the time-domain. We also review combinations of fluorescence lifetime with other imaging modalities to capture multi-dimensional and correlative information from a single sample. Throughout the review, we focus on applications in biomedical research. We conclude with a critical outlook on current challenges and future opportunities in this rapidly developing field.