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Mosquitoes carry a number of deadly pathogens that are transmitted while feeding on blood through the skin, and studying mosquito feeding behavior could elucidate countermeasures to mitigate biting. Although this type of research has existed for decades, there has yet to be a compelling example of a controlled environment to test the impact of multiple variables on mosquito feeding behavior. In this study, we leveraged uniformly bioprinted vascularized skin mimics to create a mosquito feeding platform with independently tunable feeding sites. Our platform allows us to observe mosquito feeding behavior and collect video data for 30-45 min. We maximized throughput by developing a highly accurate computer vision model (mean average precision: 92.5%) that automatically processes videos and increases measurement objectivity. This model enables assessment of critical factors such as feeding and activity around feeding sites, and we used it to evaluate the repellent effect of DEET and oil of lemon eucalyptus-based repellents. We validated that both repellents effectively repel mosquitoes in laboratory settings (0% feeding in experimental groups, 13.8% feeding in control group, p < 0.0001), suggesting our platform's use as a repellent screening assay in the future. The platform is scalable, compact, and reduces dependence on vertebrate hosts in mosquito research.
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The survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 µm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell-cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 µm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Humanos , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Neovascularização Patológica , Células Endoteliais da Veia Umbilical Humana , Materiais BiocompatíveisRESUMO
The rat hindlimb is a frequently utilized pre-clinical model system to evaluate injuries and pathologies impacting the hindlimbs. These studies have demonstrated the translational potential of this model but have typically focused on the force generating capacity of target muscles as the primary evaluative outcome. Historically, human studies investigating extremity injuries and pathologies have utilized biomechanical analysis to better understand the impact of injury and extent of recovery. In this study, we expand that full biomechanical workup to a rat model in order to characterize the spatiotemporal parameters, ground reaction forces, 3-D joint kinematics, 3-D joint kinetics, and energetics of gait in healthy rats. We report data on each of these metrics that meets or exceeds the standards set by the current literature and are the first to report on all these metrics in a single set of animals. The methodology and findings presented in this study have significant implications for the development and clinical application of the improved regenerative therapeutics and rehabilitative therapies required for durable and complete functional recovery from extremity traumas, as well as other musculoskeletal pathologies.
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Roedores , Caminhada , Humanos , Ratos , Animais , Fenômenos Biomecânicos , Caminhada/fisiologia , Membro Posterior/fisiologia , Extremidade InferiorRESUMO
New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenousâas opposed to overexpressedâmembrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed â¼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.
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Bicamadas Lipídicas , Nanoestruturas , Bicamadas Lipídicas/química , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Eucariotos/metabolismo , Nanoestruturas/química , Polímeros/químicaRESUMO
Modern ultrasound (US) imaging is increasing its clinical impact, particularly with the introduction of US-based quantitative imaging biomarkers. Continued development and validation of such novel imaging approaches requires imaging phantoms that recapitulate the underlying anatomy and pathology of interest. However, current US phantom designs are generally too simplistic to emulate the structure and variability of the human body. Therefore, there is a need to create a platform that is capable of generating well-characterized phantoms that can mimic the basic anatomical, functional, and mechanical properties of native tissues and pathologies. Using a 3D-printing technique based on stereolithography, we fabricated US phantoms using soft materials in a single fabrication session, without the need for material casting or back-filling. With this technique, we induced variable levels of stable US backscatter in our printed materials in anatomically relevant 3D patterns. Additionally, we controlled phantom stiffness from 7 to >120 kPa at the voxel level to generate isotropic and anisotropic phantoms for elasticity imaging. Lastly, we demonstrated the fabrication of channels with diameters as small as 60 micrometers and with complex geometry (e.g., tortuosity) capable of supporting blood-mimicking fluid flow. Collectively, these results show that projection-based stereolithography allows for customizable fabrication of complex US phantoms.
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Imagens de Fantasmas , Impressão Tridimensional/instrumentação , Estereolitografia/instrumentação , Ultrassonografia/métodos , Hemodinâmica , HumanosRESUMO
Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.
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Bicamadas Lipídicas/química , Proteínas de Membrana/química , Mitocôndrias/química , Membranas Mitocondriais/química , Difusão Dinâmica da Luz , Proteínas de Membrana/genética , Membranas Artificiais , Mitocôndrias/genética , Concentração Osmolar , Polieletrólitos/química , Polímeros/química , Cloreto de Sódio/químicaRESUMO
Neurodegenerative disorders are among the most common diseases in modern society. However, the molecular bases of diseases such as multiple sclerosis or Charcot-Marie-Tooth disease remain far from being fully understood. Research in this field is limited by the complex nature of native myelin and by difficulties in obtaining good in vitro model systems of myelin. Here, we introduce an easy-to-use model system of the myelin sheath that can be used to study myelin proteins in a native-like yet well-controlled environment. To this end, we present myelin-mimicking nanodiscs prepared through one of the amphiphilic copolymers styrene/maleic acid (SMA), diisobutylene/maleic acid (DIBMA), and styrene/maleimide sulfobetaine (SMA-SB). These nanodiscs were tested for their lipid composition using chromatographic (HPLC) and mass spectrometric (MS) methods and, utilizing spin probes within the nanodisc, their comparability with liposomes was studied. In addition, their binding behavior with bovine myelin basic protein (MBP) was scrutinized to ensure that the nanodiscs represent a suitable model system of myelin. Our results suggest that both SMA and SMA-SB are able to solubilize the myelin-like (cytoplasmic) liposomes without preferences for specific lipid headgroups or fatty acyl chains. In nanodiscs of both SMA and SMA-SB (called SMA(-SB)-lipid particles, short SMALPs or SMA-SBLPs, respectively), the polymers restrict the lipids' motion in the hydrophobic center of the bilayer. The headgroups of the lipids, however, are sterically less hindered in nanodiscs when compared with liposomes. Myelin-like SMALPs are able to bind bovine MBP, which can stack the lipid bilayers like in native myelin, showing the usability of these simple, well-controlled systems in further studies of protein-lipid interactions of native myelin.
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Maleatos , Bainha de Mielina , Animais , Bovinos , Humanos , Bicamadas Lipídicas , Lipossomos , Polímeros , EstirenoRESUMO
We present two openly accessible databases related to the assessment of implicit motives using Picture Story Exercises (PSEs): (a) A database of 183,415 German sentences, nested in 26,389 stories provided by 4,570 participants, which have been coded by experts using Winter's coding system for the implicit affiliation/intimacy, achievement, and power motives, and (b) a database of 54 classic and new pictures which have been used as PSE stimuli. Updated picture norms are provided which can be used to select appropriate pictures for PSE applications. Based on an analysis of the relations between raw motive scores, word count, and sentence count, we give recommendations on how to control motive scores for story length, and validate the recommendation with a meta-analysis on gender differences in the implicit affiliation motive that replicates existing findings. We discuss to what extent the guiding principles of the story length correction can be generalized to other content coding systems for narrative material. Several potential applications of the databases are discussed, including (un)supervised machine learning of text content, psychometrics, and better reproducibility of PSE research.
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Logro , Identificação Psicológica , Relações Interpessoais , Autoimagem , Teste de Apercepção Temática/normas , Adulto , Alemanha , Humanos , Masculino , Motivação , Psicometria , Reprodutibilidade dos Testes , Fatores Sexuais , Inquéritos e QuestionáriosRESUMO
Sacrificial templates for patterning perfusable vascular networks in engineered tissues have been constrained in architectural complexity, owing to the limitations of extrusion-based 3D printing techniques. Here, we show that cell-laden hydrogels can be patterned with algorithmically generated dendritic vessel networks and other complex hierarchical networks by using sacrificial templates made from laser-sintered carbohydrate powders. We quantified and modulated gradients of cell proliferation and cell metabolism emerging in response to fluid convection through these networks and to diffusion of oxygen and metabolites out of them. We also show scalable strategies for the fabrication, perfusion culture and volumetric analysis of large tissue-like constructs with complex and heterogeneous internal vascular architectures. Perfusable dendritic networks in cell-laden hydrogels may help sustain thick and densely cellularized engineered tissues, and assist interrogations of the interplay between mass transport and tissue function.
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Vasos Sanguíneos/citologia , Carboidratos/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Vasos Sanguíneos/fisiologia , Proliferação de Células , Desenho de Equipamento , Hepatócitos/citologia , Humanos , Hidrogéis/química , Consumo de Oxigênio , Perfusão , Impressão Tridimensional , Engenharia Tecidual/instrumentaçãoRESUMO
There is currently a substantial volume of research underway to develop more effective approaches for the regeneration of functional muscle tissue as treatment for volumetric muscle loss (VML) injury, but few studies have evaluated the relationship between injury and the biomechanics required for normal function. To address this knowledge gap, the goal of this study was to develop a novel method to quantify the changes in gait of rats with tibialis anterior (TA) VML injuries. This method should be sensitive enough to identify biomechanical and kinematic changes in response to injury as well as during recovery. Control rats and rats with surgically-created VML injuries were affixed with motion capture markers on the bony landmarks of the back and hindlimb and were recorded walking on a treadmill both prior to and post-surgery. Data collected from the motion capture system was exported for post-hoc analysis in OpenSim and Matlab. In vivo force testing indicated that the VML injury was associated with a significant deficit in force generation ability. Analysis of joint kinematics showed significant differences at all three post-surgical timepoints and gait cycle phase shifting, indicating augmented gait biomechanics in response to VML injury. In conclusion, this method identifies and quantifies key differences in the gait biomechanics and joint kinematics of rats with VML injuries and allows for analysis of the response to injury and recovery. The comprehensive nature of this method opens the door for future studies into dynamics and musculoskeletal control of injured gait that can inform the development of regenerative technologies focused on the functional metrics that are most relevant to recovery from VML injury.