RESUMEN

The identification, management, and prevention of concussion across all competitive sports and athletic populations has been a notable topic of research over the last decade. Soccer is no exception, with over a billion participants worldwide. In soccer, 3 distinct subsets of head injuries are often the contributors to concussion: head-to-equipment, head-to-surface, and head-to-player collisions. Recognition of concussion is crucial, and ideally made on the sideline during competitive play. Recently updated screening tools include the SCAT6 and ChildSCAT6, which are widely utilized at all levels of play. Management of concussion is divided into on-field and in clinic management. Initial management includes removal from the field of play. There is increased emphasis on earlier incorporation of exercise prescription as a means to improve recovery in the concussed athlete. While few objective diagnostic tests exist to identify concussion, many are in development, most notably advanced imaging and biomarker modalities.

Asunto(s)

Traumatismos en Atletas , Conmoción Encefálica , Fútbol , Humanos , Conmoción Encefálica/diagnóstico , Conmoción Encefálica/terapia , Fútbol/lesiones , Traumatismos en Atletas/diagnóstico , Traumatismos en Atletas/terapia

RESUMEN

Introduction: Bladder cancer is a common neoplasia of the urinary tract that holds the highest cost of lifelong treatment per patient, highlighting the need for a continuous search for new therapies for the disease. Current bladder cancer models are either imperfect in their ability to translate results to clinical practice (mouse models), or rare and not inducible (canine models). Swine models are an attractive alternative to model the disease due to their similarities with humans on several levels. The Oncopig Cancer Model has been shown to develop tumors that closely resemble human tumors. However, urothelial carcinoma has not yet been studied in this platform. Methods: We aimed to develop novel Oncopig bladder cancer cell line (BCCL) and investigate whether these urothelial swine cells mimic human bladder cancer cell line (5637 and T24) treatment-responses to cisplatin, doxorubicin, and gemcitabine in vitro. Results: Results demonstrated consistent treatment responses between Oncopig and human cells in most concentrations tested (p>0.05). Overall, Oncopig cells were more predictive of T24 than 5637 cell therapeutic responses. Microarray analysis also demonstrated similar alterations in expression of apoptotic (GADD45B and TP53INP1) and cytoskeleton-related genes (ZMYM6 and RND1) following gemcitabine exposure between 5637 (human) and Oncopig BCCL cells, indicating apoptosis may be triggered through similar signaling pathways. Molecular docking results indicated that swine and humans had similar Dg values between the chemotherapeutics and their target proteins. Discussion: Taken together, these results suggest the Oncopig could be an attractive animal to model urothelial carcinoma due to similarities in in vitro therapeutic responses compared to human cells.

RESUMEN

Suids, both domesticated and wild, are found on all continents except for Antarctica and provide valuable food resources for humans in addition to serving as important models for biomedical research. Continuing advances in genome sequencing have allowed researchers to compare the genomes from diverse populations of suids helping to clarify their evolution and dispersal. Further analysis of these samples may provide clues to improve disease resistance/resilience and productivity in domestic suids as well as better ways of classifying and conserving genetic diversity within wild and captive suids. Collecting samples from diverse populations of suids is resource intensive and may negatively impact endangered populations. Here we catalog extensive tissue and DNA samples from suids in collections in both Europe and North America. We include samples that have previously been used for whole genome sequencing, targeted DNA sequencing, RNA sequencing, and reduced representation bisulfite sequencing (RRBS). This work provides an important centralized resource for researchers who wish to access published databases.

Asunto(s)

Genoma , Genómica , Humanos , Porcinos , Animales , Genoma/genética , Análisis de Secuencia de ADN , Secuenciación Completa del Genoma , ADN

RESUMEN

Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access to specialized nanofabrication tools. These densely packed, highly ordered arrays are used to capture liposomes and bacterial outer membrane vesicles on the basis of their surface biomarkers, with a maximum of one particle per array dot, resulting in densely packed arrays of particles. Despite the high particle density, the underlying antibody or toxin array ensured that neighboring individual particles are optically resolvable. Provided target particle biomarkers and suitable capture molecules are identified, this approach can be used to generate high density arrays of a wide variety of small biological particles, including other types of EVs like exosomes.

Asunto(s)

Exosomas , Vesículas Extracelulares , Membrana Externa Bacteriana , Lípidos , Liposomas

RESUMEN

Bladder cancer (BC) is the 10th most common neoplasia worldwide and holds expensive treatment costs due to its high recurrence rates, resistance to therapy and the need for lifelong surveillance. Thus, it is necessary to improve the current therapy options and identify more effective treatments for BC. Biological models capable of recapitulating the characteristics of human BC pathology are essential in evaluating the effectiveness of new therapies. Currently, the most commonly used BC models are experimentally induced murine models and spontaneous canine models, which are either insufficient due to their small size and inability to translate results to clinical basis (murine models) or rarely spontaneously observed BC (canine models). Pigs represent a potentially useful animal for the development of personalized tumors due to their size, anatomy, physiology, metabolism, immunity, and genetics similar to humans and the ability to experimentally induce tumors. Pigs have emerged as suitable biomedical models for several human diseases. In this sense, the present perspective focuses on the genetic basis for BC; presents current BC animal models available along with their limitations; and proposes the pig as an adequate animal to develop humanized large animal models of BC. Genetic alterations commonly found in human BC can be explored to create genetically defined porcine models, including the BC driver mutations observed in the FGFR3, PIK3CA, PTEN, RB1, HRAS, and TP53 genes. The development of such robust models for BC has great value in the study of pathology and the screening of new therapeutic and diagnostic approaches to the disease.

RESUMEN

We show that photosensitized phospholipid oxidation, initiated by the lipid-conjugated fluorophore TopFluor-PC, causes defects, namely, membrane tubes and vesicle-like structures, in supported lipid bilayers (SLBs). Lipid oxidation is detrimental to the integrity of the lipid molecules; when oxidized, they undergo a conformational expansion, which causes membrane tubes to protrude from the SLB. Lipid oxidation is verified by FT-IR spectroscopy, and area expansion is observed in Langmuir trough experiments. Upon growing to a critical length, the membrane tubes arising from SLBs rapidly undergo transition to vesicle-like structures. We find a correlation between the maximum tube length and the diameter of the resulting vesicle, suggesting the conservation of the surface area between these features. We use geometric modeling and the measured tube length and vesicle radius to calculate the tube radius; our calculated mean tube diameter of 243 nm is comparable to other groups' experimental findings. In the presence of fluid flow, membrane tubes can be extended to tens to hundreds of microns in length. SLBs composed of saturated lipids resist light-induced tubulation, and the inclusion of the lipophilic antioxidant α-tocopherol attenuates the tubulation process and increases the light intensity threshold for tubulation.

RESUMEN

In the nervous system, a myelin sheath that originates from oligodendrocytes or Schwann cells wraps around axons to facilitate electrical signal transduction. The interface between an axon and myelin is maintained by a number of biomolecular interactions. Among the interactions are those between GD1a and GT1b gangliosides on the axon and myelin-associated glycoprotein (MAG) on myelin. Interestingly, these interactions can also inhibit neuronal outgrowth. Ganglioside-MAG interactions are often studied in cellular or animal models where their relative concentrations are not easily controlled or in assays where the gangliosides and MAG are not presented as part of fluid lipid bilayers. Here, we present an approach to characterize MAG-ganglioside interactions in real time, where MAG, GD1a, and GT1b contents are controlled and they are in their in vivo orientation within fluid lipid bilayers. Using a quartz crystal microbalance with dissipation monitoring (QCM-D) biosensor functionalized with a supported lipid bilayer (SLB) and MAG, we detect vesicular GD1a and GT1b binding and determine the interaction kinetics as a function of vesicular ganglioside content. MAG-bound vesicles are deformed similarly, regardless of the ganglioside or its mole fraction. We further demonstrate how MAG-ganglioside interactions can be disrupted by antiganglioside antibodies that override MAG-based neuron growth inhibition.

Asunto(s)

Técnicas Biosensibles , Gangliósidos/química , Membrana Dobles de Lípidos/química , Glicoproteína Asociada a Mielina/química , Sitios de Unión , Tecnicas de Microbalanza del Cristal de Cuarzo

RESUMEN

The purpose of this study is to examine the effect of the body's mass distribution to segments and the filtering of kinematic data on the estimation of vertical ground reaction forces from positional data. A public dataset of raw running biomechanics was used for the purposes of the analysis, containing recordings of twenty-eight competitive or elite athletes running on an instrumented treadmill at three different speeds. A grid-search on half of the trials was employed to seek the values of the parameters that optimise the approximation of biomechanical loads. Two-way ANOVAs were then conducted to examine the significance of the parameterised factors in the modelled waveforms. The reserved recordings were used to validate the predictive accuracy of the model. The cut-off filtering frequencies of the pelvis and thigh markers were correlated to running speed and heel-strike patterns, respectively. Optimal segment masses were in agreement with standardised literature reported values. Root mean square errors for slow running (2.5 m/s) were on average equal to 0.1 (body weight normalized). Errors increased with running speeds to 0.13 and 0.18 for 3.5 m/s and 4.5 m/s, respectively. This study accurately estimated vertical ground reaction forces for slow-paced running by only considering the kinematics of the pelvis and thighs. Future studies should consider configuring the filtering of kinematic inputs based on the location of markers and type of running.

Asunto(s)

Fenómenos Mecánicos , Carrera , Adulto , Fenómenos Biomecánicos , Peso Corporal , Prueba de Esfuerzo , Talón , Humanos , Masculino , Pelvis

RESUMEN

Gangliosides are glycolipids that are enriched on the outer surface of cell membranes. Gangliosides are receptors for a number of signaling molecules and toxins, and therefore are often incorporated into biosensors. Many of these biosensors incorporate gangliosides into supported lipid bilayers which are formed by the spontaneous rupture of unilamellar vesicles on glass or SiO2 substrates. In this work, we used quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate how the presence of the four major brain gangliosides (GM1, GD1a, GD1b, and GT1b) influences the process of supported lipid bilayer formation on SiO2 surfaces. We show that the rate of supported bilayer formation is dependent on both the charge and position of sialic acid moieties on ganglioside molecules. Additionally, Ca2+ can accelerate ganglioside-rich supported bilayer formation, but the degree of acceleration differs for vesicles containing different gangliosides. Fluorescence recovery after photobleaching measurements show that the presence of all gangliosides reduces lipid diffusion coefficients in a concentration-dependent manner, and that Ca2+ slows lipid diffusion in membranes with and without gangliosides. Finally, we use ganglioside-rich supported bilayers to measure binding constants for a GD1a-binding antibody that has similar properties to antibodies present in a variant of Guillain-Barré syndrome.

Asunto(s)

Encéfalo/metabolismo , Gangliósidos/metabolismo , Glucolípidos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Dióxido de Silicio/metabolismo , Liposomas Unilamelares/metabolismo , Animales , Gangliósidos/química , Glucolípidos/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Ovinos , Dióxido de Silicio/química , Liposomas Unilamelares/química

RESUMEN

rHIgM22 is a recombinant human monoclonal IgM designed to promote remyelination, and it is currently in Phase I clinical trials in patients with multiple sclerosis (MS). In animal models of demyelination, a single low dose of rHIgM22 stimulates oligodendrocyte maturation, induces remyelination, preserves axons, and slows the decline of locomotor deficits. Natural autoantibodies like rHIgM22 typically bind to multiple antigens with weak affinity. rHIgM22 binds to oligodendrocytes and myelin. Because the antigens for rHIgM22 is prevalent within and exclusive to central nervous system (CNS) myelin, we used CNS myelin particles in combination with surface plasmon resonance to determine the kinetic and affinity constants for the interaction of rHIgM22 to myelin. We found that both the serum and recombinant forms of the antibody bind to myelin with very small dissociation constants in the 100 pM range, which is highly unusual for natural autoantibodies. The extraordinary affinity between rHIgM22 and myelin may explain why such a low effective dose can stimulate CNS repair in animal models of demyelination and underlie the accumulation of rHIgM22 in the CSF in treated MS patients by targeting myelin.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Inmunoglobulina M/metabolismo , Vaina de Mielina/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Cinética , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie

RESUMEN

A plug and socket approach for tightening polyelectrolyte multilayers is introduced based on the use pendant ß-cyclodextrin groups. Prototypical multilayers derived from poly(sodium 4-styrene sulfonate) and ß-cyclodextrin-containing poly(4-vinylbenzyltrimethylammonium chloride) are described. Evidence for tightened multilayers has been obtained from gas permeation, swelling and quartz crystal microbalance with dissipation (QCM-D) measurements.

RESUMEN

Objectives Peripheral arterial disease most commonly presents as intermittent claudication (IC). Early evidence has suggested that extracorporeal shockwave therapy is efficacious in the short term for the management of intermittent claudication. The objective of this pilot trial was to evaluate the medium-term efficacy of this treatment. Methods This double-blind randomised placebo-controlled pilot trial randomised patients with unilateral intermittent claudication in a 1:1 fashion to receive extracorporeal shockwave therapy or a sham treatment for three sessions per week over three weeks. Primary outcomes were maximum walking distance and intermittent claudication distance using a fixed-load treadmill test. Secondary outcomes included pre- and post-exertional ankle-brachial pressure indices, safety and quality of life assessed using generic (SF36, EQ-5D-3L) and disease-specific (vascular quality of life) measures. All outcome measures were assessed at 12 months post-treatment. Results Thirty participants were included in the study (extracorporeal shockwave therapy, n = 15; sham, n = 15), with 26 followed up and analysed at 12 months (extracorporeal shockwave therapy, n = 13; sham, n = 13). Intragroup analysis demonstrated significant improvements in maximum walking distance, intermittent claudication distance and post-exertional ankle-brachial pressure indices ( p < 0.05) in the active treatment group, with no improvements in pre-exertional ankle-brachial pressure indices. Significant improvements in quality of life were observed in 3 out of 19 domains assessed in the active group. A re-intervention rate of 26.7% was seen in both groups. Conclusions These findings suggest that extracorporeal shockwave therapy is effective in improving walking distances at 12 months. Although this study provides important pilot data, a larger study is needed to corroborate these findings and to investigate the actions of this treatment. ISRCTN: NCT02652078.

Asunto(s)

Tratamiento con Ondas de Choque Extracorpóreas , Claudicación Intermitente/terapia , Enfermedad Arterial Periférica/terapia , Anciano , Índice Tobillo Braquial , Método Doble Ciego , Inglaterra , Prueba de Esfuerzo , Tolerancia al Ejercicio , Tratamiento con Ondas de Choque Extracorpóreas/efectos adversos , Femenino , Humanos , Claudicación Intermitente/diagnóstico , Claudicación Intermitente/fisiopatología , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/fisiopatología , Proyectos Piloto , Calidad de Vida , Recuperación de la Función , Encuestas y Cuestionarios , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

IMPORTANCE: Modulating the immune system does not reverse long-term disability in neurologic disorders. Better neuroregenerative and neuroprotective treatment strategies are needed for neuroinflammatory and neurodegenerative diseases. OBJECTIVE: To review the role of monoclonal, naturally occurring antibodies (NAbs) as novel therapeutic molecules for treatment of neurologic disorders. EVIDENCE REVIEW: Peer-reviewed articles, including case reports, case series, retrospective reviews, prospective randomized clinical trials, and basic science reports, were identified in a PubMed search for articles about NAbs and neurologic disorders that were published from January 1, 1964, through June 30, 2015. We concentrated our review on multiple sclerosis, Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis. FINDINGS: Many insults, including trauma, ischemia, infection, inflammation, and neurodegeneration, result in irreversible damage to the central nervous system. Central nervous system injury often results in a pervasive inhibitory microenvironment that hinders regeneration. A common targeted drug development strategy is to identify molecules with high potency in animal models. Many approaches often fail in the clinical setting owing to a lack of efficacy in human diseases (eg, less than the response demonstrated in animal models) or a high incidence of toxic effects. An alternative approach is to identify NAbs in humans because these therapeutic molecules have potential physiologic function without toxic effects. NAbs of the IgG, IgA, or IgM isotype contain germline or close to germline sequences and are reactive to self-components, altered self-components, or foreign antigens. Our investigative group developed recombinant, autoreactive, natural human IgM antibodies directed against oligodendrocytes or neurons with therapeutic potential for central nervous system repair. One such molecule, recombinant HIgM22, directed against myelin and oligodendrocytes completed a successful phase 1 clinical trial without toxic effects with the goal of promoting remyelination in multiple sclerosis. CONCLUSIONS AND RELEVANCE: Animal studies demonstrate that certain monoclonal NAbs are beneficial as therapeutic agents for neurologic diseases. This class of antibodies represents a unique source from which to develop a new class of disease-modifying therapies.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Isotipos de Inmunoglobulinas/inmunología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Animales , Humanos , Enfermedades del Sistema Nervioso/inmunología

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A) by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism.

Asunto(s)

Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Gangliósidos/metabolismo , Inmunoglobulina M/uso terapéutico , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/patología , Animales , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta Inmunológica , Epítopos/química , Gangliósidos/química , Humanos , Membrana Dobles de Lípidos/metabolismo , Ratones , Ratones Transgénicos , Microtúbulos/metabolismo , Modelos Moleculares , Neuritas/metabolismo , Neuritas/patología , Unión Proteica , Proteínas Recombinantes/uso terapéutico , Solubilidad , Médula Espinal/patología , Superóxido Dismutasa/metabolismo , Resonancia por Plasmón de Superficie , Análisis de Supervivencia , Tubulina (Proteína)/metabolismo

RESUMEN

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.

Asunto(s)

Colesterol/química , Lípidos de la Membrana/química , Microdominios de Membrana/química , Fluidez de la Membrana , Microdominios de Membrana/ultraestructura , Microscopía Fluorescente , Imagen Óptica

RESUMEN

We report on the conformal surface passivation of photonic crystal (PC) laser devices with an ultrathin dielectric layer. Air-bridge-type Γ-point band-edge lasers (BELs) are fabricated by forming a honeycomb lattice two-dimensional PC structure into an InGaAsP multiple-quantum-well epilayer. Atomic layer deposition (ALD) is employed for conformal deposition of a few-nanometer-thick SiO2 layer over the entire device surface, not only on the top and bottom surfaces of the air-bridge membrane but also on the air-hole sidewalls. Despite its extreme thinness, the ALD passivation layer is found to protect the InGaAsP BEL devices from harsh chemicals. In addition, the ALD-SiO2 is compatible with the silane-based surface chemistry, which allows us to use ALD-passivated BEL devices as label-free biosensors. The standard streptavidin-biotin interaction shifts the BEL lasing wavelength by ∼1 nm for the dipole-like Γ-point band-edge mode. A sharp lasing line (<0.2 nm, full width at half-maximum) and a large refractive index sensitivity (∼163 nm per RIU) produce a figure of merit as high as ∼800 for our BEL biosensor, which is at least an order of magnitude higher than those of more common biosensors that rely on a broad resonance peak, showing that our nanolaser structures are suitable for highly sensitive biosensor applications.

Asunto(s)

Técnicas Biosensibles , Nanotecnología/métodos , Dióxido de Silicio/química , Biotina/química , Cristalización , Diseño de Equipo , Rayos Láser , Luz , Nanoestructuras , Óptica y Fotónica , Fotones , Refractometría , Silanos/química , Estreptavidina/química , Propiedades de Superficie , Vibración

RESUMEN

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding.

Asunto(s)

Membrana Celular/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Microdominios de Membrana/química , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Dimetilpolisiloxanos/química , Imagenología Tridimensional , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Fosfatidilcolinas/química , Esfingomielinas/química , Esfingomielinas/metabolismo

RESUMEN

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Asunto(s)

Membrana Celular/química , Membrana Dobles de Lípidos/química , Análisis de Matrices Tisulares/métodos , Animales , Bovinos , Toxina del Cólera/química , Gangliósido G(M1)/química , Fosfolípidos/química , Dióxido de Silicio/química , Análisis de Matrices Tisulares/instrumentación

RESUMEN

Characterization of binding kinetics and affinity between a potential drug and its receptor are key steps in the development of new drugs. Among the techniques available to determine binding affinities, surface plasmon resonance has emerged as the gold standard because it can measure binding and dissociation rates in real-time in a label-free fashion. Surface plasmon resonance is now finding applications in the characterization of molecules for treatment of neurodegenerative diseases, characterization of molecules associated with pathogenesis of neurodegenerative diseases and detection of neurodegenerative disease biomarkers. In addition it has been used in the characterization of a new class of natural autoantibodies that have therapeutic potential in a number of neurologic diseases. In this review we will introduce surface plasmon resonance and describe some applications of the technique that pertain to neurodegenerative disorders and their treatment.

Asunto(s)

Biomarcadores/análisis , Enfermedades Neurodegenerativas/diagnóstico , Resonancia por Plasmón de Superficie , Animales , Biomarcadores/química , Humanos , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Unión Proteica

RESUMEN

Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.

Asunto(s)

Inmunoglobulina M/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Proteínas Recombinantes/farmacología , Animales , Sistema Nervioso Central/fisiología , Matriz Extracelular , Humanos , Ratones , Neuronas/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología