RESUMO
The aggregation of adeno-associated viral (AAV) capsids in an aqueous environment was investigated via coarse-grained molecular dynamics (CG-MD) simulations. The primary driving force and mechanism of the aggregation were investigated with or without single-strand DNA (ssDNA) loaded at various process temperatures. Capsid aggregation appeared to involve multiple residue interactions (i.e., hydrophobic, polar and charged residues) leading to complex protein aggregation. In addition, two aggregation mechanisms (i.e., the fivefold face-to-face contact and the edge-to-edge contact) were identified from this study. The ssDNA with its asymmetric structure could be the reason for destabilizing protein subunits and enhancing the interaction between the charged residues, and further result in the non-reversible face-to-face contact. At higher temperature, the capsid structure was found to be unstable with the significant size expansion of the loaded ssDNA which could be attributed to reduced number of intramolecular hydrogen bonds, the increased conformational deviations of protein subunits and the higher residue fluctuations. The CG-MD model was further validated with previous experimental and simulation data, including the full capsid size measurement and the capsid internal pressure. Thus, a good understanding of AAV capsid aggregation, instability and the role of ssDNA were revealed by applying the developed computational model.
Assuntos
Dependovirus , Simulação de Dinâmica Molecular , Subunidades Proteicas , DNA de Cadeia Simples , CapsídeoRESUMO
The Biophorum Development Group (BPDG) is an industry-wide consortium enabling networking and sharing of best practices for the development of biopharmaceuticals. To gain a better understanding of current industry approaches for establishing biopharmaceutical drug product (DP) robustness, the BPDG-Formulation Point Share group conducted an intercompany collaboration exercise, which included a bench-marking survey and extensive group discussions around the scope, design, and execution of robustness studies. The results of this industry collaboration revealed several key common themes: (1) overall DP robustness is defined by both the formulation and the manufacturing process robustness; (2) robustness integrates the principles of quality by design (QbD); (3) DP robustness is an important factor in setting critical quality attribute control strategies and commercial specifications; (4) most companies employ robustness studies, along with prior knowledge, risk assessments, and statistics, to develop the DP design space; (5) studies are tailored to commercial development needs and the practices of each company. Three case studies further illustrate how a robustness study design for a biopharmaceutical DP balances experimental complexity, statistical power, scientific understanding, and risk assessment to provide the desired product and process knowledge. The BPDG-Formulation Point Share discusses identified industry challenges with regard to biopharmaceutical DP robustness and presents some recommendations for best practices.
Assuntos
Indústria Farmacêutica/métodos , Preparações Farmacêuticas/química , Biofarmácia/métodos , Química Farmacêutica/métodos , Ensaios Clínicos como Assunto , Desenho de Fármacos , Humanos , Colaboração Intersetorial , Medição de Risco , Tecnologia Farmacêutica/métodosRESUMO
This paper describes the fabrication of a composite agar/PDMS device for enriching short cells in a population of motile Escherichia coli. The device incorporated ratcheting microchannels, which directed the motion of swimming cells of E. coli through the device, and three sorting junctions, which isolated successively shorter populations of bacteria. The ratcheting microchannels guided cells through the device with an average rate of displacement of (32 +/- 9) microm s(-1). Within the device, the average length of the cells decreased from 3.8 microm (Coefficient of Variation, CV: 21%) at the entrance, to 3.4 microm (CV: 16%) after the first sorting junction, to 3.2 mum (CV: 19%) after the second sorting junction, to 3.0 mum (CV: 19%) after the third sorting junction.
Assuntos
Fracionamento Celular/métodos , Escherichia coli/citologia , Ágar , Dimetilpolisiloxanos , MicrofluídicaRESUMO
This Review summarizes methods for constructing systems and structures at micron or submicron scales that have applications in microbiology. These tools make it possible to manipulate individual cells and their immediate extracellular environments and have the capability to transform the study of microbial physiology and behaviour. Because of their simplicity, low cost and use in microfabrication, we focus on the application of soft lithographic techniques to the study of microorganisms, and describe several key areas in microbiology in which the development of new microfabricated materials and tools can have a crucial role.
Assuntos
Fenômenos Fisiológicos Bacterianos , Engenharia Biomédica/métodos , Biotecnologia , Técnicas Microbiológicas/métodos , Microbiologia , Técnicas Microbiológicas/instrumentação , Miniaturização , Fenótipo , Integração de SistemasRESUMO
Near a solid boundary, Escherichia coli swims in clockwise circular motion. We provide a hydrodynamic model for this behavior. We show that circular trajectories are natural consequences of force-free and torque-free swimming and the hydrodynamic interactions with the boundary, which also leads to a hydrodynamic trapping of the cells close to the surface. We compare the results of the model with experimental data and obtain reasonable agreement. In particular, the radius of curvature of the trajectory is observed to increase with the length of the bacterium body.
Assuntos
Fenômenos Fisiológicos Bacterianos , Biofísica/métodos , Algoritmos , Bactérias/metabolismo , Aderência Bacteriana , Movimento Celular , Quimiotaxia , Simulação por Computador , Escherichia coli/metabolismo , Flagelos/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Contraste de Fase , Microscopia de Vídeo , Modelos Biológicos , Modelos Estatísticos , Modelos Teóricos , Movimento (Física) , MovimentoRESUMO
This paper describes a technique for growing filamentous cells of Escherichia coli with defined shapes, including crescents, zigzags, sinusoids, and spirals. The procedure begins with the fabrication of embossed microchambers in agarose. Cells are trapped in the chambers by placing a flat, flexible "ceiling", either a slab of agarose or poly(dimethylsiloxane), against an agarose mold on which a suspension of cells has been added; the use of agarose keeps cells hydrated and allows nutrients to diffuse into the chambers. Cells grown in microchambers in the presence of cephalexin grow into a multinucleate, nonseptate, filamentous phenotype and adopt the shape of the microchambers. The resulting cells are motile and can be released by removing the "ceiling" from the agarose microchambers and rinsing the cells into solution.
Assuntos
Escherichia coli/citologia , Nanotecnologia/métodos , Células Imobilizadas , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , SefaroseRESUMO
It is difficult to harness the power generated by biological motors to carry out mechanical work in systems outside the cell. Efforts to capture the mechanical energy of nanomotors ex vivo require in vitro reconstitution of motor proteins and, often, protein engineering. This study presents a method for harnessing the power produced by biological motors that uses intact cells. The unicellular, biflagellated algae Chlamydomonas reinhardtii serve as "microoxen." This method uses surface chemistry to attach loads (1- to 6-microm-diameter polystyrene beads) to cells, phototaxis to steer swimming cells, and photochemistry to release loads. These motile microorganisms can transport microscale loads (3-microm-diameter beads) at velocities of approximately 100-200 microm.sec(-1) and over distances as large as 20 cm.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Proteínas Motores Moleculares/fisiologia , Engenharia de Proteínas/métodos , Animais , Transporte Biológico/fisiologia , Fenômenos Biofísicos , Biofísica , Microesferas , Movimento , Fotoquímica/métodosRESUMO
The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially 'drive on the right', swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces--one an agar gel and the second PDMS--they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.
Assuntos
Escherichia coli/fisiologia , Flagelos/fisiologia , Locomoção/fisiologia , Ágar , Biofilmes/crescimento & desenvolvimento , Escherichia coli/citologia , Natação/fisiologia , Fatores de TempoRESUMO
This paper describes the use of micropatterned agarose stamps prepared by molding against PDMS masters to print patterns of bacteria on agar plates. Topographically patterned agarose stamps were inked with suspensions of bacteria; these stamps generated patterns of bacteria with features as small as 200 microm over areas as large as 50 cm2. Stamps with many small features (>200 microm) were used to study patterns of bacteria growing on media containing gradients of small molecules; stamps with larger features (>750 microm) were used to print different strains of bacteria simultaneously. The stamp transfers only a small percentage of cells that are on its surface to the agar at a time; it is thus possible to replica-pattern hundreds of times with a single inking. The use of soft stamps provides other useful functions. Stamps are easily customized to provide a range of patterns. When culture media is included in the agarose stamp, cells divide and thrive on the surface. The resulting "living stamp" regenerates its "ink" and can be used to pattern surfaces repetitively for a month. This method is rapid, reproducible, convenient, and can be used to control the pattern, spacing, and orientation between colonies of different bacteria.